Peptide Hormone Receptors 9783110850246, 9783110107593

Citation preview

Peptide Hormone Receptors

Peptide Hormone Receptors Editors M.Y Kalimi • J. R. Hubbard

W G DE

Walter de Gruyter • Berlin • New York 1987

Editors Mohammed Y. Kalimi, Ph. D. John R. Hubbard, Ph. D. Virginia Commonwealth University Medical College of Virginia Dept. of Physiology Box 551 Richmond, Virginia 23298-0001 U.S.A.

Library of Congress Cataloging in Publication Data Peptide hormone receptors. Includes bibliographies and indexes. 1. Peptide hormones-Receptors. I. Kalimi, M. Y. (Mohammed Y.), 1939- . II. Hubbard, J. R. )John R.), 1954. [DNLM: 1. Peptides. 2. Receptors, Endogenuous Substances. WK102 P4237] QP572.P4P45 1987 612'.405 87-6758 ISBN 0-89925-309-1 (U.S.)

CIP-Kurztitelaufnahme der Deutschen Bibliothek Peptide hormone receptors / ed. M. Y. Kalimi ; J. R. Hubbard. Berlin ; New York : de Gruyter, 1987. ISBN 3-11-010759-7 NE: Kalimi, Mohammed Y. [Hrsg.]

Copyright © 1987 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. Binding: Lüderitz & Bauer Buchgewerbe GmbH, Berlin. - Printed in Germany.

Foreword

It is becoming increasingly clear that receptor proteins play a vital role in the molecular mechanism, physiology and pathology of hormone action. For this reason, a great deal of research has recently been directed towards understanding these receptor molecules. This exciting work has greatly advanced our knowledge in this area, but it is scattered throughout various basic science and clinical journals in such fields as endocrinology, biochemistry, molecular biology, cell biology, and physiology. In order to consolidate and clarify the considerable body of information concerning hormone receptors "Principles of Recepterology",edited by M.K. Agarwal, was published, which focussed on steroid hormone receptors. The present volume deals with the second major category of hormone receptors, namely peptide hormone receptors. These important receptors differ from their steroid counterparts not only in their hormone specificity, but also in their intracellular location and molecular mechanism(s) of action. It is our sincere hope that this text, together with the previous one on steroid receptors, will be of value to both new investigators in the field as well as to those researchers who have already contributed by their researches to the scientific information contained in these volumes. We also wish to express our deep appreciation to the many notable scientists who have participated in the writing of the present volume. Richmond

M.Y. Kalimi J.R. Hubbard

Contents

Growth Hormone Receptors John R. Hubbard

1

Prolactin Receptors:

The Status of Knowledge and Current Concepts

Concerning the Mechanism of Action of Prolactin Raphael J. Witorsch, Jitendra Dave, Robert A. Adler Glucagon Receptor:

63

Structure and Function

Richard T. Premont, Ravi Iyengar

129

Insulin Receptor Sam J. Bhathena

fjg

Adrenocorticotropin Hormone (ACTH) Receptors Nicole Gallo-Payet

287

The Epidermal Growth Factor (EGF) Receptor Venkant R. Mukku, John L. Kirkland, Tsu-Hui Lin, Russell B. Lingham, George M. Stancel

335

Cholecytokinin (CCK) Receptors Michel A. Morency, Ram K. Mishra

385

Vasopressin Receptors Lawrence E. Cornett

437

Parathyroid Hormone Receptors R. A. Nissenson, R. F. Klein

481

Somatomedin Receptors Paul B. Kaplowitz, Steven D. Chernausek

519

LH/hCG Receptors Thomas T. Chen, DeAnna Day Hatmaker

561

VIII Oxytocin Receptors:

A n Overview

Wendell W . Leavitt, John M. Burns, Abraham Alecozay

615

The Calcitonin Receptors Robert W . Downs, Jr

639

Receptors for Angiotensin II C o l l i n Sumners, Melvin J. Fregly

663

Author Index

715

Subject Index

717

GROWTH HORMONE RECEPTORS

John R. Hubbard Departments of Orthopedic Surgery and Biochemistry Brigham and Women's Hospital Harvard Medical School Boston, MA 02115 U.S.A.

I. Introduction II. Measurement of Growth Hormone Receptors III. Relationships Between Receptor Binding and Biological Actions of Growth Hormone IV. Regulation and Clinical Aspects of Growth Hormone Receptors V. Growth Hormone Receptor Purification VI. Growth Hormone Receptor Characterization VII. Preparation and Use of Antibodies to Growth Hormone Receptors VIII. Future Trends in Growth Hormone Receptor Research

I.

INTRODUCTION

Growth hormone (somatotropin or somatotropic hormone) is a protein of about 21,500 daltons, which has many physiological and metabolic functions (1-8).

Growth hormone (GH) promotes the

growth of nearly all body tissues in

vivo .

This occurs by

increased cell size, cell number and extracellular matrix synthesis.

Metabolically, GH (directly, or indirectly) exerts the

following actions: a. increases synthesis of protein, RNA and DNA b. decreases use of carbohydrates for energy and promotes glycogen production c. increases use of fats for energy d. increases retention of calcium and phosphate

Peptide Hormone Receptors © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

2 Many biological effects of GH mimic

ill

vitro .

in

vivo

have been difficult to

For example, the anabolic action of GH on

cartilage and muscle are not clearly evident in systems (2,6).

.in

vitro

This discrepancy has led to the "somatomedin

hypothesis", which states that many effects of GH

in

vivo

(particularly on skeletal growth) occur indirectly via GH-dependent generation of somatomedin (or insulin-like growth factor) which acts directly on cartilage and other tissues (1-13). Somatomedin appears to be produced primarily in the liver, however, the mechanism of somatomedin generation is not completely understood.

Although the somatomedin hypothesis is

commonly accepted, the physiological role(s) and action(s) of somatomedin

iji

vivo

have not been clearly demonstrated.

In

addition, Isaksson et al. (14) reported that human GH injected directly into the proximal growth plate accelerated longitudinal bone growth.

Because of the many important biological effects of growth hormone, elucidation of its mechanism(s) of action is of significant scientific and clinical interest.

Like other peptide

hormones, GH binds to high affinity receptors on target tissues. This binding has been shown to be specific, saturable and reversible.

Hormone-receptor interaction is believed to trigger

a series of biochemical and molecular events which eventually elicit a response at the cell, tissue and organism levels (1-3).

3 Since the interaction of

GH with its receptor appears to be a

vital step in the molecular mechanism of GH action, many studies have been performed to measure, regulate, purify and characterize this protein. However, numerous problems have retarded efforts to characterize these receptors. a. lack of definitive

The major difficulties include: in

vitro

responses to GH in

many systems b. little direct evidence that binding sites mediate GH responses c. cross-receptor binding of GH and prolactin to plasma membrane receptors d. heterogeneity of GH receptor molecules e. prolactin contamination in some GH preparations f. dearth of specific GH antagonists and agonists

Despite these obstacles, considerable progress has been made in GH receptor research.

Because the liver contains a high

concentration of GH receptors and is a well defined target tissue (being the site of GH-dependent production of somatomedin, ATPase, tyrosine amino transferase, ornithine decarboxylase and tryptophan pyrolase production) the hepatic receptors are perhaps the most vigorously characterized.

In this chapter, a comprehensive review of the advances and methodology in GH receptor investigation is provided, with emphasis on the most recent concepts and contributions in the field.

4

II.

MEASUREMENT OF GROWTH HORMONE RECEPTORS

Quantification, purification and characterization of GH receptors requires detection methodologies which are specific, reproducible and sensitive.

Procedures have been developed for a number of

experimental systems such as whole cells, cell membranes, and solubilized receptors.

Preparation of Labeled GH -

To conduct radioreceptor assays for the GH receptor, highly purified radiolabeled and unlabeled GH is required.

In a study

by Maes et al. (15), bovine GH was purified by the method of Dellacha and Sonenberg (16).

In most instances, the GH was

obtained from the National Institutes of Health and the National Pituitary Agency.

GH is generally labeled by iodination with

12 5 I using lactoperoxidase (17-19) and chloramine-T (20-24) methodologies.

Both procedures commonly produce specific

activities in the range of 50-100 >iCi/pg GH (15,25-27). report by

In a

Gordin and Goodman (28), a modified chloramine-T

procedure (20,23) yielded a specific activity of 220-230 uCi/ug, which averaged about 3 iodine atoms per GH molecule.

In most

procedures the labeled hormone was separated from unreacted iodine and hormone aggregates by gel chromatography (such as Sephadex G-50, G-75 or G-110).

The iodinated and polyiodinated

GH molecules have been shown to retain biological activity (29). Just prior to a binding assay, the 125 I-labeled GH is frequently repurified by gel filtration to remove free 125

I-label and damaged hormone.

5 Because of cross-receptor binding, it can be difficult to determine if a GH binding site is somatogenic or lactogenic in nature.

In addition to consideration of the lactogenic or

nonlactogenic function of particular target tissues, the source of GH used can help distinguish these receptor forms as previously described (30).

A. Liver Membrane Receptor Assays-

Liver cell membrane GH receptors have been extensively investigated.

An assay for rat hepatic microsomal membrane

receptors has been described by Baxter et al. (31), using a slightly modified method of Kelly et al. (32). M g C ^ was used to strip bound endogenous hormone from the receptors so that total receptor binding could be determined using radiolabeled GH. Briefly, microsomal membranes (1-2 mg protein) were placed in 3 ml of Tris-HCl buffer, pH 7.4.

To some samples 3.8 M MgCl 2

was added until a final concentration of 3.0 M was achieved. Samples were incubated 5 min at 21°C and then diluted with 9 ml of cold Tris-HCl buffer containing 0.1% BSA and 10 mM CaC^-

The membranes were centrifuged and the pellets washed

with 9 ml Tris-HCl and then sedimented. containing 10 mM

Tris buffer (0.6 ml)

CaCl 2 and 0.1% BSA was added, and membranes

were suspended by glass-glass homogenization.

Aliquots of 0.1 ml

were used for radioreceptor measurements.

125 In the study for Baxter and Turtle (33),

I-human GH was

incubated with rat hepatic membranes (200 ug membrane protein) in

6 300 ul of 25 m M 0.5% B S A . al.

T r i s - H C l , p H 7.4, c o n t a i n i n g 10 m M C a C l 2

S a m p l e s w e r e i n c u b a t e d a t 22°C for 16 h.

and

Maes et

(15) p e r f o r m e d b i n d i n g s t u d i e s o n liver h o m o g e n a t e s , in w h i c h 1

0.2 - 0.3 ng

I - G H w a s i n c u b a t e d w i t h 2.0 mg

(protein)

liver s a m p l e a n d 0-500 ng u n l a b e l e d G H i n 300 ul total v o l u m e of 25 m M

T r i s - H C l , pH 7.4, 0.1% B S A a n d 10 m M C a C l 2 .

w e r e i n c u b a t e d for 2 h at 2 2 ° C .

Cold Tris-HCl buffer was

t h e n a d d e d , a n d h o m o g e n a t e s w e r e t h e n p e l l e t e d by at 1 ,500 X g for 30 m i n a t 4°C. measured

for r a d i o a c t i v i t y .

Samples

centrifugation

The w a s h e d pellet-s w e r e

A p p r o x i m a t e l y 80% of

b i n d i n g w a s m e m b r a n e b o u n d , w h i l e a b o u t 20% w a s

then

receptor

solubilized.

Membrane binding assays have also been performed at

low

t e m p e r a t u r e for 1.5 h w i t h o v e r n i g h t i n c u b a t i o n a n d a t physiological

(37°C) t e m p e r a t u r e for 1.5 h

B. D e t e c t i o n of S o l u b i l i z e d

(34).

Receptors-

In s o m e s t u d i e s G H r e c e p t o r a s s a y s h a v e b e e n c a r r i e d o u t o n solubilized receptors.

In a r e p o r t by M c i n t o s h e t al.

(35),

liver m i c r o s o m a l m e m b r a n e p r o t e i n s w e r e s o l u b i l i z e d by with Triton X-100

(1 m g / m l p r o t e i n )

treatment

in T r i s - H C l , pH 7.5.

Samples

w e r e m i x e d for 30 m i n a n d t h e n c e n t r i f u g e d a t 200,000 X g for 1 IOC hr.

The s u p e r n a t a n t s w e r e a s s a y e d by i n c u b a t i o n w i t h

I-GH

(300-500 pg) in T r i s - H C l , pH 7.5 w i t h 10 m M M g C l 2 , V - g l o b u l i n (0.025%), and 0.1% B S A for 18-24 h a t r o o m Polyethylene glycol

(12.5% final c o n c e n t r a t i o n ) w a s a d d e d a n d t h e

samples were centrifuged. radioactivity.

temperature.

The p e l l e t s w e r e t h e n c o u n t e d

for

7 Similarly, Herington and Veith (25) incubated liver membranes (5-10 mg protein/ml) with 1% (vol/vol) Triton X-100 in 25 mM Tris-HCl, pH 7.4, containing 10 mM CaCl2 and 0.1% (wt/vol) BSA.

Samples were incubated at room temperature for 30 min and

then centrifuged for 2 h at 200,000 X g.

Supernatants were then

used for radioreceptor assays using 150-200 ug protein to 125 30-50,000 cpm hormone(s).

I-GH with and without excess unlabeled The incubation buffer consisted of 0.5 ml of 25 mM

Tris-HCl, pH 7.4, with 10 mM CaCl2 and 0.1% wt/vol BSA with 0.06-0.13% Triton X-100.

The reaction was stopped with 0.5 ml

cold 0.1 M NaH 2 P0 4 , pH 7.5, and then 1 ml of cold 25% (wt/vol)

polyethylene glycol.

Samples were mixed, stood for 30

min at 4°C, and then centrifuged at 1500 X g for 45 min to separate free (supernatant) hormone from bound (pellet). Spontaneous release of GH receptors from human lymphocytes (IM-9) into a soluble fraction was reported by McGuffin et al. (26). Cultured lymphocyte cells were used when they reached a stationary growth phase.

Lymphocyte pellets were washed with

cold phosphate-buffered saline solution (PBS), pH 7.0, and then o suspended (2-3 x 10

cells/ml) in PBS containing the

proteinase inhibitor iodoacetamide (0.02 M).

Cells were then

gently mixed for 90 min at 30°C, after which they were pelleted at 600 X g.

The supernatant was removed and centrifuged

at 20,000 X g for 1 h at 4°C and then for 2 h at 100,000 xg to remove particulate matter. receptor assays.

This supernatant was then used in

About 40% of original receptor binding was

recovered using this procedure. The specific binding was —9 —10 determined by incubating 10 -10 M i-human GH

8 with or without 10~ 6 M unlabeled GH for 90 min at 30°C in phosphate buffered saline (PBS).

Bound and free hormone were

separated at Sephadex G-75 (1 x 55 cm columns) chromatography at 4°C.

C. Binding Assays Using Cell Suspensions and Cell Monolayers-

GH receptors can also be measured using cell suspensions and cell culture monolayers.

A cell suspension assay for rat adipocyte

GH receptors was recently used by Gorin and Goodman (28).

Rat

epididymal fat was cut into small pieces and incubated at 37°C for 20 min in Krebs-Ringer phosphate buffer (KRP), pH 7.4, containing 1 mg/ral crude collagenase, 5.5 mM glucose and 4% BSA.

Cells were filtered through silk and washed several times

with KRP buffer containing 5.5 mM glucose and 1%

BSA.

Cells

were incubated at 37°C in KRP buffer containing 125 I-human GH with 1% BSA and 5.5 mM glucose in the presence or absence of 0.25 mM unlabeled ligand.

After incubation, 100 pi

aliquots were placed into polyethylene microcentrifuge tubes. 125 125 The bound I-GH was separated from free 1-labeled hormone by centrifuging the samples through dinonylphthalate. The containers were cut, and the cell containing upper layer was counted in a gamma camera.

Specific binding was calculated as

total binding (no unlabeled GH) minus binding in the presence of 0.25 mM unlabeled

GH.

Specific binding was commonly about 70%

of total binding.

Because, of the relative ease of obtaining blood samples from patients, GH receptor measurements using blood cells could be of

9 great use in clinical investigations.

In 1981,

Solis-Wallckermann et al. (36) reported the development of a GH receptor assay using human red blood cells.

Erythrocytes were

obtained from heparinized blood and incubated for 150 min at pH 7.5 in HEPES-NaCl containing radiolabeled GH with and without different concentrations of unlabeled ligand.

Erythrocytes were

then separated by centrifugation and the radioactivity was determined.

Recently, Kiess and Butenandt (37) characterized receptor binding in human peripheral mononuclear cells (PMC). using Ficoll-Isopaque centrifugation.

PMC were isolated

Tris-HCl buffer (25 mM at

pH 7.4 containing 120 mM NaCl, 5 mM KC1, 1.2 mM MgS0 4 , 1 mM Titriplex III, 15 mM Na-acetate, 10 mM dextrose, and 1 mg/ml BSA) was added and the cells were suspended in an overnight preincubation without alteration of cell viability.

In the

binding assay approximately 10® PMC were incubated in the Tris buffer (1 ml) containing 10-30 x 10 3 CPM

125

I-human

GH with unlabeled hormones ranging in concentration from 0-800 ng/ml.

The assay incubation was conducted for 120 min at

37 C with constant shaking.

Ice-cold Tris buffer (0.5 ml)

was added at the end of the incubation period and the cells were centrifuged at 280 X g for 10 min. counted for radioactivity.

The cell pellets were then

Binding was shown to be

hormone-specific, reversible and time-dependent (37).

Billestrup and Martin (38) used cell culture monolayers in a study on GH receptors of rat insulinoma RIN-5AH cells.

Briefly,

confluent 60 mm cell culture dishes seeded with RIN-5AH cells

10 were washed with 0.01 M HEPES buffer, pH 7.4, containing 2.5 mM NaH 2 P0 4 , 0.13 mM NaCl, 4.7 mM KC1, 1.24 mM MgS0 4 , 2.5 mM CaCl 2 , and 1% human serum albumin (HEPES buffer).

The

125 monolayer was incubated with about 0.14 ng

I-human GH

(80-90 uCi/ug) generally for 1 h at 37°C in a shaking water bath.

Some samples also received 100 ug GH/ml to determine

nonspecific binding. cold HEPES buffer.

The cultures were then washed 6 times with Cells were removed from the plate by 2+ 2+

incubating with 0.05% trypsin in Ca

- and Mg

-free

Hanks' Balanced Salt Solution with 0.3 mM EDTA for 5 min at 37°C.

Samples were then counted for radioactivity.

Again

specific binding was estimated by subtracting nonspecific binding from total binding. D.

In Vivo Receptor Measurements-

In 1984, Roguin et al. (39) compared the biological activities of chemically modified GH to their binding activities.

The

in

in

vivo

vitro

and

in

binding assay was a

modification of the procedure described by Turyn and (40).

vivo

Dellacha

Anaesthetized Long-Evans female rats were injected with

125

6 I-GH (10 c.p.m.) with or without unlabeled GH or

chemically modified GH into the jugular vein.

Liver samples were

taken 20 min after injection, washed with 0.15 M NaCl, blotted dry and weighed.

Radioactivity was then determined.

While both

active and inactive forms of GH were equally effective in their isolated cell and cell liver membrane binding assays, only the derivatives which increased body weight appeared to bind to receptors

iji

vivo .

Since serum clearance of all GH forms was

11 s i m i l a r , d i f f e r e n c e s in b i o l o g i c a l

iri

vivo

w e r e n o t d u e t o a l t e r a t i o n of h o r m o n e levels. therefore, suggest that

in

vivo

binding Their

receptor binding

correlate closer with biological activity than methods

III.

activities results,

assays

iji

vitro

(40).

RELATIONSHIPS BETWEEN RECEPTOR BINDING AND

ACTIONS OF GROWTH

BIOLOGICAL

HORMONE

Demonstration that receptor binding correlates with a c t i o n s of a h o r m o n e s e r v e s to s u p p o r t the c o n c e p t

biological that

d e t e c t a b l e r e c e p t o r s are i n v o l v e d in the m e c h a n i s m of action.

hormone

W h i l e t h i s h a s b e e n s h o w n for G H r e c e p t o r s i n s o m e

s y s t e m s , t h i s is a n a r e a t h a t n e e d s c o n s i d e r a b l y

more

investigation.

One o f the e a r l i e s t G H r e c e p t o r s t u d i e s w a s in 1973, w h e n Tsushima and Friesen

(41) c o m p a r e d the b i o l o g i c a l p o t e n c i e s

of

five d i f f e r e n t G H s a m p l e s to t h e i r a b i l i t y t o d i s p l a c e 125

I-GH from heptic plasma membrane preparations

from 125

rabbits.

The hormone samples were found to displace

in p r o p o r t i o n to t h e i r b i o l o g i c a l

activities, supporting

I-GH the

relationship between receptor binding and biological actions GH. T h e low level of G H r e c e p t o r s i t e s in e p i p h y s i a l

cartilage

is

c o n s i s t e n t w i t h the lack of d i r e c t m e t a b o l i c e f f e c t s of GH o n cartilage

in

vitro

(42).

of

12 The relationship between receptor binding and biological action of human GH was also examined using cloned rat insulinoma RIN-5AH cells (38).

GH was shown to increase insulin concentration 80%

in these cells, as well as DNA, protein and cell number. Half-maximal effect was found at 10 ng/ml GH, which was approximately the amount needed for half-maximal displacement of 125 I-GH in cell receptor binding studies.

Maximal biological

response occurred with 100 ng/ml GH, which was the concentration needed for maximal high affinity receptor occupancy.

Their

results, therefore, suggest a stoichiometric relationship between GH receptor binding and induction of insulin concentration in RIN-5AH cells.

Their studies also indicated that this biological

action of GH occurred independent of somatomedin production. Insulin-deficient diabetes mellitus is characterized by growth retardation and low sensitivity to GH in many tissues.

In a

study by Baxter et al. (43) streptozotocin-induced diabetic animals were shown to have a reduced number by hepatic GH receptors which was reversed by insulin therapy.

Similarly, a

decreased level of GH receptors was produced by fasting-induced hypoinsulinemia in rats.

While indirect, these experiments

suggest a general correlation between low GH receptor levels and diminished for GH response in diabetic animals.

As previously mentioned in "measurement of growth hormone receptors", in a study by Roguin et al. (39) the biological potency of GH was altered by chemically introducing different numbers of modified residues in GH molecules.

While

ill

vivo

binding assays correlated with the ability of the GH preparation

13 to increase body weight in rats, other

in

vitro

methods using

isolated hepatocytes or hepatic membrane preparation appeared to be far less discriminating.

In other studies, a correlation between receptor binding and biological

action of GH was not apparent.

Cultured preadipose

3T3 cells differentiate into adipose cells in response to GH.

In

a study by Nixon and Green (44) the GH receptors of 3T3 cells of differing susceptibility to GH-mediated transformation was examined.

Both the readily c o n v e r t e d

and i n s u s c e p t i b l e

cells

4

bound approximately 10

GH molecules per cell and had a Kd of

-9 about 10

M.

GH internalization and degradation rates were

also similar.

Thus differences in the biological response of

preadipose 3T3 cells were not clearly apparent, though discriminating mechanisms may have occurred in post-receptor binding events. The relationship between receptor binding and GH action has also been studied in rat adipocytes (45).

Isolated adipocytes showed

insulin-like responses to human GH 3 h after excision, but were refractory by the 4'th hour.

About 20,000 specific GH receptors

were measured per cell in both GH responsive and non-responsive time points.

While studies on the relationship receptor binding and GH action are somewhat conflicting, the "receptor hypothesis" of GH action is widely accepted.

Development of new GH-sensitive

systems may greatly aid research in this area.

in

vitro

In addition,

studies where GH receptor levels and biological action(s) did not

14

correlate may be due to differences or alterations in many other non-receptor properties of the cells.

IV.

REGULATION AND CLINICAL ASPECTS OF GROWTH HORMONE RECEPTORS

It is increasingly evident that GH receptors exist in a dynamic state of flux and regulation.

Because these receptors appear to

be essential to the molecular mechanism of GH action, modulation of these proteins could significantly alter biological responses to GH.

Investigation of the regulatory influences on this

protein is, therefore, of significant experimental and possibly clinical interest.

In this section, information concerning the

regulation (particularly endocrine, age, sex and chemical modulators) of the GH receptor is discussed.

In addition, the

relationship between GH receptor levels and clinical disorders is considered.

A.

Regulation by Growth Hormone-

Many hormones have been shown to regulate their own receptors. For example, insulin down-regulates its receptor in many systems (46-48).

Investigation of the possible control of GH receptors

by the hormone ligand is therefore of considerable interest.

As

with insulin, the GH receptor appears to be down-regulated in some systems (49-54).

For example, Lesniak and Roth (52) found

that human GH depressed IM-9 lymphocyte receptor levels.

The

degree of down-regulation was dose-dependent with 50% reduction

at 2 X 10 ^ M

(5.0 ng/ml, the

in

vivo

basal concentration

of GH) and 80% loss at 20 ng/ml at 30°C steady state condition.

This reduction in receptor binding appeared to be due

to decreased receptor concentration rather than alteration of receptor affinity or cell number.

Removal of GH from the media

restored receptor levels with 50% recovery in 6-8 h and full replenishment by 24 h.

Restoration of the receptors seemed to

require protein synthesis as recovery was significantly inhibited -4 by 10

M cycloheximide (52).

Similar results with IM-9

lymphocytes were reported by Rosenfeld and Hintz (54), who used this phenomenon to develop a radioassay for human GH.

In their

study, 10% receptor loss occurred with 1.25 ng/ml GH, and a 50% decrease was found with 6-8 ng/ml GH (54).

It is of interest to

note that this down-regulation also occurred in response to ovine and human placental lactogen and ovine prolactin (55). Down regulation of fibroblast GH receptors was also observed in human fibroblasts (53).

A 24 h preincubation of cultured

fibroblasts with human GH reduced receptor binding approximately 20% at 5 ng/ml and 55% at 500 ng/ml GH (53).

In many other biological systems, GH appears to actually induce its own receptor levels. al. (56) showed that high

For example, a study by Herington et GH levels produced by transplantable

GH producing tumors correlated with increased rat liver GH receptor binding.

However, injection of 100 or 500 pg of bovine

GH/day for 5-10 days caused no apparent change in GH binding sites.

In 1978, Furuhashi and Fang (57) studied the relationship

between hepatic GH receptors and serum GH levels in normal and

16 GH^ tumor secreting rats.

GH receptors were found to be

elevated in tumor-bearing rats (with high GH levels), suggesting that GH induces its own receptor.

The increased binding was due

to an enhanced number of binding sites, with little or no change in the affinity constant (Ka).

In normal animals, GH receptor

binding correlated with rat serum GH levels (58).

Further evidence that GH enhances hepatic receptors was reported by Vezinhet et al. (58) and Posner et al. (59) using rabbit and sheep.

In their studies, hypophysectomy greatly reduced GH

receptor levels

in both experimental animals.

When

hypophysectomized sheep were injected with 1 mg/kg ovine or bovine GH every other day for 19-21 days, the GH receptor levels were significantly enhanced.

Receptor binding reflected an

alteration of GH receptor concentration, not changes in affinity for the ligand.

However, Herington (30) found no clear relationship between rat hepatic receptor binding and known age-dependent patterns of GH levels, suggesting that endogenous GH may not exert significant control over its own receptor in normal

in

vivo

conditions.

In an investigation by Baxter et al. (60), GH levels were raised about 200-fold in MtT/Wl5 pituitary tumor-bearing Wistar-Furth rats.

Initial liver GH receptor measurements were unchanged in

male rats and decreased by over 75% in female animals in the tumor bearing animals.

However, desaturating the GH receptors by

incubation with 3.2 M MgCl- for 5 min, resulted in GH

17

receptor level measurements 2-3 fold higher in tumor-bearing animals compared to controls.

While this study supports the

contention that GH induces its own receptor, alteration of the receptor levels may have been due to prolactin which is also secreted by MtT/Wl5 tumors.

Further investigation of GH regulation of its receptor was conducted by Baxter et al. (31) using implanted osmotic minipumps to release rat GH or ovine GH into female rats.

Again, M g C ^

treatment was used to release endogenous hormone from receptor sites so that total receptor levels could be measured. At hormone release rates of 150-400 ¿ig rat GH/day the GH receptor levels were enhanced 2-3 fold.

Unlike rat GH, ovine GH infused 75-400

ug/day did not consistently effect GH receptors.

Baxter and

Zaltsman (61) showed that enhancement of GH binding sites by infusion of 200 pg rat GH/day for 7 days occurred in both normal and hypophysectomized rats.

In a recent study by Gorin and Goodman (63), GH receptors were resolved into 3 molecular weight bands by SDS gel electrophoresis (as described in section VI - growth hormone receptor characterization).

After hypophysectomy, GH receptor binding was

reduced approximately 50%, however, no change in the relative proportion of the 3 species was observed.

Recently, Gause and Eden (64) showed that the mode of GH replacement greatly influenced GH receptor modulation in rat adipocytes.

As in many other tissues, hypophysectomy greatly

reduced receptor binding.

The hypophysectomized rats were given

18 T^ a n d c o r t i s o n e t h e r a p y w h i c h o n l y p a r t i a l l y b i n d i n g levels.

restored

G H w a s a d m i n i s t e r e d as 2 i n j e c t i o n s / d a y , 4

i n j e c t i o n s / d a y or by a n o s m o t i c m i n i p u m p i n g s y s t e m

(64).

Rats

i n j e c t e d w i t h G H t w i c e a d a y s h o w e d little a l t e r a t i o n in r e c e p t o r levels.

O n the o t h e r h a n d , G H i n j e c t e d 4 t i m e s a d a y or by

o s m o t i c m i n i p u m p s s i g n i f i c a n t l y e n h a n c e d G H r e c e p t o r l e v e l s if a s s a y e d u p t o 6 h (but n o t 12 h)

post-GH-treatment.

E n d o c r i n e r e g u l a t i o n of n o n - h e p a t i c G H r e c e p t o r s h a s a l s o investigated.

As previously mentioned, hypophysectomy

GH r e c e p t o r b i n d i n g in rat a d i p o c y t e s

(64).

not r e s t o r e d by c o r t i s o n e a c e t a t e t r e a t m e n t

been

depressed

This decrease (50 p g / 1 0 0

g-day),

b u t w a s e n h a n c e d t o a b o u t 50% r e c o v e r y by c o r t i s o n e a c e t a t e T4

(1 p g / 1 0 0 g - d a y ) t r e a t m e n t

In 1983, S t e w a r t et al. r e c e p t o r in m a n .

plus

(64).

(62) s h o w e d t h a t G H c o u l d i n d u c e its o w n

In t h e i r s t u d y , h u m a n G H t r e a t m e n t w a s

administered to GH-deficient children.

2 1/2 h A f t e r

l y m p h o c y t e G H r e c e p t o r s w e r e f o u n d to be e l e v a t e d o v e r and at 5 h t h e y w e r e i n c r e a s e d a l m o s t 3 - f o l d o v e r low values.

was

injection 4-fold, initial

Children injected with chorionic gonadotropin showed no

a l t e r a t i o n of r e c e p t o r

B. E f f e c t of o t h e r

binding.

(non-GH)

hormones-

In a d d i t i o n to G H , s e v e r a l o t h e r h o r m o n e s i n f l u e n c e G H

receptors.

(Many of t h e s e s t u d i e s h a v e u t i l i z e d t h e rat h e p a t i c s y s t e m . ) 1976, H e r i n g t o n et al.

(56) r e p o r t e d t h a t r a t h e p a t i c

receptors

In

19 were enhanced by a 10-12 day treatment with ug/day).

p-estradiol (25

This finding would seem to support other studies which

reported enhanced binding in females (65,66).

However, Furuhashi

et al. (57) found little or no relationship between serum estrogen, corticosterone or prolactin levels and rat liver GH receptor binding.

Interestingly, they also reported an inverse

correlation between GH receptors and rat serum testosterone levels (57).

Baxter and Turtle (33) investigated the effect of diabetes on rat liver GH receptor levels.

In their studies, GH receptors were

reduced about 50-80% in streptozotocin-induced diabetic rats. Insulin treatment significantly restored GH receptor levels. Immunoreactive GH levels were unaltered by streptozotocin, indicating that changes in GH levels did not cause the alteration in receptor binding in the diabetic animals.

Insulin status had

little or no effect on the affinity constant, which was reported 9 -1 at 5.6 x 10 M

(33).

It is also interesting to note

that 3-day fasting in rats caused a 67% decrease in immunoreactive insulin, which was correlated with a fall in hepatic GH receptor binding.

While many interpretations are

possible, it was speculated that hypoinsulinemia produced the fall in GH receptors. Many of the biological actions of GH have been attributed to the generation of somatomedin (1-14).

Studies have, therefore, been

conducted to determine if GH-mediated regulation of GH receptors might be due to somatomedin effects.

In recent studies (31,61)

20 using GH infusion into normal rats, serum GH concentrations and hepatic GH receptors were increased, while somatomedin-C was unaltered or lowered in these normal animals.

These results

suggest that somatomedin is probably not responsible for changes in hepatic GH receptor levels.

C.

Influence of Sex, Age and Pregnancy-

A number of studies have shown an age, sex and pregnancy dependence of GH receptor levels.

Overall, puberty and pregnancy

are characterized by significant increases in GH receptor binding.

While binding does not increase in males after puberty,

females usually show an increase in adulthood.

Examples of

specific findings in these areas of regulation are discussed below.

In 1974, Kelly et al. (65) reported that GH binding sites on liver membranes were higher in female rats (compared to male controls) of all age groups ranging from 10 days to adult (over 40 days).

While binding in female rats increased with age, the

GH receptor binding in male animals remained almost constant.

In

a more recent investigation, DeHertogh et al. (66) found that rat liver GH binding was greater in females between 50-120 days, but binding was about equal to males at ages of 40 days or less. Binding in female samples steadily increased (from about 6-8 fmol/mg protein at 8 days of age to over 60 fmol/mg protein at 120 days) with age.

In the male animals binding rose up to 30-40

days of age (about 6 fmole/mg protein at 8 days to almost 40

21 fmol/mg protein at 35 days), appeared to decrease between 35 and 50 days (down to about 15 fmoles/mg protein), and finally increased again reaching about 30 fmole/mg protein at 120 days. A study by Baxter and Zaltsman (61) showed that induction of GH receptors by infusion of GH occured in both sexes.

In a recent study by Husman et al. (67), no difference in hepatic receptors was found in MgCl 2 -treated samples from male and female rats.

The apparent dissociation constants (Kd) were also -9 essentially identical with 0.24 x 10 M in female and 0.27 x -9 10 M in male rat preparations (67).

Herrington (30) reported that receptor binding (before and after MgCl2 _ treatment) to bovine GH was not significantly different in male and female rats until attaining adulthood; at which time receptor binding was significantly higher in female animals.

The early work of Kelly et al. (65) also showed that rat liver membrane GH binding sites were slightly greater than controls during early pregnancy, and was over 200% of nonpregnant female control values from 20-day pregnant rats.

Similarly, Herington

et al. (56) reported that the hepatic membranes from pregnant rats had about twice the number of GH binding sites than from female controls.

In a recent study (67), 2-4 enhanced binding in

pregnant rat hepatic preparations was found before and after MgCl2~treatment (to remove endogenous ligand).

The Kd of

9 -1 pregnant rat preparation was 0.44 x 10 M , while that of Q nonpregnant rat samples was 0.27 x 10 m~1

22 D. Starvation-

In 1982, Postel-Vinay et al. (68) reported that hepatic GH receptors were low in starved rats.

Thus only 45% of control

binding was observed in microsomal membranes and 52% of control in plasma membrane preparations.

This result was independent of

GH levels since immunoactive GH concentration was the same in fasted and normal rats.

Similar results were reported by another

group ( 69) .

Recently, Gorin and Goodman (73) showed that the relative proportion of 3 different molecular weight forms of the receptor (discussed in section VI - Growth Hormone Receptor Characterization) was not altered by fasting.

23

•Growth hormone Insulin

(31,56-64)

(33)

Estradiol

(56)

Cortisone acetate plus T 4 Females (sex-dependence) Pregnancy

t

(64) (65,66)

(56,67)

GROWTH HORMONE RECEPTOR BINDING LEVEL

I

•Growth hormone

(49-54)

Human placental lactogen (55 Testosterone Starvation

Fig. 1

(57)

(68,69)

Physiological Regulators of Growth Hormone Receptor Binding Levels .

The physiological factors which

increase ( ^ ) and decrease (4r ) GH receptor binding are shown above.

Reference numbers are in

parentheses. Growth hormone has been shown to increase and decrease receptor binding depending on the tissue examined

(*).

24 E.

Chemical modulators-

Several chemical reagents have been shown to alter GH (70) receptor binding.

Such studies can lend insight into:

a) how receptor binding could be altered by physiological regulators b) how receptor binding might be chemically/ pharmacologically regulated

dji

vivo

and

in

vitro c) the chemical nature of the GH receptor and hormone-receptor interaction

In 1976, Van Obberghen et al. (70) investigated the influence of microfilament and microtubule modifying agents on human lymphocyte (IM-9) GH receptors.

When lymphocytes were incubated

with 10 pg/ml cytochalasin A, B, and D, receptor binding was reduced 60%.

This loss of binding was due to a reduction in the

number of binding sites and was not reversible by removal of cytochalasins.

On the other hand, the anti-microtubule reagents

vincristine, colchicine and vinblastine had no apparent effect on the GH receptor binding properties.

These results therefore

suggested that microfilaments, but not microtubules, were involved in expression of GH receptors on the surface of human lymphocytes (70).

In another study, the effect of plant lectin concanavalin A (Con A) on rabbit and rat hepatic GH receptor binding was studied

(71).

Con A depressed binding (about 30%) in particulate and

soluble microsomal membrane preparations in a concentration-dependent manner.

The Con A competitor, o£.-methyl -

mannoside prevented the action of Con A on receptor binding properties.

Con A appeared to have little or no effect on

receptor binding affinity, but depressed the number of binding sites possibly by binding directly to the receptor protein (71).

In a report by Tsim and Cheng (72), the thiol-reactive agent p-chloromercibenzene (1 mM) had no effect on rat hepatic GH receptor binding properties.

On the other hand, this reagent

completely inhibited prolactin binding to hepatic receptors.

Martal et al. (73) chemically modified highly purified GH preparations to study structural components on the GH molecule that are required for receptor binding.

Methylation, ethylation,

quanidination and acetimidination all significantly disrupted binding to liver homogenates.

The lysine or arginine groups at

positions 41, 64, 70 and 115 were implicated as residues that may be important in hormone-receptor interaction.

In another study, methoxylglycyl residues were introduced into the GH molecule to determine their influence on receptor binding (39).

Carboxylate groups were chemically reacted with glycine

methyl ester and water-soluble carbodi-imide.

While modified GH

molecules containing up to 7-8 methoxylcyl residues appeared to have similar potency as native GH in hepatocyte binding assays.

iri

vitro

membrane and

Only forms (3 methoxyglycl residues)

26 which retained biological activity in a growth assay displaced labeled GH in an

in

vivo

binding method.

In a study by Blossey (74), GH binding to rabbit liver membranes was slightly reduced by dithiothreitol, p-mercaptoethanol and N-ethylmelaimide, while enhanced by 20 mM L-cysteine.

Membranes

treated with phospholipase A^, C and D bound hormone similar to controls, while DNase and RNase slightly enhanced binding. Neuraminidase appeared to have no effect on binding, whereas «-and ¿3-galactosidase greatly reduce binding.

A schematic diagram

showing the influence of various chemicals and enzymes on GH receptor binding is shown in Fig. 2.

27

GROWTH HORMONE RECEPTOR BINDING

No Effect

Increase -DNase(74)

-microtubule modifiers (70)

-RNase(74)

-Thiol-reactive

Decrease -microfilament modifiers (70) -reducing agents (74)

p-chloromerci-

-Concanavalin A (71)

benzene (72)

-p-galactosidase (74)

-Phospholipase A 2 ,C, and Neurominidase (74)

Fig. 2.

Chemical and Enzymatic Modulators of Growth Hormone

Receptor

Binding.

The effect of chemical and enzymatic

modulators of GH receptor binding (increase, decrease or have no effect) are shown above.

Reference numbers are in parentheses.

F. Clinical Aspects of Growth Hormone Receptors-

The clinical significance of GH receptor levels and modulation is largely unexplored.

One possible correlation that has been

investigated involves Laron-type dwarfism which is characterized by short stature but high blood levels of GH (1,2,75,111).

The

circulating GH in these dwarfs appears to be active in other systems, and exogenous GH does not enhance somatomedin levels or increase growth rates.

A tissue-level defect was therefore

28 postulated for the cause of this dwarfism.

In a rather

preliminary but provocative clinical study, essentially no GH receptor binding was detected in liver biopsies from two patients with Laron-type dwarfism (75).

On the other hand, liver samples

from all 6 healthy subjects demonstrated a significant number of specific binding sites. The liver from Laron dwarf patients did 125 possess

binding sites for

I-insulin.

These results

support the contention that Laron-type dwarfs may have defective hepatic GH receptors which could account for depressed production of somatomedin (75).

Since GH depresses GH receptor levels in

some (not all) tissues, exogenous GH may not provide help to short children due to receptor reduction.

On the other hand,

short children with low levels of endogenous GH may be more responsive to GH-treatment due to availability of functional receptors (112) . V.

GROWTH HORMONE RECEPTOR PURIFICATION

Purification of GH receptors is important for:

a. chemical characterization of the protein

(including

amino acid composition(s) and sequence(s), subunit analysis, chemical modifications such as phosphorylated residues, and determination of microheterogeneity) b. understanding the physicochemical nature of GHreceptor interaction c. generation of specific antibodies to the GH

29

receptor for further analysis such as subcellular location (see Section VII - preparation and use of antibodies to growth hormone receptors).

Purification of this protein has been difficult, in part due to the problem of separating GH receptors from prolactin receptors. At high concentrations prolactin is able to displace nearly all GH from its liver receptor, and likewise GH is capable of displacing some of the prolactin from prolactin-specific membrane receptors (16).

Radioimmunoassays have indicated that

cross-contamination of the hormones was not the cause of this phenomenon, but rather there sharing.

was a degree of receptor site

In mammary tissue the prolactin receptor appears to be

much more specific (76,77).

After detergent solubilization of the receptor, affinity chromatography using covalently linked GH (usually human) has proven to be one of the most effective procedures in purification of the GH receptor.

Affinity chromatography using Concanvalin A

is also effective since the carbohydrate portion of the receptor binds to this lectin.

For example, Tsushima et al. (110) showed

that GH receptors would bind Concanavalin A, and then could be selectively eluted using methy1-glucoside.

GH receptor purification attempts utilizing affinity chromatography were initiated in the 1970's. Gottsmann et al. (78) solubilized rabbit liver microsomes with 1% Triton X-100 (at 37°C for 30 min). 125

Receptor binding was detected using

I-labeled human GH ligand.

After removal of the Triton by

30 Sephadex G-200 chromatography, receptor purification was performed using affinity chromatography in which 4 mg of human GH was chemically attached to 1 g of CNBr-activated Sepharose 4B. Elution of the receptor was carried out with 4 M ammonium thiocyanate.

Mcintosh et al. (35) solubilized microsomes from livers of pregnant rabbits using Tris-HCl, pH 7.5 containing Triton X-100 (1 mg/mg protein). was solubilized

Approximately 70-80% of the receptor binding

(35).

In order to prepare an affinity column,

human GH (in 0.1 M NaHCO^, pH 8.6) was chemically coupled to the N-hydroxy-succinimide ester of 3,3'-diaminodipropylamino-succinyl agarose (Bio-rad, Affi-Gel 10). 4 h at 4°C glycine (100 ml, 1M) was added.

After mixing for

The affinity gel

was then washed with 6 M guanidine hydrochloride (200 ml), 8 M urea (500 ml), and 0.1 M NaHCC>3 (2000 ml, pH 0.5).

Columns

were equilibrated with 50 mM Tris-HCl, pH 7.0-9.5, containing 0.1% Triton X-100 and 10 mM MgCl 2 observed at pH 7.5.

Maximal binding was

The receptor was eluted using 5 M M g C ^

with a recovery of about 67%.

Unfortunately, these early purification attempts (35,78) did not clearly separate GH receptors from prolactin receptors.

In 1979,

Waters and Friesen (76) reported a technique to correct this problem. The pregnant rabbit liver system was chosen because of the advantage of having a particularly high level of GH receptors (76,79).

The 3-stage purification procedure first utilized human

GH affinity columns.

Separation of prolactin-specific receptors

from GH-specific receptors was achieved by use of Triton, which

31

increased the

of the prolactin-specific receptor 5-fold (5

x 1 0 9 M _ 1 to 2 x 1 0 1 0 M _ 1 ) , while slightly -9 -1 decreasing the

of the GH receptor from 3 x 10

M

9 -1 to 2 x 10 M

.

Under these conditions the affinity of

prolactin and GH receptors for GH differed 10-fold, so that the GH receptor could be eluted from the affinity column with 4 M urea, while the prolactin-specific receptor required 5 M MgC^.

Purification was then continued using preparative

isoelectric focusing and Sepharose 6B gel chromatography

(76).

The GH affinity gels were prepared coupling human GH (hGH) or bovine GH (bGH) to Affi-Gel 10 at pH 8.5 using the procedures provided by Bio-Rad (76).

The reaction was quantitated by use of

125 I-labeled hGH or bGH in the coupling mixture (50 mg samples).

After coupling overnight at 4°C, 5 ml of

ethanolamine-HCl

(1 M, pH 8.5) was added per 10 g of gel to stop

the coupling reaction. 22°C.

This mixture was incubated for 2 h at

The gel (30 ml samples) was then washed with

NaHC0 3 (2000 ml, 0.1 M), 8 M urea (500 ml in 0.1 M NaHC0 3 ), NaHC0 3 (1000 ml, 0.1 M), 5 M guanidine-HCl ml in 0.1 M NaHC0 3 ), NaHC0 3

(300

(1000 ml, 0.1 M), NaCl (1000

ml, 2 M), sodium acetate/acetic acid, pH 4.0 (1000 ml, 0.05 M), NaHCOj ( 1000 ml, 0.1 M), NaH2PC>4 (1000 ml, 0.2 M), Tris-HCl (2000 ml, 0.025 M with 0.1% Triton X-100, pH 7.4) (Tris/Triton), MgCl 2 (500 ml, 5 M in the Tris/Triton X-100 solution) and Tris/Triton X-100 (1000 ml).

To remove "labile"

bound hormone, the gel was incubated for 2 h at 22°C with 2 vol/vol of a rabbit liver soluble membrane preparation in the presence of the proteinase inhibitors Trasylol (5000 Kallikrein

32 inactivity units) and phenylmethanesulfonyl fluoride (0.3 mM) . The gel was then washed with 6 M urea, 5 M MgCl 2 in Tris/Triton X-100 and the Tris/Triton X-100 (2000 ml).

The gel

was washed again with Tris-Triton X-100 just before addition of Triton solubilized samples.

Liver membranes prepared by the

procedure described by Tsushima and Friesen (41).

Membranes were

suspended in 25 inM Tris-HCl buffer at pH 7.4 with a protein concentration of 5-10 mg/ml.

Receptors were solubilized by

addition of Triton X-100 or Triton X-305 (1% v/v final) and mixed for 10 min at 25°C.

In bulk scale purification

(starting

with 250 to 500 g of pregnant rabbit liver) the membranes were initially treated twice with Triton X-305 since it solubilizes fewer membrane proteins than Triton X-100.

Five volumes of

Triton-extract were added per 1 volume of affinity gel and mixed in 10 mM MgCl 2 , Trasylol (5000 Kallikrein inactivity units/100 ml extract), and 0.3 mM PhCH2SC>2F.

After

incubation for 2 h at 22°C the gel was put into a column and washed with over 150 volumes of cold Tris/Triton X-100 solution. The affinity gel was slowly eluted with 3 volumes of 4 M urea in Tris/Triton X-100 solution and samples were collected.

The gel

was then treated with 5 volumes of 6 M urea in Tris/Triton X-100, Tris/Triton X-100 and finally 3 volumes of Tris/Triton X-100 containing 5 M MgCl 2 .

Urea eluted samples (as well as the

others in preliminary studies) were then dialyzed 2 times at 4°C against

Tris/Triton X-100 solution for binding studies

and further purification.

The affinity step yielded

approximately 70-fold purification and SDS gels revealed about 15 protein bands.

33 Further purification by preparative isoelectric focusing showed most GH receptor binding in the pH 4-5 range (76). carried out in 0.1% Triton X-100 for 48 h at 4°C.

Focusing was Binding

assays were performed after adjusting the pH to 7.5 with 2 M NaOH or 2 M HC1 (76) .

The final major purification step utilized preparative Sepharose 6B gel chromatography.

Samples (20 ml aliquots) were filtered on

118 x 2.55 cm columns using a Tris/Triton X-100 buffer.

The

chromatrography was run on a 60 cm head with a flow rate of 17 ml/h at 4°C.

An 8,000-fold purification of the GH receptor

was achieved by Waters and Friesen (76). .

Receptor techniques yielding still greater purification in simpler more efficient systems are still being sought.

One

technique of potentially great usefulness is monoclonal antibody (to GH receptors) affinity columns.

Such a procedure was

initiated by Simpson et al. (80) as described in Section VII (preparation and use of antibodies to growth hormone receptors).

While these studies have been very important in GH receptor research, large scale purification of receptors from various animal and tissue sources is greatly needed in future investigations.

34 Table I Growth Hormone Receptor Purification Studies

Investigators

Year

Receptor Source

Reference Number

Gottsman et al.

1976

rabbit liver

78

Mcintosh et al.

1976

pregnant rabbit liver

35

Waters & Friesen

1979

pregnant rabbit 1 iver

76

Simpson et al.

1983

rabbit liver

80

VI.

GROWTH HORMONE RECEPTOR CHARACTERIZATION

The growth hormone receptor(s) from various animals and tissues has been characterized by a variety of biochemical techniques. In this section the methodologies used to characterize purified and partially purified GH receptors will be discussed.

While

still uncertain, it should be emphasized that multiple receptor forms may exist.

Thus the specific animal, tissue and ligand may

effect data concerning GH receptor properties.

Evidence for GH

receptor heterogeneity comes from antibody studies (see section VII), characterization of receptor forms from different sources, and differential binding of different GH forms.

For example,

receptor binding characteristics of rat GH, 22k human GH and 20k human GH was compared (81).

While each of these forms of GH were 125

equally effective in inhibiting

I-rat GH binding to rabbit

hepatic receptors, the 20k human GH and rat GH were much less

35

potent than the 22k human GH in blocking 22k human GH to the same membranes.

125

I-labeled

This, difference was not due to

lactogenic receptors, but rather the 20k human GH and rat GH were believed to bind to a smaller subset of GH receptors.

In a recent review by Hughes et al. (104) a model was presented in which 3 classes of rabbit liver GH binding sites were identified according to their abilities to bind different GH molecules.

GH receptor-1 was a low capacity binding protein

which bound human GH, rat GH, rabbit GH and a 2 0k varient human GH with similar affinities.

GH receptor-2 was characterized by

high affinity for human GH and low affinity for the other GH molecules.

This second receptor accounted for 85-90% of GH

receptor binding capacity in the rabbit liver membranes.

The

third binding site was identified as a prolactin receptor, which binds human GH and the 20k varient, with low affinity for rat and rabbit GH.

A. General GH Receptor Binding Characteristics-

The basic characteristics of GH to its receptor(s) has been investigated in many systems.

For example, Gavin III et al. (82)

showed that GH receptor binding in isolated rat adipocytes was reversible and time, temperature and pH dependent.

Optimal

specific binding occurred in about 40 min at 37°C, pH 7.4 (82).

Linear Scatchard plots showed a Ka of approximately

9 -1 10 M

with about 15,000 binding sites per cell.

The

adipocyte receptor did not significantly discriminate between

36 rat, monkey, porcine and bovine GH.

Specificity was shown by the

lack of binding to human placental lactogen and prolactin.

In a recent study by Gorin and Goodman

(28) the rate of turnover

of rat adipocyte GH receptors was investigated.

In this report,

preincubation of fat cells with either 20 pg/rnl cycloheximide protein synthesis inhibitor) or 200 jig/ml puromycin

(a

(an inhibitor

of translation) caused a steady loss of specific GH receptor binding following first order kinetics.

Loss of GH receptor

binding had a half-life of approximately 45 min, which was unaltered by the presence or absence of GH.

When fat cells were

treated wth 0.1 mg/ml trypsin for 10 min, receptor binding was destroyed.

However, binding sites returned to near normal

2 h after trypsin removal.

levels

This recovery could be prevented by

addition of cycloheximide after trypsin was removed.

125 Binding of

I-GH to human peripheral mononuclear cells

(PMC) was shown to be maximal at 1 2 h incubation Tr c at 37°C Saturation was found with 25 mg

I-GH per 10

(37).

PMC.

Half-maximal receptor binding inhibition was observed at 12-25 ng unlabeled GH in incubations containing about 10 Tris-HCl buffer.

PMC in 1 ml

The binding was not very sensitive to potassium

concentration or pH, while sodium, calcium, and magnesium ion concentration significantly altered GH binding. 125 Maximal binding of

I-human GH to human

occurred in 2 h at 30°C (53).

fibroblasts

30 ng/ml GH produced

half-maximal binding in these cells.

Scatchard

analysis

indicated a single class of receptor sites with an affinity

37 9 -1 constant of 1.07 x 10

M

.

50% dissociation occured in

about 1.5 h at 3 0°C and 3 h at 15°C.

In addition they

found no apparent change in specific binding with alteration of pH from 7.4-8.9 (53) . 12 5 The time and temperature dependent binding of

I-labeled

human GH to the insulin-secreting cell line RIN-5AH has also been studied (38).

A steady state binding was achieved in 60 min at

37° and 120 min at 24°C.

Approximately 80% dissociation

occured in GH-free media after about 120 min. receptor binding occured with 3 x l O ^ M GH.

Half-maximal The receptor

also bound to rat GH and human placental lactogen, but with less affinity than the human GH.

Scatchard analysis suggested about

2,700 high affinity receptor sites per RIN-5AH cell. GH receptors of rat liver microsomes and golgi fractions were recently characterized by

Husman et al. (67).

Binding was shown

to be protein, time and temperature-dependent, with maximal binding at 15-20 h in microsomal membranes and 15-16 h in golgi fractions at 22°C.

Receptors appeared to be somatogenic, as

50% binding was inhibited by 5-130 ng bovine, rat or human GH, while a much greater amount (500 ng) of rat prolactin was required for 50% displacement.

Treatment of membranes with 3 M

MgCl2, to remove endogenous ligand, enhanced binding 2- to 3-fold.

Subcellular fractionation experiments showed about 20-

to 25-fold higher concentration of receptors in golgi/endosomal preparations compared to total membrane fraction.

Only low

receptor binding was located in lysosomal fractions and

38 non-golgi/endosomal microsomes contained 2-fold enhanced

receptor

binding.

A n i n v e s t i g a t i o n by B u r s t e i n e t a l .

(83) u s i n g m i x e d

recombinants

of h u m a n G H a n d c h o r i o n i c s o m a t o m a m a n o t r o p i n i n d i c a t e d t h a t the initial

134 r e s i d u e s of G H are i n v o l v e d in

interactions.

hormone-receptor

Similarly, an N-terminal fragment

(Mr of

15,000

d a l t o n s ) of h u m a n G H w a s s h o w n to b i n d t o r e c e p t o r s o n IM-9 human lymphoblastic cells

(84).

T a b l e II s u m m a r i z e s some of

various species and tissues where GH receptors have been identified.

the

39 Table II Growth Hormone Receptors Identified in Various Animals and Tissues

Tissue Source

Liver

Animal Source

Reference No.

rat

33,67

rabbit

78

pregnant rabbit

35,76

mouse

106

sheep

105,107

human

108

rat

28,82,86

sheep

107

Lymphocytes

human

62,84

Fibroblasts

human

53

Thymocytes

bovine

109

mouse

109

Insulinoma

rat

38

Peripheral mononuclear eel Is

human

37

Adipose

40 B. Gel filtration-

Gel filtration has been used by many groups to purify and characterize GH

receptors from various sources.

McGuffin et al.

(26) estimated a molecular weight of 200,000 or more for human lymphocyte GH receptors using Sephadex G-200 chromatography of soluble receptors prepared without the use of detergent.

In a

study by Gottsmann and Werder (78), pregnant rabbit liver 125 I-human GH binders were calculated at Mr of about 250,000 and 500,000 on 3 x 80 cm Sephadex G-200. Using a 90 x 1.5 cm Sepharose 6B column, and an elution buffer consisting of 50 mM Tris-HCl, 0.1% Triton and 10 mM MgCl at pH 7.6, Mcintosh et al. (35) calculated Triton solubilized rabbit liver GH receptor Mr to be about 200,000 daltons.

However, due

to association of Triton with the receptor, the Mr of Triton-solubilized receptors may be overestimated

(85).

In a

more recent study by Waters and Friesen (76), the rabbit liver GH receptor was separated from possible contamination and cross-receptor binding from prolactin receptors. Molecular size was estimated using a 118 x 2.55 cm Sepharose 6B column eluted with 0.025 M Tris-HCl containing 0.1% Triton X-100, pH 7.4.

The

column was run with 60 cm head and a flow rate of 17 ml/h at 4

°C (76).

The 8,000 fold purified receptor was calculated to

have a Mr of 300,000 daltons and Stokes radius of 62 A.

A

smaller peak of less than 40,000 daltons was also noted, which could represent dissociated subunits or a receptor cleavage fragment.

41

C.

Electrophoresis-

Electrophoretic analysis has indicated that the GH receptor may contain subunits or is attached to non-receptor proteins.

In a

recent study by Gorin and Goodman (63) rat adipocyte GH receptors 125 were studied by SDS gel electrophoresis.

I-labeled human

GH was cross-linked to intact adipocytes using the bifunctional coupler disuccimimidyl suberate (1 mM). solubilized in 1%

Samples were then

SDS in the presence or absence

of DTT

(usually 100 mM) before applying to 7.5% or 5% polyacrylamide gels.

Electrophoresis separation of proteins was conducted for

4-5 h at 30 mA constant current.

Gel proteins were then

visualized by staining with 0.05% coomassie blue R in 25% propanol-7% acetic acid.

Gels125 were destained with 5%

isopropanol-7% acetic acid. were detected

I-Hormone-receptor complexes

on destained gels by autoradiography.

In the

absence of DTT, 3 radioactive bands were observed with Mr of 56,000, 130,000 and 250,000 on 7.5% gels. 125 unlabeled GH during binding of

The presence of excess

I-GH to adipocytes resulted

in the absence of all three binding species.

Taking into account

the molecular weight of GH (about 22,000 daltons) the receptor proteins averaged Mr of approximately 32,000, 108,000 and over 230,000 (63).

The presence of a reducing agent diminished the

high molecular weight band and enhanced the 130,000 species suggesting that 130,000 molecular weight subunits could be generated from the high molecular weight receptor.

About 42% of

the radioactivity was found in the high molecular weight species, 39% in the 130,000 dalton binder and 19% in the low molecular weight form in the absence of DTT.

When DTT was present 25% of

42 the high molecular weight activity shifted to the 130,000 dalton band.

When the samples were analyzed on 5% gels the high molecular weight band was resolved into 2 bands of 240,000 and 310,000 daltons.

The inclusion of protein inhibitors, N-ethyl maleimide

or sulfhydryl alkylating reagents in the preparation of the receptor did not alter their results, supporting the contention that multiple molecular weight binding species were not artefactually generated (63).

In a later study, Gordin and

Goodman (28) showed that the 56,000, 130,000 and 250-300,000 Mr species were reduced with a similar half-life after treatment of adipocytes with cycloheximide.

Carter-Su et al. (86) also studied the biochemical characteristics of rat adipocyte GH receptors using SDS gel electrophoresis.

Receptors were covalently labeled with

125 I-GH by incubating cells with 0.4 mM disuccinimidy1 suberate for 15 min at 15°C.

The cross-linking reaction was

stopped by addition of excess buffer (10 mM Tris, 0.25 M sucrose, ImM EDTA, pH 7.8).

Autoradiographs of SDS gels revealed a major

band at Mr=134,000 when samples were reduced.

This peak could be

eliminated when samples were incubated with a large excess of unlabeled GH (but not insulin or prolactin).

At low

concentrations of reductant molecular weights of 135,000 and 270,000 daltons were observed, suggesting that the receptor may contain intrachain disulfide bonds.

As the reducing agent was

increased the Mr=270,000 form was reduced with a corresponding increase in the Mr=134,000 species.

After accounting for the

43 weight of GH, the receptor protein (in reduced form) was calculated to be about Mr=112,000.

These results therefore agree

quite well with studies by Gorin and Goodman (63).

125 Disuccinimidy1 suberate was used to covalently couple

I

human GH (5 nM) to rat hepatocytes for biochemical analysis (87). 7.5% Polyacrylamide gel electrophoresis indicated complexes of Mr=220,000 and 300,000.

Reduction with 100 mM dithiothreitol led

to the generation of a Mr=130,000 form with concommitant reduction in the higher molecular weight species.

Subtracting

the molecular weight of GH from this complex, the major reduced binding protein had a molecular weight of about 100,000. In a later study on rat hepatic GH receptors, Yamada and Donner (88) observed multiple binding protein complexes with molecular weights of 300,000, 220,000, 130,000, 65,000 and 50,000 daltons. Unlike the larger complexes, prolactin inhibited binding to the 65,000 and 50,000 dalton species. After accounting for the hormone itself, the receptor proteins were calculated to have Mr of 280,000, 200,000 and 100,000.

In a study (76) using highly purified rabbit liver GH receptors, 3 bands were observed on mercaptoethanol reduced SDS slab gels with the major protein at about 80,000 Mr.

The proteins were

visualized by staining with 0.2% Coomassie blue in 50% trichloroacetic acid.

Destaining was conducted using

methanol/acid acid/water.

A second investigation of rabbit liver

receptors showed major SDS bands at 56,000, 68,000, and 76,000 Mr (74) .

44

A summary of electrophoretic estimations of GH receptor(s) molecular weights is shown in Table III.

Table III

Electrophoretic Estimations of Growth Hormone Receptor Molecular Weights

Reference Number

63

Receptor Source

rat adipocyte

Mr of Growth Hormone Receptor Not Reduced

Reduced

32,000

32,000

108,000*

108 ,000*

240,000*

230,000

310,000* 86

rat adipocyte

112,000

112,000

250,000 87

rat liver

200,000

100,000

280,000

88

rat liver

100,000 200,000 280,000

76

rabbit liver

80,000

74

rabbit liver

56,000 68,000 76,000

•designates the major receptor form

100,000

45 D.

Isolectric Focusing-

Waters and Friesen (76) characterized highly purified rabbit liver GH receptors by analytical slab gel isoelectric focusing. Gels consisted of riboflavin-polymerized 3.5% acrylamide, 0.1% methylene bisacrylamide and 0.1% Triton X-100. 3.5 to 9.0 was used.

Samples were applied to the area

corresponding to pH 7.0. h at 8 watts.

A pH gradient of

Focusing was conducted in 4 C for 4

0.5 cm samples were taken from the gel and eluted

at 4°C in 0.1 M Tris/Triton buffer, pH 7.5.

The pi of the

Triton X-100 solubilized receptors was approximately 4.6 with considerable charge heterogeneity.

Interestingly, treatment of

the receptor preparation with neuraminidase for 30 min at 37°C at pH 5.85 caused a shift in the pi to about 6.2 with slightly less charge heterogeneity, suggesting that the receptor is a sialoglycoprotein and that differential sialic acid content contributed to the observed heterogeneity seen with isoelectric focusing.

Further evidence that the GH receptor is a glycoprotein was indicated by the decreased number of IM-9 lymphocyte receptor sites resulting from treatment with tunicamycin, an antibotic which blocks

E.

N -glycosylation (89).

Two-dimensional gel electrophoresis-

Two-dimensional gel electrophoresis is a very powerful tool in characterizing proteins.

In a study by Carter-Su et al. (86),

two-dimensional gel electrophoresis was used to study the

46 adipocyte GH-receptor complexes. with 60 ng/ml

125

Rat adipocytes were incubated

I-human GH for 2 h at 37°C.

Cells were

incubated with the cross-linking agent, ethylene glycol bis (succinimidyl succinate) prior to plasma membrane preparation. Samples were reduced with 10 mM dithiothreitol and solubilized with 1% sodium dodecyl sulfate.

Analysis revealed that the

mononeric 22,000 Mr human GH molecule bound to a membrane receptor protein of approximately 112,000 Mr in reduced form assuming a stoichiometry of 1:1 for hormone and receptor.

While

this molecular weight is larger than that reported using pregnant rat and rabbit liver (90), rabbit liver (90,91) and rabbit mammary glands glands (90), these smaller molecular weight proteins may represent lactogenic GH receptor molecules which bind prolactin with even higher affinity than GH.

Prolactin rat

liver receptors of about 60,000 and 37,000 Mr have previously been reported

VII.

(92,93).

PREPARATION AND USE OF ANTIBODIES TO GROWTH HORMONE

RECEPTORS

Preparation of specific antibodies to the GH receptor(s) is of great importance for investigating the structure, function, heterogeneity, location and molecular mechanism of the GH receptor.

Initial experiments generated polyclonal antibodies,

and later monoclonal techniques yielded antibodies of much greater specificity to allow more definitive interpretation of results.

47 A.

Polyclonal Antibodies to GH Receptors-

In 1978, Tsushima (94) reported generation of polyclonal antibodies against rabbit liver GH receptors.

However, the

purity and specificity of this receptor preparation (purified by chromatography on concanavalin-A Sepharose, DEAE-cellulose, and Sepharose 6B) was unclear, particularly in light of hormone cross-binding of prolactin and GH receptors (94,95).

Later, Waters and Friesen (95) produced antibodies in guinea pigs to rabbit liver GH receptors.

The receptors were purified using

a receptor-specific affinity chromatograph technique (76,95).

In

this procedure (95), female guinea pigs were innoculated dorsally at 10-20 intradermal sites with 10-50 >ig samples of purified rabbit liver GH receptor preparations in 2 volumes of Freund's adjuvant (total volume of 1.5 ml). 14-day intervals.

Injections took place at

Following 3 or more injections the animals

were bled every 14 days.

Crude Y -globulin samples were prepared

by ammonium sulfate precipitation using 1 volume of saturated ammonium sulfate in 0.05 M sodium phosphate buffer, pH 7.4.

The

immunoactivity precipitated in the 20-40% ammonium sulfate fraction.

Precipitates were washed 2 times and dialyzed against

10 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl. Th antisera generated blocked binding of

1 9S i ovine GH and

125 I-ovine prolactin to liver membrane receptors, but did not inhibit binding of 125 I-ovine prolactin to rabbit specfic 125 mammary gland GH receptors,

I-insulin to human placental

membrane and rabbit liver membranes, or ^^i-bovine follitropin to porcine testicular cell membranes.

Similarities

48 of immunological determinants of hepatic GH receptors from rabbits, mouse, human, and sheep species were indicated in inhibition studies using this antisera. B.

Monoclonal Antibodies to GH Receptors-

In 1983, Simpson et al. (80) utilized hybridoma technology

to

generate specific monoclonal antibodies to rabbit liver GH receptors.

GH receptors were purified using human GH affinity

columns and Sepharose 6B chromatography and Friesen (76).

by the method of Waters

Female CB5F^/j mice were immunized with 26

ug GH receptors injected s.c. in Freund's Complete Adjuvant every 7 days up to the 28th day.

Animals were then injected i.p. with

260 ug GH receptor 4 times every 14 days.

Mice were killed 3

days after the final injection and the spleens were aseptically excised.

In polyethylene glycol fusion procedures, 25 x 10^ P3x20 g myeloma cells were added to about 10

isolated spleen cells

and pelleted by centrifugation (80).

RPMI/1640 (2 ml) containing

7.5% (vol/vol) dimethyl sulfoxide and 50% (wt/vol) PEG were added to the pellet.

The cells were then carefully resuspended for30

sec and allowed to stand for another 30 sec.

While gently

agitating the cells, 10 ml of fresh media were added an ml at a time over a 90 sec period.

After allowing the cells to stand for

2-3 min, the cells were centrifuged and then resuspended in 5 ml of medium.

In cloning procedures, 50 ul of cells were placed in

four 24-well plates with 1 ml HAT medium containing approximately 10^ spleen cells obtained from mice that had not been

49 immunized (as feeders).

RPMI/1640 (0.5 ml) containing

aminopterin, hypoxanthine, and thymidine was added on days 5-7 when needed.

After 10-11 days hybridomas were confluent, and 0.5

ml of media were removed for analysis of antibody production. Positive wells were cloned twice by limiting dilution.

About

10 7 monoclonal cells in 0.5 ml medium were injected i.p. into CB6F-^/j mice (previously injected i.p. with 0.5 ml 2,6,10,14-tetra- methylpentadecane) to prepare ascite fluid.

The

125 antibody activity was detected by inhibition of binding to rabbit liver membranes.

1-human GH

At a dilution of 1:10,000

ascitic fluid approximately 50% inhibition was obtained, and 95% inhibition was observed at higher concentrations (80). Experiments using protein A-Sepharose and ELISA assays for mouse Ig subclasses indicated that the antibody was an IgE type protein. Simpson et al. (80) used the monoclonal antibodies to prepare an immunoaffinity column for GH receptor purification studies.

For

preparation of this affinity gel, 82 mg of antibody protein was precipitated with saturated (NH^^SO^ and then chemically coupled to 10 ml of activated Sepharose-4B using procedures provided by Pharmacia.

The antibody affinity gel (1

ml) was put into a disposable pipette tip with a Tris 0.1% Triton X-100 buffer.

250 ml of unpurified solubilized receptor

preparation were added to the gel and incubated for 48 h at 4°C.

The antibody activity could be dissociated with 0.1 M

glycine, producing about 10-fold purification.

The production of monoclonal antibodies to affinity purified rabbit liver GH receptors (76) was also reported by Barnard et al. (96).

In their procedure male BALB/C mice were given three

20 jug (emulsified in complete Freund's adjuvant) injections of purified rabbit GH receptor protein every 2 weeks.

Two weeks

later, an iv boost injection of 10 ;ug in saline was given.

Four

days later, the serum of these immunized mice, at 1:800 dilution 125 was able to inhibit 50% of

I-human GH binding to membrane

receptors. The splenic lymphocytes from 3 mice were mixed in culture medium (RPMI-1640 containing 15% fetal calf serum, 2 x 10 _ 3 M glutamine, 100 IU/ml penicillin and 100 ug/ml streptomycin), g About 10

splenic white blood cells (SWBC) were fused with 25

x 10 6 NS-1 myeloma in a 42% (wt/vol) of PEG/RPMI-1640 with 15% (vol/vol) dimethyl sulfoxide. pelleted.

The cells were washed and

0.05 ml of cell suspension were added to 24-well

plates containing 10 6 feeder cells (BALB/C SWBC) in 1.0 ml of -4 -5 culture medium containing 10 M hypoxanthine, 1.6 x 10 M thymidine, 4 x 10 ^M aminopterin, and 2-mercaptoethanol

(HAT medium).

4x10

Cells were incubated at 37°C

and refed on day 5 and then when needed.

To screen for antibody

production Barnard et al. (96) used the immunoprecipitation method described by Waters and Friesen (95). antibody were subcloned by limiting dilution.

Clones producing The best antibody

producers were injected into pristene-primed BALB/C mice in orde to generate ascitic fluid.

The antibodies were extracted from

the ascitic fluid by ammonium sulfate precipitation or DEAE chromatography.

125 I-antibody purity was determined by

51

quantitation of the maximum degree of precipitability of 125

I-antibody using specific antimouse immunoglobulin G.

In a recent study, four monoclonal antibodies raised against rabbit liver GH receptors and one to rat liver receptors were used as probes for investigating structural heterogeneity of GH receptor molecules (97).

Using these antibodies to

immunoprecipitate solubilized receptors and inhibit binding 125 of

I-ovine GH to membrane binding sites and solubilized

receptors , Barnard et al. (97) proposed three types of GH receptors in rabbit hepatic plasma membranes.

Type 1 receptors

were postulated to be involved in the anabolic action of GH and reacts with all the monoclonal antibodies tested.

Type 2 binding

sites did not possess the epitope for the anti-(rat GH receptor) antibody in the GH-binding region of the molecule and was believed to be the cytosolic GH receptor, although it was found in the plasma membrane.

A third binding site (type 3) was lost

during purification procedures and did not contain the epitope for an anti-(rabbit GH receptor) monoclonal antibody.

The rabbit

plasma membrane appears to contain approximately 30% type 1, 50% type 2, and 20% type 3 receptors (97).

As can be seen in Table

IV below, antibody work on GH receptors is still rather limited. Generation of antibodies to GH receptors from sources other than rabbit liver (particularly human sources) should be initiated.

52 Table IV

Generation of Antibodies to Growth Hormone Receptors

Reference Number

C.

Type of Antibody

Source of Receptor

94

polyclonal

rabbit liver

95

polyclonal

rabbit liver

80

monoclonal

rabbit liver

96

monoclonal

rabbit liver

97

monoclonal

rabbit liver

Monoclonal Antibodies to GH-

Retegui et al. (98) used monoclonal antibodies to human GH to study hormone-receptor interaction.

They found that the Fab

fragments of three monoclonal antibodies blocked hormone binding to IM-9 human lymphoid and pregnant rabbit liver receptors. Similar inhibitory potencies were obtained in both the liver and lymphocyte systems indicating that both binding sites with the same area of the GH molecule.

may react

Their inhibitory

antibodies also reacted to synthetic peptides corresponding to residues 19-128, 73-128 and 98-128 suggesting that the amino portion of the GH molecule participates in receptor binding (98). Monoclonal antibodies against human GH were also used by Cadman et al. (99) to study hormone-receptor interactions.

A concern

with these studies was that the antibodies would not identify

53 sites on the GH molecule that were specific for GH and prolactin receptors, possibly due to overlapping binding regions on the GH molecule for these two receptors (85).

VIII.

FUTURE TRENDS IN GROWTH HORMONE RECEPTOR RESEARCH

Although considerable progress has been made in the study of GH receptors, many basic science and clinical questions are yet to be resolved and pose an exciting challenge to the research community.

For example, few studies have demonstrated a clear

relationship between GH-receptor interaction and biological responses to GH.

The role and importance of the receptor protein

is therefore mostly speculative.

In addition, little is known

about the precise chemical nature (such as amino acid sequence and 3-dimensional conformation) of GH receptors and whether these differ significantly between species and tissues.

The sequence

of molecular events ranging from control factors influencing expression of the GH receptor gene, to transcription of the GH receptor RNA and post-translational modifications are all yet to be elucidated.

The chemical interaction between GH and its receptor is also a subject of interest.

The key functional groups on each molecule

and the nature of their orientation with one another is only beginning to be explored.

Since the GH receptor is located in

the cell membrane, investigation of how the membrane environment influences GH receptor properties could prove valuable.

The fate

of GH and receptor molecules after interaction has occurred must

54 also be studied and could lend insight into molecular mechanism on turnover of these proteins.

While many groups have begun to explore regulators of GH receptors, the actual physiological and pathophysiological role these factors play

in

vivo

is largely unknown.

It is quite

likely that many other endocrine and paracrine regulators of GH receptor expression are yet to be discovered.

In addition, the

synergistic and antagonistic interplay between these modulators has not been investigated.

The mechanism by which these

regulators alter binding levels is essentially unexplored. Control may occur at synthesis, degradation, chemical modification or internalization.

For each of these possible

mechanisms, modulating enzymes can be envisioned which are as yet unidentified and characterized.

Another aspect of regulation

which is to be rigorously investigated is how GH receptor modulation may be involved in various disease states.

Elucidation of how the GH receptor fits into the biochemical mechanism of GH action is of great interest.

That is, how does

the binding of GH to its receptor lead to molecular, biochemical, cellular, physiological and anatomical changes of the target tissues?

A great gap in understanding still exists concerning

the events that occur after GH-receptor interaction.

Unlike some

other peptide hormones which cause the production of specific effector second messengers such as cAMP, a definitive second messenger generated after GH-receptor interaction has not been found.

Our understanding of the role of the receptor in GH

mechanism must therefore continue to evolve.

For example, a

55 recent report by Fletcher and Greenan (100) discussed the role of human chorionic gonadotropin (hCG) receptor occupancy.

In their

study, they observed that cells did not have to bind hCG to have a hormone-like response if they contacted a cell which had hormone-receptor interaction.

Whether similar processes exist

for GH-receptor interaction are not known.

Studies of

receptor-mediated second messengers must continue to be explored, since hormone-receptor interaction is surely only the first step in a cascade of molecular processes which regulate cell and tissue activity.

In light of he tyrosine-specific protein kinase

activities associated with certain recptors, growth factors and viral oncogenes, studies on GH receptor-depedent covalent modifications may produce important information about receptor mehanism

(85,101-103).

Two recent achievements may be of particular importance in resolving many questions about the GH receptor.

First the

purification of GH receptors is of significant importance for chemical characterization studies and generation of specific antibodies to the receptor protein.

Secondly, the greatly

expanded preparation and use of monoclonal antibodies to the GH receptor is likely to be of great importance in future research. The monoclonal system can provide essentially unlimited amounts of highly specific antibodies for GH receptor study.

It is

possible for example, that these antibodies could be used in the development of radioimmunoassays for easier measurement of GH receptors, as well as used in GH receptor localization, purification and characterization.

56 ACKNOWLEDGEMENTS

I would like to thank Danette R. Mattmueller and Dr. James J. Steinberg for their critical reading of this review, and Terri Sayles for preparation of the manuscript.

REFERENCES 1.

Williams R.H. In: Textbook of Endocrinology, W.B. Saunders Co., Philadelphia.

2.

Hubbard, J.R., M.Y. Kalimi, R.J. Witorsch. In: Review of Endocrinology and Reproduction, The Renaissance Press, Inc., Richmond (in press).

3.

Guyton, A.C. 1976. In: Textbook of Medical Physiology, W.B. Saunder Co., Philadelphia.

4.

Hubbard, J.R., J.P. Liberti. 1982. Endocrinology

5.

Hubbard, J.R., J.P. Liberti. 1981. Biochim. Biophys. Acta 332-338.

6.

Isaksson, O.G.P., S. Eden, J.O. Jansson. 1985. Ann. Rev. Physiol. £7 , 483-499.

7.

Alzuler, N. 1974. In: Handbook of Physiology (Knobil, E. , Sawyer, W.H., eds.) Vol. 4, pp 233-252, Am. Physiol. Soc., Washington, D.C.

8.

Daughaday, W.H. 1981. In: Endocrine Control of Growth W.H., ed.) pp 1-24, Elsevier, New York.

9.

Salmon, W.D., Jr., W.H. Daughaday. 1957. J. Lab. Clin. Med. 825-836.

110 , 1483-1488. 673 ,

(Daughaday, 49 ,

10.

Hubbard, J.R., J.P. Liberti. 1980. Biochim. Biophys. Acta 207-214.

627 ,

11.

VanWyk, J.J., L.E. Underwood. 1978. In: Biochemical Actions of Hormones (Litwack, G., ed.) Vol 5, Academic Press, New York.

12.

Philips, L.S., R. Vassilopaulou-Sellin. 1980. N. Engl. J. Med. 307 , 371-380.

13.

Liberti, J.P., M. Miller. 1980. J. Biol. Chem.

14.

Isaksson, O.G.P., J.O. Jannson, I.A.M. Gause. 1982. Science 1237-1239.

255 , 1023-1029. 216 ,

57 15.

Maes, M., R. DeHertogh, P. Watrin-Grangen, J.M. Ketelslegers. 1983. Endocrinology 113 , 1325-1332.

16.

Dellacha, J.M.,M. Sonenberg. 1964. J. Biol. Chem.

17.

Ketelslegers, J.M., K.J. Catt. 1974. J. Clin. Endocrinol. Metab. 39 , 1159-1162.

18.

Thoreil, J.I., B.G. Johansson. 1972. Biochim. Biophys. Acta 363-369.

19.

Shiu, R.P.C., P.A. Kelly, H.B. Friesen. 1973. Science 971.

20.

Lesniak, M.A., J. Roth, P. Görden, J.R. Gavin III. 1973. Nature (New Biol) 241 , 20-21.

21.

Hunter, W.M., F.C. Greenwood. 1962. Nature

22.

Greenwood, F.C., 114.

23.

Roth, J. 1975. In: Methods in Enzymology, part B. Vol 37, pp 223, Academic Press, New York.

24.

Apinyocharti, P., R.C. Baxter. 1979. Clin. Chem.

25 , 161-163.

25.

Herington, A.C., N.M. Vieth. 1977. Endocrinology

101 , 984-987.

26.

McGuffin, W.L., J.R. Gavin III, M.A. Lesniak, P. Görden, J. Roth. 1976. Endocrinology 98 , 1401-1407.

27.

Baxter, R.C., J.M. Bryson, J.R. Turtle. 1980. Endocrinology 1176-1181.

28.

Gorin, E., H.M. Goodman. 1985. Endocrinology

116 , 1796-1805.

29.

Goodman, H.M.,L.K. Levy. 1983. Endocrinology

113 , 2017-2023.

30.

Herington, A.C. 1982. Horm. Metabol. Res.

31.

Baxter, R.C., Z. Zaltsman, J.R. Turtle. 1984. Endocrinology 1893-1901.

32.

Kelly, P.A., G. Leblanc, J. Djiane. 1979. Endocrinology 1631-1638.

33.

Baxter, R.C., J.R. Turtle. 1978. Biochem. Biophys. Res. Commun. 84 , 350-357.

34.

Gerasino, P., J. Djiane, P.A. Kelly. 1979. Mol. Cell. Endo. 11-23.

35.

Mcintosh, C., J. Warnecke, M. Nieger, A. Barnes, J. Kobberling. 1976. FEBS Lett. 66 , 149-154.

239 , 1515-1520.

251 ,

180 , 968-

194 , 495-496.

W.M. Hunter, J.S. Glover. 1983. J. Biochem.

89 ,

107 ,

1_4 , 422-424. 114

104 ,

,

36.

Solis-Wallckermann, J., 0. Robinson, M. Caballero, R. Barbosa, E.S. Canaley, C.Y.A. Zarate. 1981. Rev. Invest. Clin. _33. , 37-43.

37.

Kiess, W., 0. Butenandt. 1985. J. Clin. Endo. Met. 746.

38.

Billestrup, N., J.M. Martin. 1985.

39.

Roguin, L.P., J.M. Delfino, N. Vita, J. 224 , 535-540.

40.

Turyn, D., J.M. Dellacha. 1978. Endocrinology

41.

Tsushima, T., H.G. Friesen. 1973. J. Clin. Endocrinol. Metab. 334-337.

42.

Kan, K.W., R.L.Cruess, B.I. Posner, H.J. Guyda, S. Solomon. 1983. J. Endocr. 103 , 125-131.

43.

Baxter, R.C.,J.M. Bryron, J.R.Turtle. 1980. Endocrinology 1176-1181.

44.

Nixon, T., H.Green. 1983. J. Cell Physiol.

45.

Grichting, G., L.K. Levy, H.M. Goodman. 1983. Endocrinology 111-1120.

46.

Gavin, J.R.,J. Roth, D.M. Neville, P. DeMeyts, N. Buell. 1974. Proc. Natl. Acad. Sei. USA J 1 > 84-88.

47.

Blackard, W.G., P.S. Guzelian, M.E. Small. 1978. Endocrinology 103 , 548-553.

48.

Vigneri, R. , N.B. Pliam, D.C. Cohen, V. Pezzino, K.Y. Wong, I.D. Goldfine. 1978. J. Biol. Chem. 253 , 8192-8197.

49.

Picard, F., M.C. Postel-Vinay. 1984. Endocrinology

50.

Messina, J.L., J.L. Kostyo, S. Eden. 1982. 64'th Annual Meeting of the Endocrine Society, San Francisco, CA, pp 99 (Abstract).

51.

Smal, J., J. Closset, G. Hennen, P. Demeyts. 1985. Biochem. J. 225 , 283-289.

52.

Lesniak, M.A., J. Roth. 1976. J. Biol. Chem.

53.

Murphy, L.J., E. Vrhovseh, L. Lazarus. 1983. Clin. Endocrinol. Metab. 57 , 117-1124.

54.

Rosenfeld, R.G., R.L. Hintz. 1980. J. Clin. Endocrinol. Metab. 50 , 62-69.

55.

Lesniak, M.A., P. Görden, J. Roth. 1977. Metab. 44 , 838-849.

56.

Herington, A.C., L.S. Phillips, W.H. Daughaday. 1976. Metabolism 25, 341-353.

Endocrinology

60^ , 740116 , 1175-1181.

A.C. Paladini. 1984. Biochem. 103 , 1190-1195. 37 ,

107 ,

115 , 291-296. 113 ,

114 , 1328-1333

251 , 3730-3729.

J. Clin. Endocrinol.

59 57.

Furuhashi, W., V.S. Fang. 1978.

Endocrinology 103 , 2053-2060.

58.

Vezinhet, A., B. Posner, B. Patel, J. Charrier. 1980. Reprod. Nutr. Develop. 20 , 289-294.

59.

Posner, B.I., B. Patel, A. Vezenhet, J. Charrier. 1980. Endocrinology 107 , 1954-1958.

60.

Baxter, R.C., Z. Zaltsman, J.R. Turtle. 1982. Endocrinology

61.

Baxter, R.C., Z. Zaltsman. 1984. Endocrinology

62.

Stewart, C., S. Clejan, F. Fugler, T. Cheruvanky, P.J. Collipp, 1983. Arch. Biochem. Biophysics 220 , 309-313.

63.

Gorin, E. , H.M. Goodman. 1984. Endocrinology

64.

Gause, I., S. Eden. 1986. Endocrinology

65.

Kelly, P.A., B.I. Posner, T. Tsushima, H.G. Friesen. 1974. Endocrinology 95 , 532-539.

66.

DeHertogh, M., P. Watrin-Granger, J.M. Ketelslegers. 1983. Endocrinology 113 , 1325-1332.

67.

Husman, B., G. Andersson, G. Norstedt, J.A. Gustafsson. 1985. Endocrinology 116 , 2605-2611.

68.

Postel-Vinay, M.C., E.C. Cohen-Tanugi, J. Charrier. 1982. Mol. Cell. Endo. 2j3 , 657-669.

69.

Grichting, G., V. Coiro, H.M. Goodman. 1983. 65th Annual Meeting of the Endocrine Society, San Antonio, TX pp 216, Abst. #542.

70.

Van Obberghen, E., P. DeMeyts, J. Roth. 1976. J. Biol. Chem. 6844-6851.

71.

Herington, A.C., D. Elson. 1979. Mol. Cell. Endo.

16 , 91-97.

72.

Tsim, K.W.K.,C.H.K. Chong. 1984. Mol. Cell. Endo.

¿8 , 61-66.

73.

Martal, J., N. Chene, P. delaLlosa. 1985. FEBS Letters 295-297.

74.

Blossey, H.L. 1979. Horm. Metab. Res.

75.

Eshet, R. , Z. Laron, A. Pertzelan, R. Arnon, M. Diatzman. 1984. Isr. J. Med. Sei. 20 , 8-11.

76.

Waters, M.J., H.G. Friesen. 1979. J. Biol. Chem.

77.

Shiu, R.P.C., H.G. Friesen. 1974. Biochem. J.

78.

Gottsmann, M., K.V. Werder. 1976. 202, 40-41.

1020-1022.

111 ,

115 , 2009-2014.

114 , 1279-1286.

118 , 119-124.

251 ,

180 ,

1J, , 616-621.

254 , 6815-6825.

140 , 301-311.

Acta Endocrinol.

82 , Suppl.

79.

Posner, B.I., P.A. Kelly, R.P.C. Shiru, H.G. Friesen. 1974. Endocrinology 96 , 521-531.

80.

Simpson, J.S.A., J.P. Hughes, H.G. Friesen. 1983. Endocrinology 111 , 2137-2141.

81.

Hughes, J.P., E. Tokuhiro, J.S.A. Simpson, H.G. Friesen. 1983. Endocrinology 113 , 1904-1906.

82.

Gavin III, J.R., R.J. Saltman, S.E. Tollefsen. 1982. Endocrinology 110 , 637-643.

83.

Burstein, S., M.M. Grumbach, S.L. Kaplan, C.H. Li. 1978. Proc. Natl. Acad. Sei. 75 , 5391-5394.

84.

Aston, R., J. Ivanyi. 1983. EMBO J.

85.

Hughes, J.P., H.G. Friesen. 1985. Ann. Rev. Physiol.

86.

Carter-Su, C., J. Schwartz, G. Kikuchi. 1984. J. Biol. Chem. 1099-1104.

87.

Donner, D.B. 1983.

88.

Yamada, K., D.B. Donner. 1984. Biochem. J.

89.

Keefer, L.M., P. DeMeyts. 1981. Biochem. Biophys. Res. Commun. 101 , 22-29.

90.

Hughes, J.P., J.S.A. Simpson, H.G. Friesen. 1983. Endocrinology 112 , 1980-1985.

91.

Tsushima, T., H. Murakami, K. Wakai, O. Isozaki, Y. Sato, K. Shizume. 1982. FEBS Lett. 14J7 , 49-53.

92.

Borst, D.W., N.L. Sayare. 1982. Biochim. Biophys. Res. Commun. 105 , 194-201.

93.

Liscia, D.S., B.K. Vonderhaar. 1979. Proc. Natl. Acad. Sei. U.S.A. 79 , 5930-5934.

94.

Tsushima, T. 1978. In: Proceedings of the Sixth Asian and Ocean Congress of Endocrinology, Singapore Vol. I, pp 365-373, Singapore.

95.

Waters, M.J., H.G. Friesen. 1979. J. Biol. Chem.

96.

Barnard, R., P.G. Bundesen, D.B. Rylatt, M.J. Waters. 1984. Endocrinology 115 , 1805-1813.

97.

Barnard, R., P.G. Bundesen, Biochem. J. 231 , 459-468.

98.

Retegui, L.A., P. DeMeyts, C. Pena, P.L. Masson. 1982. Endocrinology 111 , 668-676.

99.

Cadman, H.F., M. Wallis, J. Ivanyi. 1982. FEBS Lett.

J. Biol. Chem.

2 , 493-497. £7 , 469-482. 259 ,

258 , 2736-2743. 220 , 361-369.

254 , 6826-6832.

D.B. Rylatt, M.J. Waters. 1985.

137 , 149-152.

61

100. Fletcher, W.H., J.R.T. Greenan. 1985. Endocrinology 1662. 101. Carpenter, G. 1983. Mol. Cell. Endocrinol.

116 , 1660-

21 ' 1-19.

102. Jacobs, S., F.C. Kull Jr., H.S. Earp, M.E. Svoboda, J.J. Van Wyk. 1983. J. Biol. Chem. 258 , 9581-9584. 103. Marx, J.L. 1984. Science

223 , 806.

104. Hughes, J.P., H.P. Elsholtz, H.G. Friesen. 1985. In: Polypeptide Hormone Receptors (Posner, B.I., ed.) pp 157-199, Marcel Dekker, Inc., New York. 105. Posner, B.I., P.A. Kelly, R.P.C. Shiu, H.G. Friesen. 1984. Endocrinology 95 , 521-532. 106. Posner, B.I. 1976. Endocrinology 107. Chan, J.S.D.,

98 , 645-654.

H.A. Robertson. 1978. Endocrinology

102 , 632-660.

108. Carr, D., H. G. Friesen. 1976. J. Clin. Endocrinol. Metab. 484-493. 109. Arrenbrecht, S. 1974. Nature

42 ,

252 , 255-256.

110. Tsushima, T., N. Sasaki, Y. Imai, F. Matsuzaki, H.G. Friesen. 1980. Biochem. J. 187 , 499-492. 111. Laron, Z., A. Pertzelan, S. Mannheimer. 1966. Isr. J. Med. Sei. 152-155. 112. Davies, T.F. 1978. J. Roy. Cell. Phycns.

1,2 , 379-397.

2 ,

PROLACTIN RECEPTORS: THE STATUS OF KNOWLEDGE AND CONCEPTS CONCERNING THE MECHANISM OF ACTION OF PROLACTIN

CURRENT

Raphael J. Witorsch Department of Physiology Medical College of Virginia Virginia Commonwealth University Richmond, Virginia 23298 Jitendra R. Dave Laboratory of Clinical Studies National Institute on Alcohol Abuse and Alcoholism National Institutes of Health Bethesda, Maryland 20205 Robert A. Adler McGuire Veterans Administration Medical Center Department of Medicine Medical College of Virginia Virginia Commonwealth University Richmond, Virginia 23 298

Introduction Physiological Actions of Prolactin Prolactin Receptors and Their Regulation Mechanism of Prolactin Action Concluding Comments Acknowledgements References

Introduction Perhaps more speculation exists about the mechanism of action or signal transduction of the anterior pituitary hormone prolactin than that of any other protein/peptide hormone. Part of this uncertainty is due to the fact that this hormone is rather ubiquitous and varied in its biologic actions. Moreover, in some of the principal tissues used to study prolactin receptors and mechanisms, such as liver and prostate, little is known about the fundamental role of the hormone. Furthermore in some tissues,

Peptide Hormone Receptors © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

64 such as mammary gland and prostate, prolactin responsiveness is dependent upon the presence of other hormones. Another factor that accounts for the uncertainty about prolactin signal transduction is that it apparently does not appear to act via the conventional adenylate cyclase mediated second messenger pathway. The purpose of this chapter is to provide an overview and critical analysis of the past, current, and possibly future concepts regarding the mechanism of action of prolactin. In this review we attempt to cover this vast field in three parts. In the first part we review the physiological action of prolactin and its relationship to other hormones. The second part deals with the prolactin receptor. Here we discuss prolactin radioreceptor methodology, the distribution and regulation of prolactin receptivity in mammalian target tissues, and some recent studies pertaining to the role of membrane phenomena in receptor regulation. The third part covers the concepts of prolactin signal transduction. In this section we discuss the available information and prevailing views regarding intracellular mediators for prolactin as well as the entry of prolactin into its target cells. How prolactin internalization relates to signal transduction is also be discussed. Finally, some recent work concerning the target organ proteolytic processing is reviewed as well as its possible relationship to internalization and signal transduction.

Physiological Actions of Prolactin Prolactin as a lactogenic hormone The anterior pituitary hormone prolactin (PRL) has a wide variety of effects in many different vertebrate species. Although Bern and Nicoll (1,2) and deVlaming (3) have catalogued more than one hundred reported actions of PRL, the function for which the hormone received its most widely used name derives from its effect on the mammary gland. The maturation of the mammary gland and the production of milk require a complex interaction of many hormones other than PRL.

65

As reviewed by Lyons et al. (4) and Topper (5), and more recently by Vonderhaar and Bhattacharjee(6), all of the anterior pituitary hormones participate either directly or indirectly in the development of the mammary gland and lactogenesis. Of course growth hormone (GH) in certain circumstances can be a lactogenic hormone (7) ; it might therefore serve as a "fail-safe" lactogen. Nonetheless, given the normal hormonal milieu of pregnancy and parturition, the appropriately primed mammary gland appears to produce milk in response to PRL secretion. One method of assessing this primary function has been to measure stimulation of the production of the milk protein, casein, by PRL (8) . As recently reviewed by Rosen (9) , measurement of casein mRNA in response to PRL has begun to provide clues to the mechanism of PRL's action in the mammary gland. Using midpregnant rat mammary glands in chemically defined culture medium containing insulin and Cortisol, Rosen and colleagues demonstrated that PRL caused a moderate increase in casein mRNA synthesis and a dramatic increase in the half-life of casein mRNA (10). One of the enzyme subunits of lactose synthetase, alpha-lactalbumin, is also stimulated by PRL (11). As demonstrated by Vonderhaar (12) , triiodothyronine is important to demonstrate PRL's effect on this specific milk protein. The mRNA for alpha-lactalbumin also appears to be stimulated by PRL (13). The effect of PRL on the other components of milk have not been as comprehensively studied. It has been postulated that PRL controls lipoprotein lipase in the mammary gland and peripheral adipose tissue, switching activity depending on the lactational state of the animal (14) . Falconer and Rowe demonstrated that PRL decreases sodium uptake into mammary glands (15), a possible mechanism to control milk sodium concentrations. Since milk calcium is mainly protein bound, PRL may indirectly increase this cation by stimulating protein synthesis. PRL's osmoregulatory effects (vide infra) suggest that the hormone may regulate the water content of milk. Recent investigations of PRL's effects on immune function (vide infra) were preceded by evidence (16) that PRL augments the migration of plasma cells that synthesize IgA to the mouse mammary gland. As with other effects of PRL, the mice

66

were primed with other hormones, in this case estrogen and progesterone, in order to demonstrate this effect. This may be the mechanism of transfer of immunoglobulins into milk. Recent work from Kleinberg's laboratory (17) and Shiu's laboratory (18) support the hypothesis that the anterior pituitary secretes mammary stimulating hormones other than PRL and GH. Kleinberg et al. (17) suggest that while in the rodent PRL is a crucial requirement for full mammary development, in the primate other pituitary factors are more important. Shiu's laboratory demonstrated that the rat pituitary tumor cell lines (GH 3 and GH.l) and normal transplanted pituitaries stimulated a human breast cancer cell line, T-47D, via a non-PRL mitogenic substance. It is clear that the anterior pituitary contains substances in addition to the classic hormones. For example a fibroblast growth factor (19) and a chondrocyte stimulating factor (20) have been found in bovine and human pituitaries, respectively. In growth hormone cells of rat pituitary gland immunoreactivity specific for human placental lactogen has been demonstrated (21). How these novel or unsuspected substances relate to PRL structurally and/or functionally is a subject of great interest and experimental activity. Two important points summarize the role of PRL in the mammary gland. One is that PRL does not exist or function by itself. As reported above, most, if not all, PRL effects in the mammary gland require other hormones. These other hormones are often under pituitary control, and their effects are consistent with the concept that for lactation, at least, the anterior pituitary is indeed a "master gland." The existence of newer mammarystimulating adenohypophysial hormones may make the concept more complex but no less true. The second point is that PRL plays a multitude of roles even in the mammary gland. It appears to affect protein, fat, and carbohydrate metabolism, ion transport, and immune function. It is not surprising, therefore, that PRL would have similar effects in other organs in mammals and in animals not having mammary glands.

67 The role of prolactin in abnormal lactation and mammary gland tumors Only in the last twenty years has the full importance of PRL in abnormal lactation been realized. The pioneering work of Frantz (22) , Friesen (23), and others demonstrated the presence of a pituitary lactogen other than GH. In 1954 Forbes et al. (24) described non-acromegalic patients with pituitary tumors who had lactation due to another putative pituitary lactogen. In 1986 it is well established that excess PRL secretion, often with galactorrhea, was the most common hormonal abnormality in patients with pituitary tumors (25). Adenomas of the lactotrope cells cause excess secretion of PRL and production of milk containing nutrients similar to those of normal lactation (26) . Other causes of augmented PRL production may also lead to abnormal lactation. Examples of this phenomenon include compression of the pituitary stalk by tumors (27), dopamine antagonist drugs such as phenothiazines (28), chest wall stimulation due to trauma or surgery (29) , and primary hypothyroidism (30). In pituitary adenoma patients, reduction of PRL secretion to normal by surgical removal of the tumor (31) or by administration of dopamine agonist drugs such as bromocriptine (32) results in cessation of the abnormal lactation. The role of PRL in mammary tumor development has been extensively reviewed elsewhere (33,34) and will not be discussed in detail here. While it is generally agreed that PRL plays an important role in mammary tumorigenesis in rodents (35) , the role of the hormone in human breast carcinoma is still unclear (9,18,33,36). Like the effects of PRL on the normal mammary gland, PRL stimulation of murine mammary tumors requires other hormones, most notably estrogen (37). Other effects of prolactin on reproduction The role of PRL in human physiology and pathophysiology other than effects on the mammary gland is under considerable investigation. The best-established non-mammary effect of excess PRL in humans and other mammals is a decrease of gonadal

68 function. The site(s) of PRL's anti-gonadal effect has been a subject of intense research. Although there is evidence that PRL may act directly on the gonad (38) , two important findings from clinical research suggest that the most important site of PRL's anti-gonadal effect is the hypothalamus. Patients bearing PRLsecreting pituitary tumors have pulsatile secretion of the gonadotropins, LH and FSH, that is abnormal in both amplitude and frequency (39,40). Reduction of excess PRL secretion by adenomectomy (39) or bromocriptine therapy (40) restores to normal pulsatile gonadotropin secretion. In men with prolactinomas, intermittent administration of exogenous gonadotropin releasing hormone (GnRH) results in a restoration of LH and FSH secretion despite persistent hyperprolactinemia (41). In addition, serum testosterone levels were returned to normal by the GnRH therapy. Thus these studies suggest that PRL's major antigonadal effect is mediated via the hypothalamus. Clinical consequences of this action are oligomenorrhea or amenorrhea and infertility in females (42) and impotence in males (43) . It is difficult to ascertain the actual prevalence of hyperprolactinemia in patients with these symptoms, but suppression of the serum PRL level to normal usually restores gonadal function in hyperprolactinemic patients. In other species, PRL may also have a profound effect on reproductive function, as well. However in some cases PRL may act as a gonadotropin, as its former name luteotropic hormone would suggest (44). As demonstrated many years ago by Everett (45) , autotransplantation of the pituitary to the renal capsule results in maintenance of the corpus luteum. Such transplantation also results in hyperprolactinemia (46) and is the basis of a widely-used model of chronic PRL excess (47). In an intact female rat, implantation of extra anterior pituitaries under the kidney capsule leads to a series of pseudopregnancies (48) . PRL may also play an important role in sexual maturation in the rat. Both adrenal steroid hormone secretion (49,50) and vaginal opening (51) may be stimulated by PRL in rodents. On the other hand, studies from Bartke and colleagues (52,53) have clearly demonstrated suppressive effects of PRL on copulatory behavior in rodents. Demonstrating a PRL-induced behavioral

69 effect of any sort in humans has been much more problematic (54). PRL also appears to play a role in reproduction in submammalian classes. For example, under certain experimental conditions PRL may increase gonadal growth in birds (55) and may alter accessory sex organs in fishes (56) and newts (57) . Early studies by Riddle, Bates, and Lahr showed that PRL stimulated brooding behavior in birds (58) . PRL has also been shown to affect parental behavior in teleosts (59). A final example of PRL's effects on procreation and rearing of the young is the basis of an important bioassay of PRL action. PRL stimulation of the pigeon crop sac to produce its "milk" has been used to assess lactogenic activity (60). Recent studies from Nicoll's laboratory (61) have utilized this classic bioassay to demonstrate the presence of a PRL-synergizing factor in serum. This factor, named "synlactin," is apparently produced in the liver of several species (62). This suggests that PRL, like GH, may at least partly act indirectly through hepatic factors. Indeed under certain conditions PRL can increase somatomedin C, the hepatic growth mediator normally stimulated by GH (63). In turn GH may have effects on synlactin. Furthermore PRL can act as a growth hormone in several species (3). Similarities of prolactin and growth hormone Of the several examples of PRL effects on growth and development, the role of PRL in premetamorphic growth in anurans has been most extensively studied. PRL increases growth in larval anurans (64) , stimulates tail collagen production (65), and has antimetamorphic effects (66). In contrast to lactation in which PRL works in concert with thyroid hormone, the effects of PRL on anuran maturation are in opposition to those of thyroid hormones (66-68) . Interestingly, both T3 and PRL appear to regulate PRL binding sites in the tadpole (68). GH has metabolic effects that are anti-insulin or insulin-like. GH is probably a family hormones rather than a single species (69) . The seemingly contradictory effects may therefore be due to the particular GH variant dominant under given conditions.

70

Similarly PRL may also appear to have anti-insulin and insulinlike effacts (70,71). Early studies in humans suggest that PRL may be diabetogenic (70) but recent work from Lewis' laboratory suggest an insulin-like effect of prolactin in adipocytes (71). In normal humans insulin-induced hypoglycemia is accompanied by increased PRL secretion (72), whereas in the rat, insulin-induced hypoglycemia leads to decreased serum PRL levels (73). Osmoregulatory effects of prolactin In many vertebrate classes, PRL has an important osmoregulatory function. As reviewed by Nicoll (74) , PRL clearly serves to eliminate excess water in teleosts, but information about osmoregulatory effects in other non-mammalian species is incomplete. The most controversial area, however, has been the putative osmoregulatory role of PRL in mammals (74) . In early studies, PRL appeared to decrease urine flow in several mammals including man (75-77) . In later studies the antidiuresis after PRL administration was attributed to vasopressin contamination of PRL powders (78-80). When vasopressin is a contaminant of PRL preparations, it may appear to be more potent. It has been suggested that the large PRL molecule protects the smaller vasopressin molecule from being degraded (80). PRL devoid of vasopressin is not antidiuretic (80,81). Adler et al. (82) studied Brattleboro rats, a strain of Long-Evans rats with hereditary hypothalamic diabetes insipidus. Implanting extra anterior pituitary glands under the kidney capsule to produce hyperprolactinemia in animals lacking vasopressin did not lead to antidiuresis. In later studies (83) utilizing normal rats, chronic hyperprolactinemia induced by pituitary grafts led to increased urine flow. Thus, in rats, as in teleosts, PRL appeared to increase water excretion. Possible prolactin effects on immune function In addition to the apparent immunoregulatory role in the mammary gland (16), recent studies suggest that PRL affects lymphocyte function. Russell and colleagues have found binding sites for PRL on circulating human lymphocytes. The Kd for these receptors

71

was approximately 10 nM, and the investigators estimated the presence of about 3 60 receptors per cell (84). In further studies from the same laboratory, PRL binding sites were identified on both T- and B-lymphocytes isolated from human spleens. The immunosuppressive agent, cyclosporine, depending upon concentration, enhanced or inhibited radioactive PRL binding to lymphocytes. The concentrations of cyclosporine used for clinical immunosuppression would inhibit PRL binding to peripheral lymphocytes (86) . It has been suggested that lymphocyte responsiveness is affected by PRL in rats, and the immunosuppressive effect of cyclosporine may be at least partially mediated by interference with PRL's action at the receptor level (87). It is also interesting to note that in the rabbit mammary gland, cyclosporine does not appear to alter PRL binding (88). A discovery in immuno-oncology that has had a significant impact to researchers in the prolactin field, is that of the Nb2 lymphoma cell line. This tumor was originally discovered by Noble, Beer, and Gout in an estrogenized rat and has been maintained by transplantation in vivo as well as a suspension culture in vitro (89) . In the presence of fetal bovine serum, these tumor cells divide in suspension culture. When horse serum is substituted for fetal bovine serum, the cells live but do not divide. Adding lactogenic hormone causes increased cell numbers in a dose-dependent fashion. The cell-line is exquisitely sensitive to these hormones, where mitogenesis is demonstrable after exposure of the cells to 10-1000 pg/ml concentrations of PRL. This mitogenic response has been the basis of a highly sensitive and convenient bioassay for the measurement of serum prolactin levels used widely (90,91). Prolactin receptors have been demonstrated on these cells, as well (92). The use of the Nb2 lymphoma as a model for examining the mechanism of action of prolactin has only very recently been appreciated as will be discussed in subsequent sections. To the best of our knowledge the potential role of prolactin as an interleukin-like substance is essentially unexamined. In the future this cell line may serve as an important interface between investigators in the fields on endocrinology, immunology, and oncology.

72

Diversity of prolactin binding sites As will be discussed in subsequent sections of this chapter, much of the studies into the mechanism of action of prolactin have revolved around an examination of prolactin binding sites or prolactin receptors. There is little question that the first interaction of prolactin with its target cell is via the binding of hormone to receptors on the cell surface. Whether subsequent steps in the transduction of the prolactin signal require the entry of the hormone into the cell or whether separate intracellular mediators are involved is at this point unresolved. In mammals, saturable binding sites for PRL have been found in mammary epithelial cells, hepatocytes, renal tubules, the adrenal cortex, gonadal cells, male sex accessory organs, brain and a variety of other tissues (93). As described above, PRL has been found to have insulin-like effects in rat adipocytes (71), and we have found binding sites for PRL on plasma membranes from rat adipocytes (R.A. Adler and V.L. Herzberg, unpublished data). To our knowledge, saturable binding sites for PRL have not been demonstrated in other mammalian tissues thought to be prolactinresponsive, such as mammalian gut and bone. The distribution of prolactin receptors in mammalian tissues will be discussed in detail in a subsequent section. Although most prolactin receptor work has been performed in mammals, prolactin receptors have also been reported in some nonmammalian vertebrates. For example, PRL receptors have been reported in the gut and bladders of amphibians (94) and the kidneys of teleosts (95) and amphibians (94). Up to stage XVIII, premetamorphic tadpoles have an increase in liver, tail fin, and kidney PRL receptors in response to PRL injection (68) . Thereafter PRL receptors decline as thyroid hormones become more important in tadpole development. This incredible diversity of PRL effects and the existence of receptors in many widely-different species is complicated by the fact that mammalian PRLs may be as potent as homologous PRL in

73

non-mammalian vertebrates (96). This suggests that there is a conserved mechanism of action of PRL or at least a highly conserved lactogenic hormone binding site. Under certain experimental conditions GH may also interact with PRL receptors (85) , suggesting the presence of lactogenic hormone receptors that respond to more than one hormone. The use of experimental hyperprolactinemia to study prolactin effects As suggested above, diverse species can serve as models of PRL actions. In mammals, however, there may be difficulties in demonstrating PRL effects. As we have discussed with regard to the osmoregulatory role of PRL, much of the difficulty has been due to the significant contamination of mammalian PRL preparations with vasopressin (74-81). Because of such problems other models have been used to study the effects of PRL which involve promoting the hypersecretion of endogenous hormone. Widely-used experimental models of chronic PRL excess include administration of dopamine antagonist drugs such as sulpiride (51), haloperidol (97) , or domperidone (98) ; transplantable pituitary tumors that secrete PRL usually accompanied by other pituitary hypersecretion (e.g., GH or ACTH, 99); and implantation of normal pituitary glands to a site remote from hypothalamic influence (47). Of the last-mentioned, two variants are often used. In one the host animals's own anterior pituitary is transplanted to the kidney capsule (45,46). The second variant leaves the host's own pituitary intact, and extra hypophyses are implanted under the kidney capsule (47,73). In both models, the animals maintain hyperprolactinemia for extended periods of time (47) . In summary, PRL has vertebrate species. that PRL can interact in carrying out these

a multitude of effects in many different There is considerable evidence to suggest with highly conserved target organ receptor diverse functions.

74 Prolactin Receptors and Their Regulation Turkington's report Sepharose mammary

beads

in 1970 that prolactin covalently linked to

stimulated

epithelial

nuclear

cells,

RNA

synthesis

suggesting

the

in

dispersed

existence

of

cell

surface receptors for the hormone, was a primary impetus in the search

for prolactin

surface

receptors

receptors

was

(100).

further

The

supported

suggestion

by

of

cell

Birkinshaw

and

Falconer's autoradiographic demonstration of prolactin binding to the

serosal

vitro and assay

surface

in vivo

using

however, membrane

assay,

that

gland (102). 1973

pseudopregnant

membranes

problems

particular

of

can

obtained

existed

from

regarding

presumably be

rabbit

mammary

Turkington developed

(101) .

due

obtained

mouse

the

to

from

a

cells

mammary

tissue,

sensitivity

the

limited

midpregnant

in

radioreceptor of

this

amount

mouse

of

mammary

Two studies published by Shiu, Kelly and Friesen in

(103) and Shiu and Friesen in 1974 (104) provided the basis

for the radioreceptor assays currently used in many laboratories today.

This

assay

mammary

glands

was

obtained

initially from

conducted

midpregnant

on membranes rabbits

that

injected intramuscularly with 10 mg of human placental and

5mg

of

hydrocortisone

development.

for

3

days

to

stimulate

from were

lactogen mammary

Mammary glands from near-term pregnant rabbits or

early lactating rabbits also appeared to be a suitable source of membranes (103, 104).

The

following

procedure

derived

and

modified

from

the

assay

developed by Friesen's group has been used in our laboratory for the last several years and has provided

optimal

and

consistent

binding values. Radioreceptor assay methodology Tissue preparation.

We have used rodent liver (female), mammary

gland and prostate.

After decapitation, the tissues are rapidly

excised,

frozen in liguid nitrogen and pulverized.

are homogenized in HC1.

The tissues

for 1 min using a Brinkmann Polytron set at six

10 vol of 0.3 M sucrose buffered to pH 7.6 with 25 mM TrisThe

supernatant

from

a

10-min

preliminary

125

x

g

75

centrifugation is recentrifuged at 15,000 x g for 20 min. The 15,000 x g supernatant is further centrifuged at 100,000 x g for The 15,000 x g and 100,000 x g pellets are used for 1 hr. prolactin binding assays. As previously reported by us and others, the 100,000 x g membrane pellet of both liver and mammary gland exhibits maximal prolactin binding activity, whereas, 15,000 x g membrane pellet of ventral and dorsolateral lobes of prostate gland exhibits maximal prolactin binding activity (103106) . Iodination of prolactin. We have routinely used the following modification of the Thorell and Johansson technique of prolactin iodination (107). Approximately 1 mCi Na 1 2 5 i (Amersham) is added to 5 ug unlabeled NIADDK ovine-prolactin and buffered with 10 ul of 0.4M sodium acetate, pH 5.6; then 1 ul of lactoperoxidase solution (2.7 mg/ml, Calbiochem grade B enzyme) is added, followed by 1 ul H 2 0 2 solution (30% diluted 1:15,000). Forty-five seconds later, 100 ul of transfer solution (0.02 Naazide, 1% KI and 16% sucrose) are added to quench the reaction. The mixture is removed, and the iodination vessel is rinsed with 100 ul transfer solution. The solutions are combined and chromatographed on a 1.5 x 90 cm Sephadex G-100 column at 4°C, previously equilibrated with a 25 mM Tris/O.1% bovine serum albumin buffer at pH 7.6. Fractions high on the descending limb of the hormone peak are used for binding assay. The specific activity of the labeled prolactin is between 60-100 uCi/ug. Prolactin Binding Assay. The membrane pellet is resuspended in 10 mM MgCl2/25 mM Tris at pH 7.6 to provide approximately 3 mg protein/ml (108). One hundred ul of membrane suspension is incubated overnight at room temperature with 75,000 to 85,000 cpm of 125I-prolactin with and without unlabeled prolactin in a final volume of 0.5 ml of buffer (10 mM MgCl2/0.1% bovine serum albumin/25 mM Tris-HCl, pH 7.6). The binding assay is terminated by adding 1.0 ml of chilled buffer. The tubes are centrifuged at 2500 rpm for 20 min. The pellets are washed with an additional 1 ml of buffer and counted in a gamma counter. Each sample is assayed in triplicate. The diminution of radioactivity by coincubation with 1 ug unlabeled prolactin represents

76 specifically

bound

hormone.

Under

these

assay

conditions

we

r o u t i n e l y o b t a i n e d 25-35% t o t a l a n d 6-12% n o n - s p e c i f i c b i n d i n g

of

a d d e d i o d i n a t e d p r o l a c t i n to t h e p r o s t a t i c a n d h e p a t i c m e m b r a n e s . Characterization prolactin ng).

with

is

performed

varying

amounts

by of

incubating

unlabeled

the

iodinated

hormone

(0

to

Consistent with many other laboratories, Scatchard

routinely

reveals

a

single

set

of

e v i d e n c e of c o o p e r a t i v e e f f e c t s .

receptor

sites

1000

analysis

without

The apparent affinity

any

constants

obtained by Scatchard analysis generally approximate about 1 to 5 nM

(109) .

Background Much and

of

information

the

information

criteria

reports

of

on the conditions

for

about

optimal

specificity

Shiu et

al.

(103)

were

hormone form.

can

be

from

and

receptor

of

the

radioiodinated competition

hormone

with

prolactin with

its

becomes

unlabelled

this duration-dependent

change

assay.

incubation

the

Friesen

is s a t u r a b l e , its

of

from

original

(104) .

They

reversible and in v i r t u a l l y

However, recent studies have indicated that with

exposure by

recovered

conditions

derived

and Shiu

reported that prolactin binding

of r a d i o r e c e p t o r

receptor,

the

progressively

less

ligand(llO).

The

the

intact

prolonged

binding

of

dissociable

mechanism

for

in r e c e p t o r a f f i n i t y h a s n o t

been

elucidated, b u t occurs w i t h other ligands, as well

(111).

Shiu

radioiodinated

and

Friesen

lactogenic with that T

(112)

compared

hormone

binding

prepared

by

properties

the

lactoperoxidase

iodinated with the chemical and

found

that

the

of

former

method

oxidizing agent provided

a

higher

specific

activity and lower nonspecific binding than the latter. apparently iodination (104).

(107)

chloramine This was

associated with the fact that the enzymatic m e t h o d is

less

harsh

Similarly,

than

Frantz

the and

use

of

a

chemical

Turkington

reported

that

lactoperoxidase iodinated lactogenic hormone retained m u c h of biological was

a c t i v i t y w h i l e t h a t of c h l o r a m i n e T i o d i n a t e d

negligible

prolactin

(113).

iodinated

by

In very

our

experience

brief

exposure

we

have

to

low

(Dave,

unpublished by

Shiu

and

observations). Friesen

as

Three suitable

iodinated traces

its

hormone

found

that

dilutions

c h l o r a m i n e T w i l l p r o v i d e l i g a n d s u i t a b l e for r a d i o r e c e p t o r shown

ligands for

of

oxidant

of

assay were

prolactin

77

radioreceptor assay, namely ovine prolactin, human prolactin, and human growth hormone (104). All three are currently in widespread use. Shiu and Friesen observed that the optimal pH range for maximal specific prolactin binding was quite narrow, between about 7.5 to 9.0. Ionic environment also affects hormone binding. At pH 7.6, Krebs Ringer bicarbonate buffer and 50mM sodium phosphate buffer resulted in significantly greater prolactin binding than 25mM Tris-HCl. That these differences reflect ionic requirements, was indicated by the fact that in Tris buffer, binding can be maximized by the addition of CaCl2 (10-25 mM) and 10 mM MgCl2 (104). These workers also examined conditions of temperature and time for prolactin receptor binding and found that at 37°C equilibrium was achieved at 3 h, at room temperature (23°C) at 5 h, and at 0°C little or no binding was observed for up to 5 hours (104). However, prolonged duration (24 to 48 h) at cold temperatures are suitable for binding assays. Shiu et al. also established specificity of this radioreceptor assay by showing that various unlabelled lactogenic hormones (ovine, monkey, human, and rat prolactin, human prolactin, and human placental lactogen) could displace iodinated prolactin from membranes in a dose dependent fashion while other protein hormones (including rat, ovine and bovine growth hormone) did not. They also showed that the ability of a lactogenic hormone preparation to displace the labelled hormone from its receptor was highly correlated with potency estimates obtained by in vitro mouse mammary gland and pigeon crop assays (103). As a result of their potency estimates, Shiu et al. suggested the radioreceptor assay as a convenient alternative to radioimmunoassay for measuring serum lactogenic hormone. In many situations this is possible, although radioreceptor assay which has a detectibility range of about 0.1 to 1000 ng of protein is generally less sensitive than PRL radioimmunoassay. Furthermore, unlike radioimmunoassay which is capable of differentiating between chemically distinct forms of lactogenic hormones (such as human growth hormone vs. human prolactin vs. human placental

78 lactogenic hormone), radioreceptor assay cannot. On the other hand, radioimmunoassay may not always distinguish between isohormones (such as intact and cleaved forms of growth hormone). As a result, radioimmunoassay and radioreceptor assays of sera or purified pituitary hormone preparations have uncovered discrepancies between immunologic and biologic estimates (114). Radioreceptor assay has been of significant value as a convenient screen of biological activity of lactogenic substances. Recently, however, a recombinant analogue of human growth hormone has been prepared that apparently exhibits some of receptor binding ability but does not exhibit lactogenic effects and antagonizes the action of human growth hormone and ovine prolactin (115). If such substances exist naturally, receptor assay alone would be of limited value. Unquestionably the major value of the radioreceptor approach has been through the insights it has provided about the mechanism of action of PRL. Purification of prolactin receptors. Initial attempts at purification of prolactin receptors involved solubilization of particulate fraction with the nonionic detergent, Triton X 100. These solubilized receptors exhibited specficity, time and temperature characteristics similar to those of membrane bound receptors. However, solubilization resulted in an apparent increase in the affinity of binding. Gel filtration revealed a molecular weight of the solubilized receptor to approximate 220,000 daltons. Further purification of the solubilized receptors involved affinity chromatography with human GH followed by elution with 5M MgCl2. The final purification was about 1500fold. Although isoelectric focusing and disc gel electrophoresis revealed several bands, receptor activity was localized to 1 or 2 bands (116). More recently prolactin receptor purification has involved solubilization of membranes with the nondenaturing zwitterionic detergent, CHAPS. This agent does not aggregate 125I-labelled prolactin as did Triton and solubilized receptors exhibited specificity and affinities similar to membrane bound receptors. Using material solubilized with CHAPS, affinity purified prolactin receptors have been resolved into a single electrophoretic band with a molecular

79

weight from 32,000 to about 37,000 (117, 118). Resolution of purified receptor covalently linked to prolactin reveal a similar molecular weight ranging from about 36,000 to 45,000 (117,119,120). These data suggest the existence of a low molecular weight prolactin binding subunit. Recently Vonderhaar and co-workers have isolated a higher molecular weight (85,000 to 90,000 daltons) form of the receptor from MCF-7 human breast cancer cells, human chorion decidua, and lactating mouse mammary gland (121). The significance of this apparent receptor heterogeneity remains to be determined. More detailed information about prolactin receptor purification has been published in several recent excellent reviews (93,117,121). Antibodies to prolactin receptors. In the course of receptor purification, partially purified material has been used as an antigen for the generation of guinea pig antisera directed against rabbit mammary gland prolactin receptors. Shiu and Friesen first showed that such antisera specifically inhibited prolactin binding and prolactin-induced responses in rabbit mammary gland explants (122,123). Antagonistic actions of these antisera against prolactin effects have also been shown in vivo (124). Djiane et al. showed that while guinea pig and sheep antisera against rabbit mammary gland prolactin receptors blocked prolactin induced increases in DNA and casein mRNA of rabbit mammary explants, both antisera in low doses mimicked prolactin action (in the absence of hormone). At high concentrations these antisera were no longer stimulatory (125). Similarly, Shiu et al. showed that, while the guinea pig antiserum to the receptor blocked prolactin-induced mitogenesis of Nb2 lymphoma cells, in the absence of prolactin, it was mitogenic in this cell line and was lactogenic in mammary gland explants. Stimulatory effects were also demonstrated with an IgG fraction of the receptor antiserum (126). These agonistic actions suggested that the hormone was not required beyond its initial interaction with its receptor. Receptor antiserum studies have also been conducted with fragments of receptor antibodies prepared from the IgG. While both divalent (Fab*2) and monovalent (Fab1) fragments block the

80

action of prolactin in mammary explants, the divalent fragment is mitogenic in mammary explants and mitogenic in lymphoma cells while monovalent fragments are not (126,127). The requirement for divalency of Fab fragments in the initiation of prolactinlike actions is interpreted to suggest that clustering of cell surface prolactin receptors are required for such effects. Recently, monoclonal antibodies have been prepared against the partially purified 32,000 dalton binding subunit of rabbit mammary gland prolactin receptor which were shown to block prolactin binding to mammary gland receptors (128,129). In another recent development anti-idiotypic antibodies raised against affinity purified antibodies to rat and ovine prolactin have been shown to recognize the prolactin receptor (130) . In vitro desaturation of prolactin receptors. One of the problems pertaining to examination of fluctuations in target hormone receptor concentration involves that portion of the receptor population that binds endogenous hormone. A large change in serum prolactin concentration independent of a change in receptors can give an erroneous picture of changing levels of hormone receptors if endogenously bound hormone is not taken into consideration. Kelly et al. developed a method of in vitro desaturation of prolactin from its receptors by exposing membranes to high concentrations of MgCl2 (3-5M). This agent was capable of removing 90-95% of radioiodinated prolactin from rabbit mammary gland or rat liver prolactin receptors. This manipulation appears to retain receptor integrity since they were able to specifically bind fresh hormone after the desaturation procedure. This procedure has been used for the estimation total receptors in tissues (131). The distribution of prolactin receptors in target tissues Mammary Gland. Since mammary gland is thought to be the principal target tissue of prolactin action, the mammary tissue from rat (132-134), mouse (113,135-139) or rabbit (103,104) has been extensively used in studies on prolactin receptors. As will be discussed in a subsequent section, intracellular receptors and

81

internalized prolactin have been demonstrated in addition to the demonstration of cell surface receptors in breast and other tissues. A variety of mammary tumors including MTW9 (140,141) and R323 0AC (132,142,143) transplantable mammary tumors in rat, DMBA (7,12-dimethylbenzanthracene) induced rat mammary tumors (134,144-149) and human breast tumors (150-153) have also been studied to determine the existence of prolactin receptors and their characteristics. In an autoradiographic study, Costlow and McGuire (144) reported that when DMBA-induced mammary tumor 125 slices were incubated with I-ovine prolactin, tumor cells exhibited higher levels of specific prolactin binding, whereas, non-specific binding was confined to connective tissue and alveolar spaces. A number of studies have reported the presence of prolactin receptors in human breast tumors. An up-dated review on studies of prolactin receptor in human tissue has been recently published (154) . Lactation is among the most studied factors known to modify mammary gland prolactin receptors. The pattern of prolactin binding changes in mammary gland following pregnancy and lactation appears to be different in rabbit from that observed in rat and mouse. Some investigators have attributed these species differences in mammary gland prolactin binding to the presence of high circulating placental lactogen during pregnancy in rat and mouse and absence of the same in rabbit (133,155,156). By competing with prolactin for its receptors, placental lactogen at higher circulating levels may occupy a large proportion of available prolactin receptors, thus decreasing the detectibility of the latter. Prolactin binding in the rat mammary gland is reported to remain low during pregnancy, followed by a rise after parturition and remaining high during the entire lactation period (133,157). However, in rabbit mammary gland, prolactin receptors first rise during mid-pregnancy. This is followed by a decline and a second rise which continues from late pregnancy through parturition and lactation (158,159). Sakai et al. (160) reported that the number, and not the affinity, of prolactin binding sites of virgin and lactating mouse mammary epithelial cells was higher than that obtained from pregnant mice. These investigators suggested that progesterone may be responsible for this decrease

82

in prolactin binding during pregnancy. A similar antagonistic effect of progesterone on prolactin binding in rabbit mammary gland has been reported (158). Various other factors like male and female sex hormones, prolactin, insulin, and polyamines have also been reported to modify prolactin binding in normal and neoplastic mammary gland (134,143,145,161,162). The detailed account of these and other studies has been provided in a number of reviews (163-165). Ovary. As mentioned above, prolactin exerts a variety of actions on ovarian function. Along with gonadotropins and estrogens, prolactin is known to play a key role in the maintenance and formation of a functional corpora lutea. The presence of prolactin receptors in the ovary of rats, cow, pig, humans and other species was demonstrated in a number of early studies (167174) . Furthermore, a number of studies have characterized and confirmed the existence of prolactin receptors in theca, luteal and granulosa cells (168,170,175-179). Bohnet et al. demonstrated that treatment of female rats with guinea pig antiserum to prolactin receptor resulted in an increase in the number of old corpora lutea which was interpreted to reflect the luteolytic action of the hormone (180) . In a recent review by Bonifacino and Dufau the function, characterization and factors modulating ovarian prolactin receptors are discussed in great detail (181). Kidney. A decade ago, a number of studies reported the existence of specific prolactin binding sites in kidney cells of male and female rats (182,183) and the internalization of prolactin into kidney proximal tubule epithelial cells has been demonstrated immunohistochemically (184). In an autoradiographic study, Costlow and McGuire reported that kidney tubules in the cortex display specific 125I-labeled-prolactin localization, whereas the medulla was devoid of prolactin binding activity (185). Marshall et al. investigated the effects of glucocorticoids and thyroid status on prolactin binding to kidney membranes. While thyroidectomy reduced prolactin binding in the kidney membranes, adrenalectomy increased the same. Administration of thyroxine to

83

thyroidectomized and glucocorticoids to adrenalectomized animals reversed the effects of thyroidectomy and adrenalectomy on PRL binding prolactin binding in kidney membranes (186,187). sites in the kidneys of chronically hyperprolactinemic animals have been found to be somewhat increased compared to normal rats (83). The injection of exogenous PRL also appears to increase PRL binding to several rodent organs (132,159,166,188,189), the possibility that such an apparent "up-regulation" of prolactin receptors may be an immunologic artifact has been suggested (190). However, the findings that endogenous hyperprolactinemia can lead to augmented PRL binding suggests that there can be induction of PRL receptors by PRL itself. In contrast to the phenomenon of "up-regulation" which takes 1 or more days to develop, PRL also has the capacity to "down-regulate" its receptors in a matter of hours. Down-regulation will be discussed in a subsequent section. Adrenal Gland. Prolactin has been shown to modulate the nature of glucocorticoid secretion from the rat adrenal via its control of the enzyme 5-alpha reductase (50). The existence of specific prolactin binding sites in rat adrenal gland was reported in earlier studies by Marshall et al. (182). This group reported that unilateral nephrectomy and water deprivation increase serum prolactin levels and that a similar treatment significantly increases prolactin binding in adrenals. Costlow and McGuire using an autoradiographic technique confirmed the existence of adrenal prolactin binding sites and reported that adrenal medulla is almost devoid of prolactin binding activity, however, the zona reticularis and, to a lesser extent, the zona fasciculata exhibit prolactin binding activity (185). Marshall et al. also studied the effects of glucocorticoids on prolactin binding to adrenal and reported that low doses of dexamethasone produced a consistent reduction in prolactin binding (186). The binding in adrenal glands of male rats was reported to be higher than that in female rats (187). Prolactin binding was also reported by this group to be unchanged following thyroidectomy and thyroxine treatment (187). Male reproductive organs.

The evidence that prolactin plays a

84

significant role in the regulation of male reproductive function has led to a number of investigations on prolactin receptors in male reproductive organs. In 1973, Turkington et al. reported prolactin-binding activity in a number of tissues including the testes and seminal vesicles (135). Later many investigators confirmed the existence of specific prolactin binding sites in these tissues of rats and mice (191,192). Charreau et al., defined the nature of prolactin binding sites in the rat testis. These binding sites could bind either prolactin or human growth hormone and were localized exclusively in the interstitial tissue and not in the seminiferous tubules (193) . Aragona et al. also reported that binding sites for prolactin are primarily located on the Leydig cells and maximal testicular prolactin binding is observed at 4 5 days of age (194). This group also reported that bromocriptine (CB 154) treatment of male rats from days 20 to 40 decreased prolactin secretion and blocked the binding of luteinizing hormone (LH) to Leydig cells without affecting prolactin and follicle-stimulating hormone (FSH) binding, indicating that prolactin may regulate testicular LH receptors (194). These and other studies suggested that the known prolactin/LH interplay may involve a direct effect on the Leydig cells. The existence of lactogenic hormone receptors in the prostate was suggested by Sonenberg and Money in 1955. They reported that prostate gland retained radioactivity after administration of 131 I-prolactin to male rats (195) . To our knowledge, this is probably the earliest attempt to examine a peptide/protein hormone-target organ receptor relationship. The direct action of some non-gonadotropic pituitary factor on the prostate was predicted by early observations that prostatic atrophy was more marked following hypophysectomy than following castration and that androgen stimulation of prostatic growth was impaired after hypophysectomy (196,197). Subsequently evidence has accumulated from a variety of other sources that prolactin was this hypophyseal prostatotrophic factor (198). Relatively recently many laboratories have reported specific, high affinity, reversible and saturable 125I-prolactin binding to prostate gland which is primarily modulated by androgens (191,199,200). Using

85 immunohistochemical techniques, intracellular prolactin binding in normal and abnormal rat and human prostate gland has been identified (201). The use of the immunohistochemical approach to the study of prostatic prolactin receptors, the androgenprolactin interplay in prostate, and the potential role of prolactin in prostatic aging and neoplasia has recently been reviewed (201). Liver. A large number of studies on the mechanism of action of prolactin have used hepatic tissue for detailed characterization of the prolactin receptor. In fact, in a series of publications surveying the prolactin research literature, Horrobin (166) has indicated that liver is the favorite organ for prolactin binding studies, presumably because of its availability and size. Hepatic tissue obtained from male animals has been reported to be virtually devoid of prolactin receptors. The administration of estrogens or castration is known to increase prolactin binding, suggesting antagonistic action of androgens on hepatic prolactin receptors (166,202-204). Most studies on hepatic prolactin receptors have been carried out using liver from female animals. Prolactin receptor content in the liver of female mouse, rat and rabbit is absent or is very low during the postnatal through pre-pubertal period and reaches the adult level at or around puberty (205,206). In a number of physiological and pathophysiological conditions the pattern of changes in hepatic prolactin receptors is often opposite to that of mammary gland prolactin receptors. For example, in the mammary gland of mouse and rat, prolactin receptors are low during pregnancy and increase during lactation, whereas hepatic prolactin receptors are increased during pregnancy and are either low or unchanged during lactation from that of non-pregnant, nonlactating animals (205,207-209). Unlike DMBA-induced mammary tumors which have been reported to exhibit only a slight decrease in prolactin receptors following hypophysectomy (210), prolactin receptors in rat liver are rapidly decreased after the removal of pituitary (202-204,210213). Bohnet et al. reported that a combined treatment of

86 prolactin, growth hormone and adrenocorticotropic hormone is most effective in maintaining prolactin receptors in the liver of hypophysectomized rats (213). The Snell dwarf is a recessive mutant strain of mice whose pituitary gland is unable to synthesize and secrete TSH, growth hormone and prolactin. In a few earlier studies it was reported that prolactin binding is absent in the liver of dwarf mice and that administration of prolactin or pituitary grafting can induce the receptor (191,214-216). It was, thus, suggested that prolactin may modulate its own receptors in the liver. In an earlier study conducted by one of us, injection of either ovine prolactin or purified bovine growth hormone (100 ug) to dwarf mice every 8 hr for a period of 2-4 days resulted in a >20 fold increase in hepatic prolactin binding (216; Dave and Knazek, unpublished observations). We were able to achieve the induction in hepatic prolactin binding by growth hormone or prolactin administration to dwarf mice despite pre-treatment with cycloheximide for 4-days. These results suggested that increases of prolactin receptor after bovine growth hormone or ovine prolactin were not entirely due to the synthesis of new receptor protein and that this treatment in dwarf mice might have uncovered pre-existing (or cryptic) prolactin receptors which were otherwise inaccessible. This increased accessibility could be a result of some biophysical alterations in the membrane lipid bilayer. This consideration initiated a long-standing interest in the role of membrane lipid microviscosity in the modulation of prolactin receptors. Modulation of prolactin membrane microviscosity

receptors

and

its

relationship

to

The plasma membrane appears to be a dynamic matrix in which proteins and glycoproteins float (217). The possibility existed that the up-regulation of hepatic prolactin receptors in cycloheximide-treated dwarf mice could be due to physical alterations in the membrane that in some way increased the availability of cryptic receptor to ligand. The changes in availability of prolactin receptors might be associated with

87 membrane lipid microviscosity changes. For example, a decrease in membrane microviscosity or conversely, an increase in membrane fluidity might allow the receptors to move within the bilayer more easily and thereby increase their accessibility for the ligand. Therefore, a series of investigations was undertaken to further explore this relationship between membrane microviscosity and prolactin binding activity. Since the proportion of unsaturated fatty acids in membrane is one of the determinants of lipid microviscosity, hepatic plasma membrane lipids were altered in vivo by feeding rats a diet deficient in essential fatty acids (EFA). Maintenance of C3H female mice on an EFA deficient diet for 32 weeks produced a progressive loss exceeding 50% in hepatic prolactin binding as well as a marked increase in membrane microviscosity. Furthermore, treatment of EFA deficient mice with exogenous prolactin did not reverse this loss of hepatic PRL binding (218). Consistent with this apparent effect of EFA, in vitro treatment of mouse liver homogenates with arachidonic acid, which is derived from EFA, exhibited a dose-dependent increase (maximally about 75%) and then decrease in specific prolactin binding activity (219) . Since phospholipase A2 generates arachidonic acid from membrane phospholipids, the effect of this enzyme in vitro was also tested on female mouse hepatic membranes. Phospholipase A 2 also produced a dose-related increase (about 50%) and then decrease in prolactin binding. The effect of bradykinin, an activator of membrane phospholipase A 2 , was also tested in the above in vitro system and produced a dose dependent increase (about 25%) and decline in prolactin binding (219). Scatchard analyses of prolactin binding activity after arachidonic acid, phospholipase A 2 , and bradykinin treatment revealed that the increased prolactin binding activity produced was due to increased receptor number rather than affinity (219). In addition, decreases in membrane microviscosity were associated with the above increases in prolactin receptor number (219). The

effects

of

exposure

of

mouse

liver

homogenates

to

88 prostaglandins on prolactin binding and microviscosity were next explored, since arachidonic acid is a well-known precursor of these compounds. Of the prostaglandins of the '2' series, only prostaglandin I2 (prostacyclin) showed a dose-dependent increase (of about 50%) and then decrease in prolactin binding. This increase in prolactin binding corresponded to a decrease in membrane microviscosity (220). The dose-dependent biphasic changes in PRL binding and membrane microviscosity observed for prostacyclin, as well as for arachidonic acid, phospholipase A 2 , and bradykinin are unexplained but may reflect specific biochemical changes in the membrane. To confirm the involvement of prostaglandins in the modulation of prolactin binding activity, the effects of treatment with indomethacin, an inhibitor of prostaglandin synthesis, was explored. Indomethacin treatment in vivo produced a reduction in the number of hepatic prolactin receptors in both male and female mice in a dose dependent fashion concomitant with an increase in membrane microviscosity (221). Treatment of normal mice with exogenous prolactin produced a biphasic dose-dependent change in prolactin receptors (a stimulation and then a return to baseline) reminiscent of that discussed above for prostacyclin and the other agents associated with the prostaglandin cascade. The change in PRL receptors was inversely related with changes in membrane microviscosity (222). Treatment of rats with a stimulatory dose of PRL could not completely reverse the effects of indomethacin treatment on prolactin receptors (221). In these experiments with mice treated with indomethacin and/or prolactin, an inverse relationship was revealed between the molar ratio of phospholipids to cholesterol and membrane microviscosity (221,222). The above findings suggested that prolactin might influence the synthesis of membrane phospholipids. This possibility was explored by treating male mice with exogenous prolactin for 1 or 2 days and measuring the levels of prostaglandins in hepatic membranes as well as prolactin receptor activity and membrane microviscosity. In addition to the expected time-dependent increase in prolactin binding and decrease in membrane

89 microviscosity, there was a time-dependent increase in both prostaglandin E and F2a (223) . Subsequent studies in pregnant increased prolactin binding, decreased mice showed that microviscosity and increased prostaglandin synthesis and membrane phospholipid:cholesterol ratios were all correlated with changes in endogenous serum lactogenic hormone levels (prolactin and placental lactogen) until mid to late pregnancy. Toward late pregnancy and throughout lactation, all of these parameters returned to control levels (209). This reversal was attributed to the progressive increases in lactogenic hormones and the biphasic nature of prolactin mediated effects on membrane microviscosity, membrane phospholipid:cholesterol ratios and its own receptors (217). The above relationship between prolactin binding, membrane microviscosity and prostaglandins have been confirmed in studies performed in another organ, the prostate of the male rat. Indomethacin treatment to male rats produced a marked reduction in the number of prolactin binding sites and corresponding increase in microviscosity of membranes from ventral and dorsolateral lobes of the prostate (224). In a subsequent study, treatment of rats with ovine prolactin produced a dose-dependent increase in prolactin binding site concentration in both lobes of the prostate with corresponding decreases in membrane microviscosity. The dorsolateral lobe was proportionately more responsive to exogenous hormone than the ventral lobe (189). This latter finding is consistent with physiologic data of others suggesting that the lateral prostate is a preferential target for the hormone (198) . The findings up to this point may be summarized as follows: 1) an inverse correlation appears to exist between membrane microviscosity and prolactin receptor number; 2) modulation of membrane microviscosity and, hence, prolactin receptors may occur via the phospholipase A2 mediated production of prostaglandins; 3) prolactin within the physiologic range appears to up-regulate its own receptor in liver and prostate via this prostaglandin mediated alteration of membrane microviscosity, thus increasing the availability of PRL receptors.

90 Another approach to the association between prolactin receptors and membrane fluidity involved testing the effect of aliphatic alcohols in vitro, known fluidizers of membranes, on prolactin receptor levels in prostatic membranes. Varying concentrations of ethanol, 1-propanol, or 1-butanol produced a dose dependent increase and then decline in specific prolactin binding. Changes in prolactin binding were inversely related to changes in membrane microviscosity (225). Thus alcohol-induced increased fluidity of membranes had the capacity of unmasking cryptic PRL receptors. In a subsequent study, alcohol manipulation was used to gain insight into the age-dependent changes in prolactin binding activity in prostates of Sprague-Dawley male rats. Immature rats (24-25 days old) exhibit the highest prostatic prolactin binding activity. The prostatic prolactin binding capacity of mature rats (80-90 day old) was 50% while that of aged rats (550-610 day old) was 25% that of immature rats. Relative to immature rats, membrane microviscosity of mature rats and aged rats were increased 72% and 142%, respectively. In vitro ethanol treatment failed to unmask cryptic PRL receptors in immature rats but produced a dose-dependent increase and then decrease of PRL binding in both mature and aged adults. Aged rats exhibited a more pronounced (170%) increase in prolactin binding activity than mature adult rats (60%) . In fact, the maximal attainable levels of prolactin receptors in ethanol treated prostatic membranes from mature and aged rats were equivalent to one another and quite close in magnitude to that observed membranes from immature rats (226). These data suggest that regardless of age, the absolute concentration of prostatic prolactin receptors is quite constant and the age dependent decline in prolactin binding is determined by a corresponding increase in membrane microviscosity tending to make PRL receptors less accessible to ligand. More recently alcohol manipulation of fluidity has been used to explore, prolactin binding at the subcellular level. As will be discussed later, prolactin binding activity of Golgi-rich

91

membranes of a variety of tissues including rat prostate and rat liver is 4-6 fold higher than that of plasma membranes. Golgi membranes of rat ventral prostate appear to be about 2.5 times more fluid than plasma membranes which may in part account for this difference in PRL receptor levels. Ethanol exposure of Golgi membranes failed to increase either their receptor levels or membrane fluidity while plasma membranes were responsive to ethanol both in terms of increasing prolactin binding or decreasing membrane microviscosity. Essentially identical results were obtained comparing Golgi and plasma membranes of female rat liver (227) . The data suggest that the pool of PRL receptors subject to modulation is in the plasma membrane while those in the Golgi region are relatively unresponsive to fluidity mediated modulations. This preferential response of plasma membranes is also demonstrable under physiological conditions. Bromocriptineinduced hypoprolactinemia in rats produced a decrease in prolactin binding and lipid fluidity in plasma membrane enriched No effect was observed in fractions of prostate and liver. Golgi enriched fractions. In vitro ethanol treatment fluidized and increase prolactin binding in prostatic and hepatic plasma membranes but had little effect in Golgi membranes. A proportionately greater (2-fold) increase in prolactin binding and fluidity was observed in plasma membranes from bromocriptinetreated rats than form control rats. The results suggest that physiologic changes in endogenous prolactin will preferentially influence prolactin receptor activity in plasma membranes over those in Golgi membranes and appears to do so via membrane fluidity changes (228) . Other local influences on prolactin receptor activity have been observed, such as the stimulatory effects of dextran coated charcoal treatment on prostatic membranes(105) and the direct inhibitory effects of dihydrotestosterone (105) and of the glucocorticoidglucocorticoid receptor complex (229) on prostatic and hepatic membranes, respectively. Whether these are mediated via microviscosity related alterations in the availability of cryptic prolactin receptors remains to be determined.

92

Other investigators have also provided evidence of prolactin receptor modulation via an alteration in the availability of cryptic receptors in the membrane. Similar to the fluidizing effect of alcohol discussed above, Vonderhaar and co-workers have shown that treatment of membranes with the non-denaturing zwitterionic detergent CHAPS produced marked increases in the number of prolactin receptors (118,121). This same group has also shown that mouse lactating mammary gland membrane contains the enzyme machinery for the formation of phosphatidyl choline via the methylation of phosphatidyl n-ethanolamine. This reaction is associated with an increased prolactin receptor concentration and an alteration in membrane microviscosity (121,231). Costlow and co-workers have demonstrated the unmasking of cryptic PRL receptors in intact mammary cancer cells in culture via energy depletion (232). Exposure of such cells to low doses of prolactin (0.1 to 0.5 ng/ml) will produce a sustained (6 day) increase in prolactin receptor content while high doses (ug/ml) of the hormone produce a down-regulation of receptors (233). Finally the effects of energy depletion (234) and phospholipid methylation (231) on increasing the availability of cryptic receptors appears to occur at the plasma membrane level consistent with the data reported above for alcohol fluidization and bromocriptine (227,228). Taken together the data suggest that sensitivity of a target to prolactin is modulated on the cell surface via alterations in the availabilty of cryptic receptors.

Mechanism of Prolactin Action The search for a second messenger Since many protein/peptide hormones generate the second messenger, cyclic AMP, it was assumed that prolactin acted via a similar mechanism. However, little evidence exists for a cyclic AMP-mediated mechanism of prolactin action (13 5). The search for a second messenger for prolactin has been elusive. Houdebine and co-workers published a series of papers from 1981-1984 in which

93 evidence was provided that a small peptide intracellular mediator was generated from rabbit mammary plasma membranes after This prolactin interaction with its cell surface receptor. mediator then interacted with nuclei resulting in the increased transcription of messenger RNA for casein and alpha-lactalbumin (117,235). Unfortunately, there has been recent difficulty in obtaining a consistent demonstration of this second messenger and, therefore, these investigators in 1985 published a retraction of their claims (236). Investigations into defining the mechanism of action of prolactin have employed the following approaches using primarily rodent and rabbit mammary explants in culture: 1) attempts to identify putative intracellular mediators by measuring biochemical changes in tissue exposed to prolactin; 2) measurements of secretory (milk protein, lipid, RNA synthesis), proliferative (DNA synthesis) , and other biochemical changes in explants exposed to agonists and antagonists of putative mediators; 3) examination of the above parameters after exposure to pharmacologic agents intended to alter endogenous levels of putative mediators. Falconer and co-workers suggested that the lactogenic effect of prolactin may be linked to the activation of the Na/K pump after they demonstrated that prolactin activates a ouabain-sensitive Na-K ATPase, decreases tissue Na and increases K, and ouabain blocked prolactin stimulation of fatty acid and protein synthesis (15,237,238). In 1980 Rillema reviewed the existing data from his and other laboratories on the biochemical events associated with prolactin action in mouse mammary gland explants. The following phenomena were emphasized. Some data existed suggesting that cyclic GMP may be stimulatory while cyclic AMP might be inhibitory in mammary gland. Prolactin appears to require calcium for the stimulation of its principal actions. Prolactin actions also appear to involve phospholipase activation which leads to the generation of prostaglandins. Arachidonic acid, a prostaglandin precursor, and certain prostaglandins mimicked the action of prolactin while indomethacin, a prostaglandin synthesis inhibitor, blocked prolactin action. Finally, prolactin stimulated tissue exhibits increased levels of

94

polyamines as well as activated enzymes associated with polyamine synthesis, such as orinithine decarboxylase, and polyamines could under certain conditions mimic prolactin actions (239). Based on these observations and related reports, Rillema proposed a cascade model for the signal transduction of prolactin induced casein biosynthesis. As a result of prolactin interaction with cell surface receptors, plasma membrane phospholipase is activated. This leads to the conversion of membrane phospholipids to prostaglandins via its intermediary, arachidonic acid. Prostaglandins stimulate membrane associated guanylate cyclase, thus enhancing the rate of cyclic GMP synthesis. Cyclic GMP then stimulates cyclic AMP phosphodiesterase activity which reduces the level of cyclic AMP. Reduction in cyclic AMP leads to an enhanced RNA synthesis. Prolactin also stimulates polyamine synthesis via an independent pathway, and spermidine in some way interacts with the enhanced RNA synthesis to promote casein biosynthesis (239) . Recently Rillema and coworkers have published additional studies which tend to support the involvement of reduced cyclic AMP as a stimulatory component (240), calcium as an obligatory co-factor (242,243), ornithine decarboxylase activation as an event in prolactin action (240,241), prolactin-induced phospholipase A 2 activation and prostaglandin generation (242,244), and suggesting phosholipase C a possible link between prolactin-induced activation, the subsequent generation of diacyl glycerides which in turn activate ornithine decarboxylase via protein kinase C (241). These investigators have also recently provided evidence of prolactin-induced increases in the phosphorylation of RNA, phosphoproteins, and phospholipids (245). On the other hand, other investigators have expressed reservations about the involvement of Na/K ATPase, cyclic nucleotides, prostaglandins, polyamines, and calcium in the generation of the prolactin message. Among the reasons for this uncertainty are: 1) high doses of prolactin were required to produce some of the biochemical changes associated with putative

95

mediator formation (9) ; 2) lack of reproducibility of certain observations (246,247); 3) findings that were inconsistent with the above concepts (248); 4) the actions of selected inhibitors of putative mediator generation (such as ouabain) may be toxic and, therefore, acting via nonspecific mechanisms (249); 5) none of the putative mediators have been able to adequately replace prolactin as a stimulant of casein messenger RNA accumulation (9,247,250). The fact that none of the above agents were able to convincingly mimic the action of prolactin with regard to mRNA transcription suggests that none of them are the intracellular mediator. Alternatively, if the mechanism of action of prolactin involves a cascade of chemical reactions or a coordinated interplay of a multiplicity of components rather than a single messenger, as Rillema suggests (239), it is not surprising that a single agent failed to replace PRL. There may also be alternate explanations for some of the above observations. For example, if as suggested above, phospholipase A 2 activation is involved in prolactin receptor up-regulation (218-225), the observations implicating prostaglandin involvement may reflect cell surface or hormone receptivity phenomena rather than signal transduction per se. The lack of reproducibility of various findings between laboratories, although disconcerting, may be explained on the basis of the complexity of the test systems used in studies of prolactin signal transduction, namely rabbit, rat, and mouse mammary explants. Prolactin responses in mammary explants generally require exposure of the tissue to insulin and glucocorticoid (and exposure to estradiol, at least in vivo) as well as with prolactin (6) . Furthermore, addition of thyroid hormone to the incubation medium selectively enhances prolactininduced increases in alpha-lactalbumin synthesis (6) . Manipulation of the levels of any one of these hormones (as well as the addition of progesterone, which is inhibitory) might influence the magnitude of the prolactin-induced effect. The interplay between prolactin and these other hormones is not completely understood and appears to be rather complex. For example, insulin is required for the accumulation of casein mRNA,

96

and glucocorticoid is capable of enhancing prolactin-induced accumulation of casein mRNA by retarding the degradation of the RNA (6,9). Thyroid hormones appears to enhance prolactin induction of alpha-lactalbumin mRNA synthesis (6) . Since the action of prolactin in mammary gland is subject to modulation by hormones or hormonally modified intracellular mechanisms, it is very difficult to differentiate a specific effect of prolactin from some, as yet unexplained, interplay with other factors. Another source of complexity stems from the fact that prolactin produces a variety of responses and these have not been consistently used by the laboratories interested in prolactin signal transduction. Furthermore, evidence exists to suggest that different expressions of prolactin action (such as casein biosynthesis vs. lipid biosynthesis) may involve different intracellular mechanisms (242). At this point in time there is a need to examine the mechanism of action using a simpler system. The Nb2 lymphoma cell line appears to be a suitable model for further elucidation the mechanism of prolactin action. It is exquisitely sensitive to and highly specific for lactogenic hormone, apparently requires no other hormones, and as far as we know, exhibits a single response, proliferation. Recent studies with this model have suggested biochemical events that are associated with prolactin-induced mitogenesis in this cell line that are similar to those observed previously in mammary explants. Among these are induction of ornithine decarboxylase and the generation of polyamines (251,252), and the possible involvement of phospholipase A 2 activation, and prostaglandin generation (253). Gertler et al. have also recently shown that the mitogenic effect of lactogenic hormone on Nb2 lymphoma cells is enhanced by the addition of the tumor promoter 12-0tetradecanoyl-phorbol-13-acetate (TPA) as well as several other 4 beta phorbol diesters. Since a principal biochemical effect of TPA is activation of protein kinase C, the involvement of phospholipase C activation and diacylglycerol release from inositol phospholipids has been suggested as a component of the mechanism of action of the hormone(254).

97

Recent studies with this cell line also suggest that prolactin induces modifications in phosphatidylcholine metabolism, namely increases in choline uptake into phosphatidylcholine, increases in phosphatidylcholine content, and marked reductions in phosphatidylcholine turnover (255). Fleming et al. recently reported that human GH was capable of stimulating a very rapid and marked accumulation of c-myc transcripts in Nb2 lymphoma cells. This is a common form of mitogen induced gene expression. Unlike other cells where c-myc transcripts are maintained after brief mitogen exposure, the transcripts rapidly decline after hormone withdrawal from Nb2 lymphoma cells. This apparently explains why prolonged exposure to lactogenic hormone is required for mitogenic effects in this cell line (256). Although components of prolactin signal transduction may be at hand, the sequence of events and the interaction of the various components have yet to be elucidated. A single messenger may not be found. However, promise certainly exists for identifying the rate limiting steps and characterizing the principal components of the mechanism. Perhaps when our understanding of the mechanism of action of prolactin has been elucidated on the Nb2 lymphoma, it will be appropriate to reexamine the mammary gland. For additional perspectives on prolactin signal transduction the reader is referred to the recent review of Vonderhaar (121). Internalization of prolactin In view of the almost universal acceptance of the concept that protein/peptide hormones generated an intracellular second messenger (257), it was not until the mid-1970's that the possibility that such hormones entered cells was given widespread and serious attention. Several lines of evidence were responsible for the interest in protein/peptide hormone internalization at that time. Among these were the demonstration of binding sites for a variety of hormones in subcellular fractions other than plasmalemma (258), the demonstration that exogenous hormones bound to intracellular organelles or were visualized internally (258), and the convincing demonstration of receptor mediated endocytosis of low density lipoprotein and its

98 role in cholesterol homeostasis (259). Particular interest in prolactin internalization was stimulated at this time by the failure to find a second messenger for this hormone and a lack of understanding as to its mechanism of action. Light microscopic immunoperoxidase studies indicated the existence of intracellular endogenous prolactin in secretory cells of lactating rat mammary gland (260) and revealed binding sites for exogenous rat prolactin confined to the Golgi region of ventral prostate epithelial cells (261). Receptors for lactogenic hormone were demonstrated in a variety of subcellular fractions of the rat liver, the Golgi vesicles being the most enriched fraction having about 6 times the receptor concentration of plasmalemma (262) . As alluded to previously, this apparent sequestration of prolactin binding sites to Golgi structures is consistent with numerous observations indicating an enrichment of ligand receptor sites in Golgi fractions in a variety of tissues (258) . Posner, Bergeron, Khan and co-workers at McGill University initiated studies on the receptor mediated uptake of insulin and prolactin in rat liver in vivo by measuring radioactivity bound to hepatic cell fractions at various times after intravenous administration of 125I-labeled hormone. Two temporally-distinct phases of prolactin uptake by female rat liver were observed, an initial phase (within 5 minutes of injection) in which hormone bound transiently to a fraction containing small uncoated vesicles and a more prolonged phase (15-30 minutes) into the combined Golgi light and intermediate fractions comprised of lipoprotein containing vesicles. Radioactivity eluted from these fractions appeared to be intact hormone based upon its chromatographic behavior and ability to bind receptors (263). The pattern of prolactin uptake revealed in these early studies were similar that observed previously with insulin (264). Recent studies have revealed that intracellular prolactin receptors in rat liver are heterogeneous. Khan et al. measured the distribution of lactogenic hormone binding to Golgi light and Golgi intermediate fractions and individual lysosomal

99

subfractions (LI to L4) relative to the distribution of Golgi and lysosomal marker enzymes, galactosyltransferase and acid phosphatase, respectively. The light and intermediate Golgi fractions exhibited high lactogenic hormone binding along with elevated galactosyltransferase activity and low acid phosphatase activity. The LI subfraction was also prolactin receptor enriched, but was low in galactosyltransferase and had significant acid phosphatase activity (acid phosphatase activity was highest in the L2 subfraction) . The LI subfraction, which also contained lipoprotein, was tentatively designated as "unique" vesicles, distinct from either Golgi vesicles or lysosomes (265). Percoll gradient density analysis of Golgi intermediate fractions also revealed two populations of receptors, one that cosedimented with the Golgi marker, galactosyltransferase, and the other which was more dense migrating with acid phosphatase. The latter component was also considered by Khan et al. to be "unique" vesicles (265). Ferland et al. reported virtually the same relative distribution of prolactin receptors and acid phosphatase activity in individual lysosomal fractions of rat liver and designated LI as "prelysosomes" while L2 which also had considerable prolactin binding activity was considered to be mature lysosomes (266). With the increased recognition of the complexity of intracellular prolactin receptors, the time course of the uptake of prolactin by rat liver after in vivo injection of radioactive hormone was re-evaluated by Khan and co-workers. Golgi intermediate fractions were subfractionated on Percoll density gradients. At 5 minutes postinjection the radioactivity was associated with elements of low density that were rich in galactosyltransferase. At 10 minutes, radioactivity shifted to elements of higher density enriched with acid phosphatase. At subsequent times the radioactivity in all fractions diminished (264). Ferland et al. reported very similar uptake kinetics and distribution of radiolabeled prolactin in rat liver in vivo using different methods of isolating fractions. Radioactive hormone association with plasma membrane peaked at 5 minutes and then bound preferentially in the Golgi fraction, with radioactivity being very high at 5 minutes but reaching a peak at 15 minutes

100

(corresponding to peak hormone uptake by significant radioactivity was found in reached peak levels at 30 minutes. observed for radioactivity incorporated

liver). At 15 minutes the LI fraction but Similar kinetics were into the L2 fraction

(266).

Although both of the above studies were interpreted to suggest a sequence of uptake of hormone into Golgi enriched vesicles followed by a transfer of hormone to L fractions, they do not exclude the possibility that there are two pathways of internalization of hormone, one leading to the Golgi associated membranes and the other, delayed in time, leading to the lysosomal compartment. Basset et al. recently provided data consistent with this latter possibility by comparing the hepatic uptake of radiolabelled rat prolactin in vivo in male and female rats. In females prolactin was primarily localized to low density membranes associated with galactosyltransferase while about 10-15% of the trace was associated with higher density membranes containing the lysosomal marker enzyme, N-acetyl-Bglucosaminidase. In normal male rats, prolactin localized solely to the latter fraction. Treatment of male rats with estrogen produced a distribution of incorporated prolactin that was similar to that of females (267). Recently Bergeron et al. employed the quantitative radioautographic morphologic method they developed to monitor the uptake of 125I-labelled prolactin by rat liver. In female rats, where receptor mediated uptake occurred, light microscopic autoradiography revealed an initial (2 minutes) accumulation of silver grains at the hepatocyte periphery but at later times (10 and 3 0 minutes), radioactivity was clustered near the Golgi complex/bile canalicular region of the cell. At the ultrastructural level, radioactivity initially localized (within 2 minutes) in the proximity of the plasma membrane at the sinusoidal and lateral surfaces of the hepatocyte. Shortly thereafter radioactivity accumulated transiently in small smooth surfaced vesicles (considered to be endocytic vesicles and corresponding to the first phase of uptake observed previously with cell fractions). As radioactivity within endocytic vesicles

101

was declining, there was a corresponding increase in silver grains within lipoprotein containing vesicles which were closely associated with Golgi stacks. However, little or no hormone could be localized to Golgi stacks per se. Through much of the observation period approximately 15% of the radioactivity was associated with structures morphologically (but not cytochemically) identified as secondary lysosomes (268) . Endocytosis of lactogenic hormone has also been visualized by others in cultured human lymphocytes and rabbit mammary fragments using an electron microscopic quantitative radioautographic approach. These studies also revealed a sequestration of radioactivity in the Golgi region in structures identified as lysosomes (269,270). Previous radiometric and morphologic-radioautographic studies by the McGill group reveal a similar uptake for insulin by rat liver in vivo (258,264). Based upon their radiometric and radioautographic uptake findings with insulin and lactogenic hormone, they have proposed a tentative model for the uptake of these hormones into the target cell. Following interaction with the cell surface receptor, the hormone is internalized via uncoated endocytic vesicles. These vesicles fuse with endosomal vesicles (lipoprotein containing, non-lysosomal structures) which accumulate in the vicinity of the Golgi apparatus. From the endosomes, hormone is transferred to the lysosome for terminal degradation and receptors are recycled to the cell surface (264,271). "Endosomes" is a cytological term applying to all intracellular nonlysosomal components involved in the incorporation of exogenous substances (271). Recent biochemical studies by this group suggest that, although endocytosed ligand will co-migrate under certain conditions with galactosyltransferase, they bind to membranes that are distinct from Golgi vesicles and are devoid of this marker (272) . Recent sucrose density methods have been developed to subfractionate the endosomal components that bind incorporated ligand. Those subfractions previously considered Golgi associated are now referred to as early endosomes, while the "unique" ("prelysosomal") vesicles are referred to as late

102

endosomes. Unique vesicles or late endosomes have been characterized biochemically as being prolactin and insulin receptor enriched, containing lipoprotein, comigrating with acid phosphatase, and cytochemically staining weakly positive for acid phosphatase (273). It has been suggested that the unique vesicles correspond to lipoprotein containing structures on the trans aspect of the Golgi stack in liver designated previously by Novikoff as GERL. The GERL was defined as a structure that was intimately related to the Golgi saccule (G), that is part of the endoplasmic reticulum (ER) that forms lysosomes (L) (274) . More recently this region of the cell has been designated as the trans Golgi network (TGN) (275) . The TGN is thought to be either a distinct organelle or an extension of the trans cisterna of the Golgi apparatus and is identifiable by its acid phosphatase activity. The proposed function of the TGN is to sort materials for transport to the plasmalemma and lysosomes which is consistent with the putative role of the unique vesicles, of transferring ligand to the lysosomes for degradation and recycling receptors to the cell surface. Although the same general model has been considered for insulin and lactogenic hormone uptake, some significant differences have been noted. Endosomal elements accumulate twice as much prolactin as insulin. The hepatic metabolism of insulin is faster than that of prolactin. Most of this difference appears to be reflected in the rate of transfer from the plasmalemma to the early endosomal components. The half-life of prolactin in plasmalemmal associated fractions is 3-fold greater than that of insulin. For early endosomal elements the half-life of prolactin is 2-fold greater than insulin, while for late endosomal elements the half-lives of both hormones are equivalent. It is suggested that the major reason for this difference in the kinetics of uptake is because early endosomes initiate insulin degradation while prolactin degradation occurs later. Although the kinetics of prolactin and insulin processing in liver differ, the sedimentation patterns for each internalized hormone suggests that they use the same endocytic system (273) . The phenomenon of prolactin-induced "down-regulation" of its own

103

receptors exemplifies the dynamics of prolactin internalization and degradation. Djiane et al. showed that intravenous injection of large doses of bovine prolactin produced a decline in free and total prolactin receptors in both lactating rabbit mammary gland and ovariectomized female rat liver. The striking characteristic of these declines in free and total prolactin receptors was that they showed different time courses. Free receptors were maximally saturated (80% decline) within 15 minutes to 1 hour after injection while a maximal decline in total receptors (of about 50%) was observed later, 6 hours after injection (276). Down regulation has also been demonstrated by Dj iane et al. in organ cultures of lactating rabbit mammary gland. Saturation of free receptors to about 20% of control was observed 24 hours after addition of prolactin (5ug/ml) to the incubation medium and maximal decline in total receptors (about 60%) was delayed to 48 hours (277) . In a subsequent study, these investigators showed that down regulation of plasma membrane PRL receptors preceded that of Golgi-associated membrane receptors of lactating rabbit mammary gland after intravenous injection of a large dose of prolactin (278) . Such an observation is consistent with the shift of the prolactin receptor complex from the surface to the inside of the cell and its ultimate disappearance. The involvement of lysosomal degradation in prolactin-induced rapid "down regulation" of its own receptors is indicated by the effects the lysosomotropic agents, chloroquine, ammonium chloride, and methylamine on this process. When lactating rabbit mammary explants were coincubated in the presence of these agents down regulation was prevented. In the absence of exogenous prolactin, lysosomotropic agents caused an increase in prolactin receptors in rabbit mammary explants in vitro (279) and female rat liver in vivo (266) , particularly in L subfractions (266), suggesting a constant turnover of receptors. Some very recent studies by the McGill group suggest that chloroquine accumulates in late endocytic vesicles (unique vesicles) and acts by neutralizing the acidity of these organelles (280). It has been suggested that these substances be renamed as acidotropic rather than lysosomotropic agents (271).

104

Several morphological and biochemical studies indicate in a variety of tissues that although lactogenic hormone degradation is inhibited by chloroquine and other lysosomotropic agents, internalization of the lactogenic hormone to endocytic components is unimpaired (270,273,280,281). Other pharmacologic agents have been used to gain an understanding of the mechanisms involved in the internalization and processing of prolactin. Posner et al. examined the effect of pretreatment of rats with colchicine and vincristine, promoters of microtubule disaggregation, on the uptake of intravenous 125I-ovine-prolactin into subcellular fractions of rat liver. These agents had little or no effect on the amount of label incorporated into the fraction corresponding to the endocytic vesicles (258) but delayed the uptake of hormone into liver and impaired its degradation (268). Using morphometric analysis, these workers found that although the drug had no effect on internalization of label into lipoprotein containing vesicles, these hormone containing vesicles were found in the periphery of the cell and failed to orient themselves around the Golgi cisternae as in control rats. Accordingly, the authors suggest that these agents do not impair endocytosis of the hormone per se but a subsequent step in its intracellular transport (268). Available data suggest no consistent effects on internalization and processing of prolactin by cytochalasin B (247), a microfilament inhibitor, or of a variety of transglutaminase (clustering) inhibitors (258,270). The physiologic role of hormone internalization is the subject of much speculation and controversy. There is general agreement that one role of the process is to inactivate and degrade ligand while the receptor itself is scavenged and recycled to the cell surface and degraded at a slower rate (117,258,269). The accumulation of prolactin in milk (282) and in seminal fluid (283) suggests that internalization in certain tissues may be a means of transferring hormone from serosal to mucosal compartment. However, controversy exists as to whether internalization of prolactin is critical for its signal transduction. This is particularly intriguing in view of the

105

fact that the hormone has such a pronounced and prolonged sequestration inside of the cell prior to its demise. However, at the present time no evidence exists that the hormone has an intracellular locus of action although its internalization is without question. On the other hand, none of the evidence supporting the prevalent view that prolactin signal transduction occurs exclusively at the cell surface, when appropriately analyzed, is particularly compelling. The first suggestion that prolactin might act at the cell surface came from the observation that the hormone was biologically active when covalently bound to Sepharose (100). However, evidence exists that these hormones may be readily released from their covalent bonds and may even do so in a superactive form (284). The classic radioautographic observation of prolactin accumulation on the basal surface of mammary epithelial cells (101) as well as the presence of receptors in plasma membrane enriched fractions (104) is certainly overshadowed by the visualization of internalized hormone, and the demonstration of intracellular receptors (258,260). Furthermore, the demonstration of lactogenic effects of prolactin receptor antibodies (in low doses), suggestive of cell surface signal transduction (125-127), does not exclude the possibility that these agents can be internalized. In fact internalization of immunoglobulins into mammary epithelial cells is documented (285). Doubts about the importance of internalization have been raised as a result of comparing the effects of pharmacologic agents on down regulation with their effects on prolactin-induced biological responses. For example, although lysosomotropic agents markedly block down regulation, they fail to inhibit a variety of prolactin-induced effects, such as casein biosynthesis and casein mRNA accumulation (247,286). However, as reviewed above, both biochemical and morphologic data indicate that even though the degradation of the hormone is blocked, internalization of the hormone occurs. On the other hand, colchicine fails to block down regulation, but abolishes prolactin induced biologic responses (286,287). The blocking actions of colchicine may be

106

nonspecific. As demonstrated by Bergeron et al., colchicine produces a major disruption in the organization of endosomal vesicles as well as a failure for prolactin containing endosomes to accumulate in the Golgi region (268). The major architectural rearrangements in the cell or the absence of Golgi region accumulation of endosomes containing prolactin could be responsible for the actions of colchicine. Furthermore, interpretations of findings with colchicine should be tempered with caution since considerable uncertainty exists regarding the precise nature of its action (258). Finally, the observation by Ferland et al. that at physiological doses of prolactin a close association exists between the downregulation and the major biological responses in mammary explants (casein biosynthesis, casein mRNA accumulation, and DNA synthesis) (288) could very well reflect the importance internalization as a component signal transduction. Proteolytic processing of prolactin by target tissues Although it is generally accepted that internalized prolactin is terminally degraded in the target cell, virtually no information was available about the chemical nature or identity of degraded products of these hormones. Most methods employed to assess the status of internalized or bound hormone, ie., precipitablity in trichloroacetic acid, chromatographic migration and ability to bind to receptors do not detect subtle changes in the molecule. In 1981 studies were initiated in our laboratory to determine how prolactin was modified by target tissue, if at all (289). Incubation of unlabelled rat prolactin with fractions of rat ventral prostate decreased hormone immunoreactivity in a time and tissue concentration dependent fashion suggestive of hormone degradation. To verify that the prolactin was chemically altered by the tissue, a quantitative sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) radioautographic method was developed after incubation of 125I-labeled rat prolactin with subcellular fractions of ventral prostate tissue. At a pH range of 6-9, prolactin degradation occurred in 2 subcellular fractions, the cytosol and the 3 3 00xg pellet. The former

107

fraction revealed only partial proteolysis of prolactin (evidenced by fragment formation in the range of 16K to 8 K daltons) while the latter, appeared to terminally degrade the hormone (disappearance of prolactin band with no fragments visualized). However, no prolactin degrading activity was observed in the 25,000xg pellet, the fraction with highest acid phosphatase activity and presumably the one that is lysosomeenriched (289) . Therefore, the effects of acidification (pH 4 to 5) on prolactin degradation by prostatic fractions were examined. Acidification inhibited prolactin degradation in the cytosol but enhanced hormone degrading activity in the 3300 xg pellet and unmasked enormous amounts of degrading activity in the 25,000 xg pellet. Both acid-activated pellets generated substantial amounts of a 16K fragment and lesser amounts lighter fragments (12K to 8K). This activity was tentatively attributed to lysosomal proteases, since both fractions exhibited significant amounts of acid phosphatase. The acidified 25,000 xg pellet was capable of generating 150 times as much 16K fragment as the cytosol at neutral pH (290) . In the male rat the subcellular distribution of prolactin degrading activity varied from tissue to tissue. Some tissues were quite active while others were relatively quiescent. Of the various organs of the male rat, the ventral prostate was the most active in fragmenting hormone (289,290) . Since the mammary gland is the principal target for prolactin, it was of interest to determine whether or not this tissue was capable of degrading prolactin and, in particular, whether fragments were generated. The 25,000 xg pellet of 20-day lactating rat mammary gland produced an acid-activated pattern of proteolytic fragmentation that appeared identical to that of ventral prostate, but was 5-10 times as active as ventral prostate. In contrast to the distribution of this activity in ventral prostate, the acid dependent prolactin fragmenting activity of mammary gland was present in all subcellular fractions of mammary gland and predominated in the cytosol and could not be correlated with a particular subcellular marker (291) .

108

In very recent experiments acid-dependent proteolytic degradation been demonstrated in Nb2 lymphoma. The pattern of fragmentation appears identical to that of mammary gland and ventral prostate and the level of enzyme activity appears comparable to that of the ventral prostate. This activity is widespread among the subcellular fractions of the lymphoma with the highest activity being in the 25,000xg pellet and cytosol (292). In an attempt to characterize the nature of the proteases fragmenting prolactin, the effects of inhibitors of the 4 major class of proteases (serine, metallo, sulfhydryl, and aspartate) have been examined in each of the above three tissues. Each tissue exhibited sensitivity to at least two proteases but the profile of inhibitor sensitivity differed from tissue to tissue. All of the active tissues examined thus far at acid pH, however, share one common feature, their sensitivity to aspartate proteases (290-292). In all experiments, SDS-PAGE was conducted under reducing conditions (in the presence of 2-mercaptoethanol) which disrupted disulfide bonds, a procedure required for the estimation of molecular weight of electrophoretic bands. Since the intact rat prolactin molecule contains a loop of 114 amino acids formed by a disulfide bond the possibility existed that peptide fragments seen under reducing electrophoretic conditions were generated by the reducing agent and that the modification of the prolactin molecule was, in fact, more subtle. Under nonreducing electrophoretic conditions the disappearance of intact prolactin was associated primarily with the formation of a band that migrates more slowly (24 K) than intact prolactin (23 K) . Identical results were also obtained with other fractions of lactating mammary gland and the 25,000 xg pellet of ventral prostate (291). Other laboratories have also shown that cleavage in the loop of hormones of the prolactin-growth hormone family produce isohormones that migrate under nonreducing electrophoretic conditions more slowly than native hormone (as reviewed in refs. 290 and 291). It was subsequently verified that the 24 K product of prolactin was the source of the 16 K and

109

smaller fragments of prolactin by re-electrophoresis of this band under reduced conditions (291). The data indicate that the principal product of prolactin proteolysis under acid conditions was a variant of intact hormone with a cleavage site somewhere in its loop. In order to characterize this cleaved variant of prolactin chemically, biologically, and immunologically, large amounts of cleaved prolactin were generated by incubating lOOug of biological grade NIADDK rat prolactin with 25,000 xg pellets of lactating mammary gland (pH 4.5). Conventional forms of chromatography have been unable to resolve intact from cleaved prolactin. However, both products exhibited significantly different retention times on a hydrophobic interactive HPLC column. N-terminal amino acid analysis of cleaved prolactin by automatic Edman degradation revealed that the second N-terminus began at amino acid 149 of the prolactin molecule suggesting that the cleavage site is about 3/4 of the way from the N-terminus of the molecule (293). To date it is not known whether proteolytic cleavage of the prolactin molecule involves elimination of some amino acids. The biologic potency of this cleaved prolactin was assayed relative to intact hormone in two systems, radioreceptor assay (using rat prostate and liver membrane preparations) and the Nb2 lymphoma proliferative assay. The ability of cleaved prolactin to displace 125I-labeled ovine prolactin from its receptor or to stimulate the proliferation of Nb2 lymphoma cells was indistinguishable from intact rat prolactin (292-294). Using two different NIADDK anti-sera directed against rat prolactin (S-6 and S-8), approximately a 50% loss in immunoactivity was observed after cleavage of the hormone molecule (292). Our finding that cleaved prolactin has undiminished biological activity was recently confirmed by Clapp et al. using radioreceptor assay and pigeon crop assay. These investigators also reported that cleaved prolactin exhibited about a 50 % reduction in immunological activity consistent with our findings (295) . This indicates that the biological site and the

110

immunological site of prolactin reside in different regions of the molecule. The loss of immunological activity suggests that although the hormone is fully active biologically that it has undergone significant conformational changes. Evidence from the work of others suggesting that the proteolytic cleavage and/or fragmentation of hormones from the prolactingrowth hormone family may be of physiologic significance has been reviewed (289-291). The fact that cleaved prolactin is biologically active strengthens the suggestion that limited proteolysis of prolactin in target tissue has biological relevance. It is also noteworthy that two unequivocal targets for prolactin, the mammary gland and Nb2 lymphoma, and one putative target rich in prolactin receptors, the ventral prostate, possess high levels of prolactin cleaving activity. Furthermore, Clapp et al. report that lactating mammary gland has higher cleaving activity than pregnant mammary gland where prolactin action is generally inhibited (295). These workers have also shown that female rat liver has more cleaving activity than male rat liver, consistent with the well known sexual dimorphism in prolactin receptors in this tissue (295). The suggestion of physiological relevance of acid-dependent proteolytic cleavage of prolactin is strengthened by the fact that intracellular organelles, particularly the endocytic vesicle, are capable of undergoing acidification (296). The facts that prolactin appears to be cleaved in the same location within the molecule regardless of the tissue examined, that the subcellular distribution of activity is widespread and differs from tissue to tissue, and that several types of proteases are involved, suggest that the process of cleavage is performed by nonspecific multipurpose proteases in the tissue. The specificity of cleavage of the molecule appears to be* determined not by a specific "prolactinase" but by the three dimensional configuration of the molecule and a susceptible cleavage site at or near amino acid 149. The physiological role of prolactin cleavage by target tissue is not known. If, as has been suggested, that the biologically

111

active regions of hormones of the prolactin-growth hormone family reside in distinct loci within the amino acid sequences, cleavage at the target tissue level might reveal these loci (297). Alternatively, cleavage might produce a configurational change in the molecule that is critical for the biological effect. Since peptide hormones themselves appear to be derived in their organs of origin from larger precursor molecules by controlled digestion, an analogous situation could exist in target tissue (298) .

Concluding Comments This review has attempted to provide some perspective on the current state of knowledge regarding the mechanism of action of prolactin. In view of the diversity of prolactin actions in both mammalian and submammalian species, it is not surprising that there has been difficulty in elucidating the mode of signal transduction for this hormone in target tissues. Furthermore, the responsiveness of the target organ appears to be associated with changes in the biophysical state of the plasma membrane. Agents which influence membrane phenomena (such as prostaglandins) may or may not be intracellular mediators for prolactin. Considerable emphasis was placed in this review on the internalization of this hormone into target cells since much has been learned very recently in this area. The body of knowledge regarding the proteolytic processing of prolactin by target tissue was also discussed in detail. It is now apparent that not only does prolactin spend a considerable amount of time inside of the cell, but does so as a biologically active variant that has been modified in a subtle way. Unraveling the mystery of prolactin signal transduction may involve future studies of prolactin processing, target cell internalization, receptor purification and modulation and, of course, identifying the principal chemical mediators.

112

Acknowledgements The work reported herein was supported by NIH grants R01-CA 23653 (to

RJW)

and

R01—AM—22032

and

K01-AM-00707

(to

RAA),

institutional grant IN-105 from the American Cancer Society RJW)

and a merit review grant

(to RAA) .

from the Veterans

(to

Administration

The authors wish to thank Drs. Robert Vick and Vicky

Wong for assisting in the literature search.

Expert secretarial

assistance was provided by Marjorie Hobson.

References 1.

Bern, H.A., Nicoli, C.S. 1968. Ree. Prog. Horm. Res. 24. 681-720.

2.

Nicoli, C.S. 1980. Fed. Proc. 30, 2563-2566.

3.

DeVlaming, V.L. 1979. In: Hormones and Evolution (E.J.W. Barrington, Ed.) vol. 2. 561-642, Academic Press, N e w York.

4.

Lyons, W.R, Li, C.H., Johnson, R.E. 1958. Ree. Prog. Horm. Res. 14, 219-254.

5.

Topper, Y.J. 1970. Ree. Prog. Horm.

6.

Vonderhaar, B.K., Bhattacharjee. M. 1985. In: Biological Responses in Cancer (E. Mihich, Ed.) vol. 4. 125-159, Plenum Publishing Corp., New York.

7.

Kleinberg, D.L., Todd, J. 1980. Metab. 51, 1009-1013.

8.

Turkington, R.W. 1968. Endocrinology 82., 575-583.

9.

Rosen, J.M. 1981. In: Elsevier, New York.

10.

Guyette, W.A. , Matusik, R.J., Rosen, J.M. 1979. Cell 17., 1013-1023.

11.

Kleinberg, D.L., Todd, J., Niemann, W. 1978. J. Clin Endocrinol. Metab. 47, 435-441.

12.

Vonderhaar, B. 1977.

13.

Nardacci, N.J., Lee, J.W.C., McGuire, W.L. 1978. Cancer Res. 38, 2694-2699.

J.

Res. 26, 287-308.

Clin. Endocrinol.

Prolactin (R.B. Jaffe, Ed.).

Endocrinology 100.

85-126,

1423-1431.

113

14.

Zinder, 0., Hamosh, M., Fleck, T.R.C., Scow, R.O. 1974. Am. J. Physiol. 226. 744-748.

15.

Falconer, I.R., Rowe, J.M. 1977. Endocrinology 101, 181-186

16.

Weisz-Carrington, P., Roux, M.E., McWilliams, M., Phillips-Quagliata, J.M., Lamm, M.E. 1978. Proc. Natl. Acad. Sei. USA 75, 2928-2932.

17.

Kleinberg, D.L., Niemann, W., Flamm, E., Cooper, P., Babitsky, G., Valensi, Q. 1985. J. Clin. Invest. 75, 19431950.

18.

Dembinski, T.C., Leung, C.K.H., Shiu, R.P.C. 1985. Cancer Res. 45, 3083-3089.

19.

Bohlen, P., Baird, A., Esch, F. Ling, N., Gospodarowicz, D. 1984. Proc. Natl. Acad. Sei. USA 81, 5364-5368.

20.

Kasper, S., Friesen, H.G. 1986. J. Clin Endocrinol. Metab. 62. 70-76.

21.

Witorsch , R.J. 1980. J. Histochem. Cytochem. 28., 1-9.

22.

Frantz, A.G., Kleinberg, D.L. 1970. Science 170. 745-747.

23.

Hwang, P., Guyda, H., Friesen, H. 1971. Proc. Nat. Acad. Sei. USA 68/ 1902-1906.

24.

Forbes, A.P., Henneman, P.H., Griswold, G.C., Albright, F. 1954. J. Clin. Endocrinol. Metab. 14, 265-271.

25.

Daughaday, W.L. 1985. In: Williams' Textbook of Endocrinology, (J.D. Wilson and D.W. Foster, eds) 568-613, W.B. Saunders Company, Philadelphia.

26.

Kandan, S., Gopalakrishnan, V., Govindarajulu, P., Indiva, R., Malathi, T. 1983. Hormone Res. 17, 93-102.

27.

Arafah, B.M. 1986. J. Clin. 1179.

28.

Turkington, R.W. 1972. Arch. Int.

29.

Adler, R.A. 1977. Am.

30.

Ross, F., Nusynowitz, M.L. 1968. Metab. 28, 591-595.

31.

Randall, R.V., Scheithauer, B.W., Laws, Jr., E.R., Abbound, C.F., Ebersold M.J., Kao. P.C. 1985. Mayo Clin. Proc. 60. 753-762.

7th ed.

Endocrinol. Metab. 62, 1173Med. 130. 349-354.

J. Obstet. Gynecol. 127. 569-571. J.

Clin. Endocrinol.

32. Thorner, M.O. 1977. In: Clinical Neuroendocrinology, (L. Martini and G.M. Besser, Eds.) 319-361, Academic Press, New York.

33.

Nagasawa, H. 1979. Eur. J. Cancer JJ5, 267-279.

34.

Vonderhaar, B.K., Biswas, R.: In: Cellular and Molecular Biology of Experimental Mammary Cancer (D. Medina, W.R. Kidwell, G. Heppner, and E. Anderson, Eds.) Plenum Publishing Co., New York In press.

35.

Furth, J., Clifton, K.H.1957. Cancer 10, 842-853.

36.

Malarkey, W.B., Kennedy, M., Allred, L.E., Milo, E. 1983. J. Clin. Endocrinol. Metab. 56, 673-677.

37.

Welsch, C.W. 1976. J. Toxicol, and Environ. Health, Suppl. 1, 161-175.

38.

McNatty, K.P., McNeilly, A.S., Sawers, R.S. 1977. Prolactin and Human Reproduction (P.G. Crosignani C.Robyn, Eds.) 109-117, Academic Press New York.

39.

Koizumi, K., Aono, T., Koike, K., Endocrinol 107. 433-438.

40.

Klibanski, A., Beitins, I.Z., Merriam, G.R., McArthur, J.W., Zervas, N.T., Ridgway. E.C. 1984. J. Clin. Endocrinol. Metab. 58/ 1141-1147.

41.

Bouchard, P., Lagoguey, M., Brailly, S., Schaison, G. 1985. J. Clin. Endocrinol. Metab. 60, 258-262.

42.

Kleinberg, D.L., Noel, G.L. Frantz. A.G. 1977. New Engl. J. Med. 296. 589-600.

43.

Franks, S., Jacobs, H.S., Martin, N., Clin. Endocrinol. 8, 277-287(1978).

44.

Everett, J.W. 1944. Endocrinology 35, 507-520.

45.

Everett, J.W. 1954. Endocrinology 54»

46.

Chen, C.L., Amenomori, Y. Lu, K.H., Voogt, J.L., Meites, J. 1970. Neuroendocrinology 6^. 220-227.

47.

Adler, R.A. 1986. Endocr. Rev. 7, 302-313.

48.

Rothchild, I. 1965. Vitam. Horm. 23., 210-327.

49.

Gelato, M.C., Meites, J., Wuttke., W. 1978. Acta Endocrinol. 89. 590-598.

50.

Witorsch, R.J., Kitay, J.I. 1972. Endocrinology 91, 764-769.

51.

Advis, J.P., Ojeda, S.R. 1978. Endocrinology 103. 924-935.

52.

Doherty, P.C., Bartke, A., Hogan, M.P., Klemcke, H., Smith, M.S. 1982. Endocrinology 111. 820-826.

53.

Doherty, P.C., Bartke, A., Smith, M.S., Davis, S.L. 1985. Horm. Behav. 19, 111-121.

In: and

Kurachi, K. 1984. Acta

Nabarro, J.D.N. 1978.

685-690.

115 54.

B u c k m a n , M . T . , K e l l n e r , R. 1985. A m . J . P s y c h i a t r y 142. 244.

55.

M e i e r , A . H . , D a v i s , K.B. 1967. G e n . C o m p . 114.

56.

S u n d a r a r a j , B . I . , N a y y a r , S.K. 1969. G e n . C o m p . S u p p l . 2, 6 9 - 8 0 .

Endoer.,

57.

M a z z i , V . , V e l l a n o , C . , . T o s c a n o , C. 1967. G e n . E n d o e r . 8, 3 2 0 - 3 2 4 .

Comp.

58.

R i d d l e , 0., B a t e s , R . W . , Lahr., E . L . 1935. A m . J. 3, 3 5 2 - 3 6 0 .

59.

N o b l e , G . K . , K u m p f , K . F . , B i l l i n g s , V . N . 1938. 21, 3 5 3 - 3 5 9 .

60.

L y o n s , W . R . , P a g e , E. 1935. Proc. S o c . E x p . B i o l . 1049-1050.

61.

A n d e r s o n , T . R . , P i t t s , D.S., N i c o l l , C.S. 1984. G e n . E n d o e r . 54, 2 3 6 - 2 4 6 .

62.

D e l i d o w , B . C . , H e b e r t , N . C . , S t e i n y , S., N i c o l l , C . S . J. E x p . Z o o l . 238, 1 4 7 - 1 5 3 .

63.

Francis, M.J.O.,

64.

B e r m a n , R . , B e r n , H . A . , N i c o l l , C.S., S t r o h m a n , R . C . J. E x p . Z o o l . 156. 3 5 3 - 3 6 0 .

65.

Y o s h i z a t o , K., Y a s u m a s u , I. 1970. D e v e l . G r o w t h D i f f . 305-317.

66.

D e r b y , A . 1970. J. E x p . Zool. 173,

67.

E t k i n , W . , G o n a , A. 1967. J. Exp. Zool.

68.

C a r r , F . E . , J a c o b s , P . J . , J a f f e , R . C . 1981. M o l e c . a n d Cell. E n d o c r i n o l . 22, 65-76.

69.

Lewis, U.J., Singh, R.N.P., Tutwiler, G.F., Sigel, M.B., V a n d e r L a a n , E . F . , V a n d e r L a a n , W . P . 1980. R e e . Prog. H o r m . Res. 36, 4 7 7 - 5 0 8 .

70.

Beck, J.C., Gonda, A., Hamid, M.A., Morgen, R.O., R u b i n s t e i n , D., M c G a r r y , E . E . 1964. M e t a b o l i s m 13., 1 4 4 - 1 7 0 .

71.

F r i g e r i , L . G . , L e w i s , L.J., T e g u h , K., L e w i s , U . J . B i o c h e m . B i o p h y s . R e s . Comm. 134. 7 6 4 - 7 6 9 .

72.

W o o l f , P . D . , Lee, L.A., L e e b a w , W., T h o m p s o n , D., L i l a v i v a t h a n a , U . , B r o d o w s , R., C a m p b e l l , R. 1977. J. E n d o c r i n o l . M e t a b . 45, 3 7 7 - 3 8 3 .

73.

A d l e r , R . A . , S o k o l , H . W . 1983. L i f e S e i . 32,

E n d o e r . 8,

H i l l , D.J. 1975. N a t u r e 255,

242-

110-

Physiol.

Endocrinology Med.

32.

Comp. 1986.

167-168. 1964. 11,

319-328. 165.

249-258.

1986.

Clin.

2949-2956.

74.

Nicoll, C.S. 1981. In: Prolactin (R.B. Jaffe, Ed.) Elsevier, New York.

127-166,

75.

Lockett, M.F. 1965. J.

76.

Horrobin, D.F., Burstyn, P.G., Lloyd, I.J., Durkin, N. Lipton, A., Muiruri, K.L. 1971. Lancet 2., 352-354.

77.

Miller, M., VanGemert. M. 1976. Horm.

78.

Malarkey, W.B., George, J.M., Baehler, R.W. 1975. IRCS Med. Sci: Endocrine System. 2, 291.

79.

Carey, R.M., Johanson, A.J., Seif, S.M. 1977. J. Clin. Endocrinol. Metab. 44/ 850-858.

80.

North, W.G., Gellai, M., Sokol, H.W., Adler, R.A. 1981. Horm. Res. 15, 55-64.

81.

Adler, R.A., Marceau, M., Borst, D.W. , Sokol, H.W., Culp, W.J. 1982. Endocrinology 110. 674-676.

82.

Adler, R.A., Dolphin, S., Szefler, M., Sokol, H.W. 1979. Endocrinology 105. 1001-1006.

83.

Adler, R.A., Herzberg, V.L., Brinck-Johnsen, T., Sokol, H.W. 1986. Endocrinology 118, 1519-1524.

84.

Russell, D.H., Matrisian, L., Kibler, R., Larson, D.F., Poulos, B., Magun, B.E. 1984. Biochem. Biophys. Res. Comm. 121. 899-906.

85.

Posner, B.I., Kelly, P.A., Shiu, R.P.C., Friesen, H.G. 1974. Endocrinology 95, 521-531.

86.

Russell, D.H., Kibler, R., Matrisian, L., Larson, D.F., Poulos, G., Magun, B.E. 1985. J. Immunology 134. 3027-3031.

87.

Hiestand, P.C., Mekler, P., Nordmann, R., Grieder, A., Permmongkol, C. 1986. Proc. Natl. Acad. Sci. USA 83., 25992603.

88.

Mahajan, F.B., Ebner, K.E. 1985. Biochem. Biophys. Comm. 133, 753-758.

89.

Noble, R.L., Beer, G.T., Gout, P. 1980. Cancer Res. 40; 2437-2440.

90.

Gout, P.W., Beer, C.T., Noble. R.L. 1980. Canccer Res. 40, 2433-2436.

91.

Tanaka, T., Shiu, R.P.C., Gout, P.W., Beer, C.T., Noble R.L., Friesen, H.G. 1980. J. Clin. Endocrinol. Metab. 51. 1058-1063.

92.

Shiu, R.P.C., Elsholtz, H.P., Tanaka, T., Friesen, H.G., Gout, P., Beer, C.T., Noble, R.L. 1983. Endocrinology 113: 159-165.

Physiol. 181, 192-199.

Res. 7, 319-332.

Res.

117 93.

H u g h e s , J . P . , E l s h o l t z , H . P . , F r i e s e n , H . G . 1984. P o l y p e p t i d e H o r m o n e R e c e p t o r s (B.I. P o s n e r , Ed.) M a r c e l D e k k e r , Inc. N e w Y o r k .

In: 157-199,

94.

W h i t e , B.A. 111.

95.

F r y e r , J . N . 1979. G e n . Comp. E n d o e r . 39,

96.

N i c o l i , C . S . , M a y e r , G . L . , R u s s e l l , S . M . 1986. R e v . , 7, 1 6 9 - 2 0 3 (1986).

97.

D r a g o , F., S c a p a g n i n i , T h e r . 276, 2 7 1 - 2 7 8 .

98.

B e n f e n a t i , F., F e r r e t t i , C., C i m i n o , M . , V a n t i n i , L i p a r t i t i , M., M u c c i o l i , M., D i C a r l o , R . , A l g e r i , 1986. P h a r m a c o l . R e s . Comm. 18, 4 3 1 - 4 4 9 .

99.

Carlson, H.E., Lamberts, S.W.J., Brickman, A.S., Deftos, L . J . , H o r s t , R . L . , F o r t e , L.R. 1985. E n d o c r i n o l o g y 117. 1602-1607.

, N i c o l i . C.S. 1979. A m . Zool. 19, 875,

Abstract

397-403.

U. 1985. A r c h . Int.

Endocrine

Pharmacodyn.

G., S.

100. T u r k i n g t o n , R . W . 1970. B i o c h e m . B i o p h y s . R e s . C o m m u n . 1362-1367. 101. B i r k i n s h a w , M . , F a l c o n e r , I.R. 1972. J . E n d o c r . 55, 102. T u r k i n g t o n , R . W . 1971. J . C l i n . I n v e s t . , M ,

94

41.

323-334.

(1971).

103. S h i u , R . P . C . , K e l l y , P.A., F r i e s e n , H . G . 1973. S c i e n c e 968-971. 104. S h i u , R . P . C . , F r i e s e n , H . G . 1974. B i o c h e m . J . 140, 105. D a v e J . I . , W i t o r s c h , R . J . 1982. E n d o c r i n o l o g y , 2146.

301-311.

111.

2144-

106. K e e n a n , E . J . , R a m s e y , E . E . , K e m p , E . D . 1981. In: T h e P r o s t a t i c C e l l (G.P. M u r p h y , A . A . S a n d b e r g , J . P . K a r r , P a r t B 9 - 1 8 , A l a n R. L i s s , N e w Y o r k . 107. T h o r e l l , J . I . , J o h a n s s o n , B.G. 1971. B i o c h i m . B i o p h y s . 251.363-369. 108. L o w r y , O . H . , R o s e n b r o u g h , N . J . , F a r r , A . L . , R a n d a l l , 1951. J. B i o l . C h e m . 193, 2 6 5 - 2 7 5 . 109. S c a t c h a r d , G. 1949. A n n . N.Y. A c a d . S c i . , 51,

180.

Eds.) Acta,

R.J.

660-673.

110. K e l l y , P . A . , D j i a n e , J., L e b l a n c , G. 1983. P r o c . S o c . B i o l . M e d . , 172, 2 1 9 - 2 2 4 . 111. D o n n e r , D . B . , C o r i n , R . E . 1980. J. B i o l . C h e m . , 255. 9008.

Exp.

9005-

118 112. Greenwood, F.C., Hunter, W.M. 1963. Biochem. J. 89, 114123. 113. Frantz, W.L., and Turkington, R.W. 1972. Endocrinology, 21,1545-1548. 114. Subramanian, M.G., Gala, R.R. 1986. J. Clin. Immunoassay, 9, 42-52. 115. Gertler, A., Shamay, A., Cohen, V., Ashkenazi, A., Friesen, H.G., Levanon, A., Gorecky, M., Aviv, H., Hadary, D., Vogel, T. 1986. Endocrinology, 118. 720-729. 116. Shiu, R.P.C., and Friesen, H.G. 1974. J. Biol. Chem. 249.7902-7911. 117. Kelly, P.A., Djiane, J., Katoh, M., Ferland, L.H., Houdebine, L.M., Teyssot, B., Dusanter-Fourt, I. 1984. Recent Progress Horm. Res. 60: 379-439. 118. Liscia, D.S., Vonderhaar, B.K. 1982. Proc. Nat. Acad. Sei. USA, 79, 5930-5934. 119. Borst, D.W., Sayare, M. 1982. Biochem. Biophys. Res. Commun. 105. 194-201. 120. Hughes, J.P., Simpson, J.S., Friesen, H.G. 1983. Endocrinology, 112 • 1980-1986. 121. Vonderhaar, B.K.: In: The Mammary Gland: Development, Regulation and Function ( C.W. Daniel and M.C. Neville, Eds.) Plenum Publishing Co., New York. In press. 122. Shiu, R.P.C., Friesen, H.G. 1976. Biochem J., 157. 619-626. 123. Shiu, R.P.C., Friesen, H.G. 1976. Science 192, 259-261. 124. Bohnet, H.G., Shiu, R.P.C., Grinwich, D., Freisen, H.G. 1978. Endocrinology, 102. 1657-1661. 125. Djiane, J., Houdebine, L.M., Kelly, P.A. 1981. Proc. Natl. Acad. Sei. USA, lä, 7445-7448. 126. Shiu, R.P.C., Elsholtz, H.P., Tanaka, T., Friesen, H.G., Gout, P.W., Beer, C.T., Noble, R.L. 1983. Endocrinology 113. 159-165. 127. Dusanter-Fourt, I., Djiane, J., Kelly, P.A., Houdebine, L.M., Teyssot,B. 1984. Endocrinology, 114. 1021-1027. 128. Djiane, J., Kelly, P.A., Katoh, M., and Dusanter-Fourt, I. 1985. Horm. Res., 22, 179-188. 129. Katoh, M., Djioane, J., Kelly, P.A. 1985. J.Biol. Chem, 260. 11422-11429. 130. Amit, T., Barkey, R.J., Gavish, M., Youdim, M.B.H. 1986.

119 Endocrinology, 118. 835-843. 131

Kelly, P.A., Leblanc, G., Djiane, J. 1979. Endocrinology, 104.1631-1638.

132. Costlow, M.E., Buschow R.A., McGuire, W.L. 1974. Science 184.85-86. 133. Holcomb, H.H., Costlow, M.E., Buschow, R.A., McGuire, W.L. 1976. Biochim. Biophys. Acta, 428: 104-117. 134. Smith, R.D., Hilf, R., Senior, A.E. 1976. Cancer Res. 36. 3726-3731. 135. Turkington, R.W., Frantz, W.L., and Majumder, G.C. 1973. In: Human Prolactin (J.L. Pasteels and C. Robyn, Eds.) 24-47, Excerpta Medica, New York. 136. Frantz, W.L., Mclndoe, J.H., Turkington, R.W. 1974. J. Endocr. 60, 485-497. 137. Nagasawa, H., Yanai, R. 1978. Acta Endocr.88, 94-98. 138. Sakai, S., Kohmoto, K., Johke, T. 1975. Endocr. Japon. 22., 379-381. 139. Sheth, N.A., Ranadive, K.J., Sheth, A.R. 1974. Europ. J. Cancer 10, 653-660. 140. Costlow, M.E., Buschow, R.A., Richiert, N.J., and McGuire, W.L. 1975. Cancer Res. 31, 970-974. 141. Powell, B.L., Diamond, E.J., Kopaek, S., Hollander, V.P. 1977. Cancer Res. 37: 1328-1332. 142. Turkington, R.W. 1974. Cancer Res. 34, 758-763. 143. Smith, R.D., Hilf, R., Senior, A.E. 1977. Cancer Res. 37: 595-598. 144. Costlow, M.E., McGuire, W.L. 1977. J. Natl. Cancer Inst. 58. 1173-1175. 145. Costlow, M.E., Buschow, R.A., McGuire, W.L. 1976. Cancer Res. 36.3324-3329. 146. DeSombre, E.R., Kledzik, G., Marshall, S., and Meites, J. 1976. Cancer Res. 36/ 354-358. 147. Holdaway, I.M., Friesen, H.G. 1976. Cancer Res. 36, 15621567. 148. Kelly, P.A., Bradley, C., Shiu, R.P.C., Meites, J., Friesen, H.G. 1974. Proc. Soc. Exp. Biol. Med. 146. 816-819. 149. Kledzik, G.S., Bradley, C.J., Marshall, S., Campbell, G.A., Meites, J. 1976. Cancer Res. .36, 3265-3628.

120 150. Friesen, H.G. 1976. In: Cancer Research (G. St. Arneault, P. Bond, and L. Israel, Eds.) 14 3-149, Springer-Verlag, Berlin. 151. Holdaway, J.M., and Friesen, H.G. 1977. Cancer Res., 37. 1946-1952. 152. Morgan, L., Raggatt, P.R., DeSouza, I., Salih, H., Hobbs, J.R. 1977. J. Endoer. 73, 17P. 153. DeSouza, I., Hobbs, J.R., Morgan, L., Salih, H. 1977. J. Endoer. 73, 17P. 154. L'Hermite-Baleriaux, M., L'Hermite, M. 1984. In: Hormone Research in Growth and Reproduction (B.B. Saxena, K.J. Catt, L. Birnbaumer, and L. Martini, Eds.) 291-305, Raven Press, New York. 155. Kelly, P.A., Tsushima, T., Shiu, R.P.C., Friesen, H.G. 1976. Endocrinology 99.765-774. 156. McNeilly, A.S., and Friesen, H.G. 1977. J.Endoer. 509.

74.507-

157. Bohnet, H.G., Gomez, F., Friesen, H.G. 1977. Endocrinology, 101,1111-1121. 158. Djiane, J., Durand, P., Kelly, P.A. 1977. Endocrinology, 100. 1348-1350. 159. Djiane, J., Durand, P. 1977. Nature, 266. 641-643. 160. Sakai, S., Enami, J., Nandi, S., Banerjee, M.R. 1978. Mol. Cell Endoer., 12, 285-298. 161. Sheth, N.A., Tikekar, S., Ranadive, K.J., Sheth, A.R. 1976. IRCS Med. Sei. 4, 323. 162. Gandilhon, P., Melancon, R., Gandilhon, F., Djiane, J., Kelly, P.A. 1983. Anticancer Res. 3., 203-206. 163. Nagasawa, H., Sakai, S., Banerjee, M.R. 1979. Life Sei. 24. 193-208. 164. Nagasawa, H. 1979. Med. Hypotheses 51, 1117-1121. 165. Nagasawa, H. 1979. Eur. J. Cancer 15, 267-279. 166. Horrobin, D. 1975-1981. In: Prolactin, vols. 3-9, Eden Press, Montreal. 167. Saito, T., Saxena, B.B. 1975. Acta Endoer. 80, 126-137. 168. Rolland, R., Hammond, J.M. 1975. Endoer. Res. Commun. 2, 281-298. 169. Rolland, R., Gunsalus, G.L., Hammond, J.M. 1976. Endocrinology,98, 1083-1090.

121

170. Richards, J.S. Williams, J.J. 1976. Endocrinology, 99, 15711576 171. Cheng, K.W. 1976. J. Reprod. Fertil. 48, 129-135. 172. Poindexter, A.N., Buttram, V.C., Besch, P.K., Smith, R.G. 1979. Fertil. Steril., 31,27-277. 173. Davies, T.F., Katikineni, M., Chan, V., Harwood, J.P., Dufau, M.L., Catt, K.J. 1980. Nature 283, 863-865. 174. Koppelman, M.C.S., Dufau, M.L. 1982. Endocrinology, 1350-1357.

111.

175. Wang, C., Hsueh, A.J.W., Erickson, G.F. 1979. J. Biol. Chem., 2 M , 11330-11336. 176. Nolin, J.M. 1980. Biol. Reprod., 22, 417-422. 177. Dunaif, A.E., Zimmerman, E.A., Friesen, H.G., Frantz, A.G. 1982. Endocrinology, 110. 1465-1472. 178. Magoffin, D.A., Erickson, G.F. 1982. Endocrinology, 2001-2007.

111.

179. Navickis, R.J., Jones, P.B.C., Hsueh, A.J.W. 1982. Mol. Cell. Endoer., 27, 77-88. 180. Bohnet, H.G., Shiu, R.P.C., Grinwich, D., and Friesen, H.G. 1978. Endocrinology 102, 1657-1661. 181. Bonifacino, J.S., Dufau, M.L. 1984. In: Hormone Receptors in Growth and Reproduction (B.B. Saxena, K.J. Catt, L. Birnbaumer, and L. Martini, Eds.) 149-168, Raven Press, New York. 182. Marshall, S., Gelato, M., Meites, J. 1975. Proc. Soc. Exp. Biol. Med. 149. 185-188. 183. Reddy, S., and Watkins, W.B. 1975. J. Endoer. 65, 183-194. 184. Donatsch, P., Richardson, B. 1975. J. Endoer. 66, 101-106. 185. Costlow, M.E., McGuire, W.L. 1977. J. Endoer. 75, 221-226. 186. Marshall, S., Huang, H.H., Kledzik, G.S., Campbell, G.A., Meites, J. 1978. Endocrinology, 102. 869-876. 187. Marshall, S., Bruni, J.F., and Meites, J. 1979. Endocrinology, 104. 390-395. 188. Fellows, R.E., Soltysiak, R.M. 1980. In: Central and Peripheral Regulation of Prolactin Function (R.M. MacLeod, ü. Scapagnini, Eds) 159-171, Raven Press, New York. 189. Dave, J.R., and Witorsch, R.J. 1985. Am. J. Physiol., 248. E687-E693.

122 190. Hughes, J.P., Elsholtz, H.P., Friesen, H.G. 1982. Endocrinology, 111. 102-707. 191. Aragona, C. , Friesen, H.G. 1975. Endocrinology 684.

97, 677-

192. Bohnet, H.G., Friesen, H.G. 1976. J. Reprod. Fert., 48» 307316. 193. Charreau, E.H., Attramadal, A., Torjesen, P.A., Purvis, K., Calandra, R., Hansson, V. 1977. Mol. Cell. Endocr.,6, 303307. 194. Aragona, C. ( Bohnet, H.G., Friesen, H.G. 1977. Acta Endocr. (kbh), 84/ 402-409. 195. Sonenberg, M., Money, W. 1955. Ree. Prog. Horm. Res., 11. 43-82. 196. Grayhack, J.T., Bunce, P.L., Kearns, J.W., Scott, W.W. 1955. Johns Hopk. Hosp. Bull. 9£, 154-163. 197. Huggins, C., Russell, P.S. 1946. Endocrinology, 39: 1-7. 198. Yamanaka, H., Kirdani, R.Y., Saroff, J., Murphy, G.P., Sandberg, A.A. 1975. Am. J. Physiol. 229, 1102-1109. 199. Hanlin, N.L., Yount, A.P. 1975. Endocr. Res. Commun., 2., 489-502. 200. Kledzik, G.S., Marshall, S. Campbell, G.A., Gelato, M., Meites, J.1976. Endocrinology 98, 373-379. 201. Witorsch, R.J.: In: Prolactin in Neoplastic and Nonneoplastic Lesions (H. Nagasawa, Ed.) CRC Press, Boca Raton In Press. 202. Posner, B.I., Kelly, P.A., Friesen, H.G. 1972. Proc. Natl. Acad. Sei. (USA) 71, 2407-2410. 203. Kelly, P.A., Posner, B.I., Friesen, H.G. 1975. Endocrinology, 97.1408-1415. 204. Posner, B.I. 1976. Endocrinology, 99, 1168-1175. 205. Kelly, P.A., Posner, B.I., Tsushima, T. ( Friesen, H.G. 1974. Endocrinology, 95, 532-538. 206

Dave, J.R. Knazek, R.A. 1983. Mech. Ageing Dev., 23., 235243.

207. Posner, B.I. 1976. Endocrinology 98, 645-651. 208. Hayden, T.J., Bonney, R.C., Forsyth, I.A. 1979. J. Endocr. 80. 259-269. 209. Dave, J.R., Richardson, L.L., Knazek, R.A. 1983. J. Endocr., 99, 99-106.

123 210. Costlow, M.E., Buschow, R.A., McGuire, W.L. 1976. Cancer Res. 36, 3941-3943. 211. Posner, B.I., Kelly, P.A., Friesen, H.G. 1975. Science 188. 57-59. 212. Costlow, M.E., Buschow, R.A., McGuire, W.L. 1975. Life Sci. 17,1457-1465. 213. Bohnet, H.G., Aragona, C., Friesen, H.G. 1976. Endocr. Res. Commun. 3, 187-208. 214. Frantz, W.L., Mann, L.C., Welsch, C. 1977. IRCS Med. Sci., 5, 32. 215. Knazek, R.A., Liu, S.C., Gullino, P.M. 1977. Endocrinology 101, 50-58. 216. Knazek, R.A., Liu, S.C., Graeter, R.L., Wright, P.C., Mayer, J.R., Lewis, R.H., Gould, E.B., Keller, J.A. 1978. Endocrinology 103. 1590-1596. 217. Dave, J.R.: In: Actions of Prolactin on Molecular Processes (J.A. Rillema, Ed.) CRC Press, Boca Raton In Press. 218. Knazek, R.A., Liu, S.C. 1979. Proc. Soc. Exp. Biol. Med. 162. 346-350 (1979). 219. Dave, J.R., Knazek, R.R., Liu, S. C. 1981. Biochem. Biophys. Res. Commun. 103. 727-738. 220. Dave, J.R., and Knazek, R.A. 1980. Proc. Natl. Acad. Sci. (USA), 77, 6597-6600. 221. Knazek, R.A., Liu, S.C., Dave, J.R., Christy, R.J., Keller, J.A. 1981. Prostaglandins Med. 6, 403-411. 222. Dave, J.R., Knazek, R.A., Liu, S.C. 1981. Biochem. Biophys. Res. Commun. 100. 45-51. 223. Dave, J.R., Brown, N.V., Knazek, R.A. 1982. Biochem. Biophys. Res. Commun. 108.193-199. 224. Dave, J.R., Witorsch, R.J. 1983. Prostate 4, 119-128. 225. Dave, J.R., Witorsch, R.J. 1983. Biochem. Biophys. Res. Commun. 113. 220-228. 226. Dave, J.R., and Witorsch, R.J. 1984. Biochim. Biophys. Acta 772, 321-327. 227. Dave, J.R., Witorsch, R.J. 1986. Biochem. Biophys. Res. Commun. 134. 1122-1128. 228. Dave, J.R., Witorsch, R.J.: submitted for publication.

124

2 29. Dave, J.R., Witorsch, R.J., Kalimi, M.Y. 1985. Biochim. Biophys. Acta 845: 276-282. 230. Dave, J.R., Krieg, Jr., R.J., Witorsch, R.J. 1985. Biochim. Biophys. Acta. 816. 313-320. 231. Vonderhaar, B.K., Bhattacharya, A., Ahadi, T., Liscia, D.S., Andrew, E.M., Young, J.K., Ginsburg, E., Bhattacharjee, M., Horn, T.M. 1985. J. Dairy Sei., 68, 466-488. 232. Costlow M.E., Hample, A. 1982. J. Biol. Chem., 257. 69716977. 233. Costlow, M.E., Hample, A. 1984. J. Cell Physiol. 118: 247252. 234. Costlow, M.E., Rodgers, Q.E. 1986. Exp. Cell Res., 163. 159164. 235. Teyssot, B., Houdebine, L.M., Djiane, J. 1901. Proc. Natl. Acad. Sei. USA, 78, 6729-6733. 236. Houdebine, L.M., Djiane, J., Kelly, P.A. 1985. Proc. Natl. Acad. Sei. USA, 82., 3062 . 237. Falconer, I.R., Forsyth, I.A., Wilson, B.M., Dils, R. 1978. Biochem. J. 172. 509-516. 238. Falconer, I.R., Langley, J.V., Vacek, A.T. 1983. J. Physiol. 334.1-17. 239. Rillema, J.A.:

Fed.Proc. 1980. 39, 2593-2598.

240. Rillema, J.A., Wing, L.Y.C. 1982. Horm. Metab. Res. 14, 195197. 241. Rillema, J.A.1985. Proc. Soc. Exp. Biol. Med., 178. 490-494. 242. Cameron, C.M., Rillema, J.A. 1983. Prostaglandins Leukotrienes and Med. 10, 433-441. 243. Cameron, C.M., Rillema, J.A. 1983. Endocrinology 113: 15961600. 244. Rillema, J.A., Foley, K.A. 1983. Horm. Metab. Res. 15, 385388. 245. Rillema, J.A., Foley, K.A., Etindi, R.N. 1985. Endocrinology, 116. 511-515. 246. Houdebine, L.M., Lacroix, M.C. 1980. Biochimie, 62, 441-444. 247. Houdebine, L.M., Djiane, J., Kelly, P.A., Katoh, M., Dusanter-Fourt, I., Martel, P. 1984. In: Endocrinology Labrie, I. Proulx, Eds.) 203-206, Elsevier, New York.

(F.

248. Houdebine, L.M. 1980. In: Central and Peripheral Regulation of Prolactin Function (R.M. MacLeod, U. Scapagnini, Eds.)

125

189-205, Raven Press, New York. 249. Houdebine, L.M., Djiane, J. 1980. Biochimie, 62, 433-440. 250. Matusik, R.J., Rosen, J.M. 1978. J. Biol. Chem. 253, 23432347. 2 51. Richards, J.F., Beer, C.T., Bourgeault, C., Chen, K., Gout, P.W. 1982. Molec. Cell. Endocrinol. 26, 41-49 (1982). 252. Ofenstein, J.P., Rillema, J.A., Lawson, D.M. 1985. Proc. SOC. Exp. Biol. Med., 180. 236-239. 253. Ofenstein, J.P., Rillema, J.A. 1986. 68th Ann. Mtg. Endocrine Soc., Abst. no. 751. 254. Gertler, A., Walker, A., Friesen, H.G. 1985. Endocrinology, 116: 1636-1644. 255. Ko, K.W.S., Cook, H.W., Vance, D.E. 1986. J. Biol. Chem., 261. 7846-7852. 256. Fleming, W.H., Murphy, P.R., Murphy, L.J., Hatton, T.W., Matusik, R.J., Friesen, H.G. 1985. Endocrinology, 117. 2547-2549. 257. Sutherland, E.W. 1972. Science, 177. 401-404. 258. Posner, B.I., Khan, M.N., Bergeron, J.J.M. 1982. Endocrine Rev. 3, 280-297. 259. Brown, M.S., Goldstein, J.L. 1986. Science, 232. 34-47. 260. Nolin, J.M., Witorsch, R.J. 1976. Endocrinology, 99, 949958. 261. Witorsch, R.J., Smith, J.P. 1976. Endocrinology 101. 929938. 262. Bergeron, J.J.M., Posner, B.I., Josefsberg, Z., Sikstrom, R.J. 1978. J. Biol. Chem. 253. 4058-4066. 263. Josefsberg, Z., Posner, B.I., Patel, B., Bergeron, J.J.M. 1979. J. Biol. Chem. 254.209-214. 264. Posner, B.I., Khan, M.N., Bergeron, J.M. 1984. In: Polypeptide Hormone Receptors (B.I. Posner, Ed.) 61-90, Marcell Dekker, Inc., New York. 265. Khan, M.N., Posner, B.I., Verma, A.K., Khan, R.J., Bergeron, J.J.M. 1981. Proc. Nat. Acad. Sci. USA 79, 4980-4984. 266. Ferland, L.H., Djiane, J., Houdebine, L.M., Kelly, P.A. 1984. Endocrinology 115, 1842-1849. 267. Basset, M., Smith, G.D., Peters, T.J. 1984. Biochim. Biopys. Acta, 769, 79-84.

126 268. Bergeron, J.J.M., Resch, L., Rachubinski, R., Patel, B.A., Posner, B.I. 1983. J. Cell Biol. 96, 875-886. 269. Barazzone, P., Lesniak, M.A., Görden, P. , van Obberghen, E., Carpentier, J.L., Orci, L. 1980. J. Cell. Biol. 87, 360369. 270. Kayne, S., Raymond, M.N., Dusanter-Fourt, I., Houdebine, L.M., Ollivier-Bousquet, M. 1983. Eur. J. Cell Biol. 30. 244-253. 271. Bergeron, J.J.M., Cruz, J., Khan, M.N., Posner, B.I. 1985. Ann. Rev. Physiol. 47., 383-403. 272. Kay, D.G., Khan, M.N., Posner, B.I., Bergeron, J.J.M. 1984. Biochem. Biophys. Res. Commun., 123. 1144-1148. 273. Bergeron, J.J.M., Searle, N., Khan, M.N., Posner, B.I. 1986. Biochemistry 25, 1756-1764. 274. Novikoff, A.B. 1976. Proc. Natl. Acad. Sei. USA 7J3, 27812787. 275. Griffiths, G., Simons, K. 1986. Science, 234. 438-443. 276. Djiane, J., Clauser, H., Kelly, P.A. 1979. Biochem. Biophys. Res. Commun. 90, 1371-1378. 277. Kelly, P.A., Djiane, J., De lean, A. 1980. In: Central and Peripheral Regulation of Prolactin Function (R.M. MacLeod, U. Scapagnini) 173-188, Raven Press, New York. 278. Djiane, J., Houdebine, L.M., Kelly, P.A. 1981. Proc. Soc. Exp. Biol. Med., 168, 378-381. 279. Djiane, J., Kelly, P.A., Houdebine, L.M. 1980. Molec. Cell. Endocrinol. 18, 87-98. 280. Khan, R.J.. Khan, M.N., Bergeron, J.J.M., Posner, B.I. 1985. Biochim. Biophys. Acta 838. 77-83. 281. Hizuka, N., Görden, P., Lesniak, M.A., Carpentier, J.L., Orci, L. 1982. Endocrinology 111: 1576-1580. 282. Gala, R.R. 1980. Life Sei. 26, 783-788. 283. Sheth, A.R., Mugatwala, P.P., Shah, G.W., Rao, S.S. 1983. Fertil. Steril., 26, 905-907. 284. Topper, Y.J., Oka, T., Vonderhaar, B.K., Wilchek, M. 1983. Biochem. Biophys. Res. Commun., 6j>, 793-798. 285. Moncharmont, B., Su, J.L., Parikh, I. 1982. Biochemistry, 21, 6916-6921. 286. Houdebine, L.M., Djiane, J. 1980. Molec. Cell. Endocrinol., 12, 1-15.

127 287. H o u d e b i n e , L.M. 1980. E u r . J . C e l l . B i o l . , 22, 288. D j i a n e , J . , H o u d e b i n e , L . M . , K e l l y , P.A. E n d o c r i n o l o g y , 110. 7 9 1 - 7 9 5 .

755-760.

1982.

289. C o m p t o n , M . M . , W i t o r s c h , R . J . 1983. P r o s t a t e , 4,

119-128.

290. C o m p t o n , M . M . , W i t o r s c h , R.J. 1984. E n d o c r i n o l o g y , 484. 291. W o n g , V . L . Y . , C o m p t o n , M . M . , W i t o r s c h , R . J . 1986. B i o p h y s . A c t a , 881. 1 6 7 - 1 7 4 . 292. W o n g , V . L . Y . 1 9 8 6 . P h . D . T h e s i s , V i r g i n i a University. 293. V i c k , R . S . 1 9 8 6 . P h . D . T h e s i s , V i r g i n i a University. 294. V i c k , R . , W o n g , V . , W i t o r s c h . S o c . , A b s t . N o . 12 3.

115.

476-

Biochim.

Commonwealth

Commonwealth

1986. 6 8 t h A n n . M t g .

Endo.

295. C l a p p , C., M a y e r , G . L . , H e b e r t , N . J . 1 9 8 6 . 6 8 t h A n n . M t g . E n d o . S o c . , A b s t N o . 122. 296. Y a m i s h i r o , D . J . , M a x f i e l d , F.R. 1984. J . C e l l u l a r 26, 2 3 1 - 2 3 8 .

Biocheml

297. N i a l l , H . D . 1972. In: Prolactin and Carcinogenesis, Fourth T e n o v u s W o r k s h o p (A.F. B o y n s , K. G r i f f i t h s , E d s . ) , 13-21, Alpha Omega Alpha, Cardiff. 298. K r i e g e r , D . T . , M a r t i n , J . B . 1981. N e w E n g l a n d J . M e d . , 8 7 6 - 8 8 5 (1981)

304.

GLUCAGON RECEPTOR: STRUCTURE AND FUNCTION

Richard T. Premont and Ravi Iyengart Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030; P r e s e n t address: Department of Pharmacology, Mount Sinai School of Medicine, New York, New York 10029)

Introduction Pharmacological Characteristics of the Glucagon Receptor Radiolabeling of Glucagon Glucagon Binding Assays Factors Influencing Glucagon Binding Structure of the Glucagon Receptor Crosslinking Protease and Endoglycosidase Mapping Physicochemical Analysis Approaches to Purification Glucagon Receptor Function Adenylyl Cyclase System Activation of N s Functional States of the Glucagon Receptor Regulation of the Glucagon Receptor Internalization Desensitization Future Directions Acknowledgements References

Peptide Hormone Receptors © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

130 Introduction Glucagon

is a peptide

hormone

of

29

amino

secreted by the a cells of the pancreas.

acids

produced

and

It is synthesized as a

larger preprohormone, and glucagon is cleaved from this precursor post-translationally. member

of

the

other

members

Glucagon is a prominent and well

gastrointestinal include

(GI)

vasoactive

family

studied

of hormones,

intestinal

peptide

whose (VIP),

gastric inhibitory peptide (GIP), and secretin, the substance for which the term "hormone" was coined. Glucagon

is

released

hypoglycemia,

mediated by blood for

the

adipose

the

this

glucose

maintenance

fasting.

from

although

of

pancreas

secretion

mainly

in

response

seems not to be

levels, and is primarily available

glucose

to

directly

responsible

during

periods

of

The major sites of glucagon action are the liver and tissues.

Through binding

to its specific

cell-surface

receptor, glucagon induces glycogenolysis and gluconeogenesis, as well

as

inhibiting

hepatic

degradation and ketosis. tic

hormone

amount

of

organism.

pair,

and

promoting

protein

Insulin and glucagon form an antagonis-

which,

available

lipogenesis

at

the

energy,

in

systemic

the

form

level, of

control

glucose,

for

the the

At the intracellular level, these hormones affect the

balance of intermediary metabolism by altering the fate of acetyl CoA; while insulin exerts predominantly anabolic effects, and is in addition a general mitogen, glucagon produces more responses. in

catabolic

It now appears that many of the complications arising

insulin-deficient

actions of glucagon. gon, see Unger

diabetes

are

often

due

to

the

unopposed

For an overview of the physiology of gluca-

(1), or

for a more comprehensive treatment,

see

the volume edited by Unger and Orci (2). The specific

effect of glucagon binding

to its plasma

membrane

receptor is the stimulation of the enzyme adenylyl cyclase, which converts MgATP to the intracellular second messenger 3',5' cyclic AMP (cAMP). tion

of

the

Increased concentrations of cAMP promote the activacAMP-dependent

protein

kinase

(protein

kinase

A,

131

formerly phosphorylase kinase kinase) through specific binding to its regulatory subunit. This results in the dissociation of the regulatory subunit and the concomitant activation of the catalytic subunit. The free, active catalytic subunit modulates the activity of its specific substrate enzymes by transferring phosphate from ATP to particular serine or threonine sites on these substrates. Thus, the glucagon-induced signal sets in motion a cascade of specific enzyme in/activations, eventually affecting those enzymes which do the actual work of glycogenolysis, etc. While the specific "hormonal" effects of glucagon are mediated through increasing the concentration of cAMP (the only known function of which is to activate cAMP-dependent protein kinase), at least one effect is not: the glucagon-dependent regulation of cell surface glucagon receptor levels. The glucagon receptor-adenylyl cyclase system has, for more than two decades, been one of the most fruitful models in the study of hormone action. In the late 1950's, studies on the similarity between glucagon and epinephrine action in the liver led Rail and Souther land (3) to the discovery of cAMP, and gave rise to the concept of intracellular "second messengers". When in 1971, Rodbell and colleagues (4-8) first characterized the hepatic glucagon receptor, their initial demonstration of the GTP requirement for glucagon stimulation of adenylyl cyclase gave birth to the now burgeoning field of signal transduction through GTP-binding regulatory proteins (N or G proteins). Analogies to this N protein-mediated hormone receptor action abound, in the actions of transducin in the visual cascade (9), in models of hormone stimulated activation of phospholipase C (10), as well as in models of insulin receptor action (11,12) (despite the fact that the insulin receptor molecule is itself a tyrosine-specific protein kinase). Of the glucagon receptor itself, quite little has been known until rather recently. Pharmacologically, work on the glucagon receptor is hampered by a lack of good agonists and antagonists, and the highly hydrophobic glucagon polypeptide itself is diffi-

132

cult to work with in several respects. In addition, most modifications of glucagon, as with the addition of a photoreactive crosslinking group, greatly reduce the binding affinity and potency. Recently, however, the use of gentler iodination conditions and HPLC purification has allowed for reproducible production of high affinity, high specific activity radioiodinated glucagon for use as a receptor probe. The availability of a defined probe and the utilization of a heterobifunctional crosslinking agent to covalently attach this probe to the glucagon receptor molecule have greatly expanded the ability to study this receptor. Glucagon-stimulated adenylyl cyclase (13) and glucagon receptor structure (14) have been recently reviewed.

Pharmacological Characteristics of the Glucagon Receptor Radiolabeling of glucagon In their initial studies of the glucagon receptor, Rodbell et al. (6) describe two methods for the radioiodination of glucagon; I2 reaction, and the chloramine-T method of Hunter and Greenwood (15). On the basis of their purification of the I2 iodlnated product and its use in stimulation of liver membrane adenylyl cyclase, they concluded that the probe was [ 1251]monoiodoglucagon and that this modification had no effect on glucagon potency. They then prepared higher specific activity species iodinated using chloramine-T, and assuming it to be monoiodinated and equipotent with non-iodinated glucagon, calculated molarity based on [125i] activity. In a later study of association and dissociation rates of their [ 125i]iodoglucagon, these researchers observed anamolously slow kinetic behavior of rat liver membrane receptors (16). Studies by others (17,18) with iodoglucagons later showed that iodoglucagons were more potent than non-iodinated glucagon in stimulating adenylyl cyclase, depending on the pH and degree of iodination. Therefore, the "monoiodoglucagon" of Rodbell et al. (6) was in fact a heterogeneous mixture of iodinated species and fragments from which no valid kinetic parameters

133

could be obtained.

It is of considerable importance to know that

the labeled glucagon used in any particular study has been carefully characterized, before accepting any conclusions based on its use. In

this

laboratory,

purified

by

glucagon

reverse-phase

is

HPLC

iodinated

to

yield

using

Iodogen

and

[125i-Tyrl0]monoiodo-

glucagon ([125I]mIG) with a specific activity of 2200 Ci/mmol (1 Ci = 37GBq)

(19).

Comparison of the HPLC elution profile of

[l25i]iodoglucagon dase

with

extreme

that

iodinated

iodinated

using chloramine-T or lactoperoxi-

using

Iodogen

has

demonstrated

susceptibility of glucagon to oxidation during

tion, and

the heterogeneity

conventional

methods

(17).

of iodoglucagons purified Also, purified

[125I]MIG

the

iodinaby

more

is quite

labile, so that even aliquots stored at -70"C show significant degradation after 30 days. to

be

iodinated

is

In this laboratory, even the glucagon

purified

by

reverse-phase

HPLC

prior

to

labeling, to minimize the production of iodinated fragments. Glucagon binding assays Various methods have been utilized to separate bound from free [ 125i]iodoglucagon, apparent

size

receptor. methods,

of

which membrane

usually

take

fragments

advantage containing

of

the

the

large

glucagon

Rodbell et al. (6) describe two centrifugation assay pelleting

labeled

membranes

through

2.5%

albumin

in

microfuge tubes or through 20% sucrose in ultracentrifuge tubes. The most commonly used assay utilizes membrane adhesion to 0.45 pm cellulose (20).

acetate

filters, as described by Goldfine et al.

Precipitation of bound label with polyethylene glycol has

also been used (21).

More recently, a wheat germ lectin-Sepha-

rose adsorption assay for the detergent solubilized receptor has been devised (22) which will be discussed in a later section. with

other

receptor

assays,

specific

binding

is

As

generally

assessed by competition with excess unlabeled hormone during the binding reaction. so

that

assays

The glucagon molecule is rather hydrophobic, for

[ 1 2 5j ] iodoglucagon

binding

have high

non-

134

specific backgrounds. To reduce this background, assays usually are performed in the presence of soluble carrier proteins, such as 1% bovine serum albumin. Binding studies using [125i]iodoglucagon are generally carried out on membranes from liver or fat cells, or on whole cells of either type. Purified liver plasma membranes for binding are most often prepared by the protocol of Neville (23), while crude NM membranes (nucleus-mitochondria fraction) prepared by lowspeed sedimentation are also frequently used. Hepatocytes are isolated using the collagenase perfusion technique of Berry and Friend (24). Binding of [ 125I]iodoglucagon to hepatic glucagon receptors is unaffected by unrelated peptide hormones, such as insulin or ACTH (6). Hepatic glucagon receptors are also able to distinguish glucagon from very similar, related hormones, such as VIP (25) and secretin (6). The hepatic receptor for vasoactive intestinal peptide (VIP) has recently been identified by crosslinking (26,27), and is structurally distinct from the glucagon receptor. Additionally, two peptides homologous to glucagon and produced as part of the glucagon preprohormone peptide, the glucagon-like peptides GLP-1 and GLP-2, have been shown to not interact with the hepatic glucagon receptor (28). Glucagon, unlike insulin, has no internal disulfide bridges and no stable three-dimensional structure. Due to its high hydrophobicity and its pseudo-palindromic sequence, however, glucagon in solution has a very strong tendency to self-associate into a trimeric form (27). To aid in the following discussion, sequence of mammalian glucagon is (30):

the

primary

amino

acid

1 2 3 4 5 6 7 8 9 10 11 12 13 14 H 2 N - His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Len-Met-Asn-Thr-OH Many studies characterizing the effects of modifications of the

135 glucagon

peptide

on

adenylyl

cyclase

have

Hruby that

(31).

receptor been

binding

and

undertaken,

on

and

stimulation

of

summarized

by

are

The fundamental finding of modification studies is

essentially

the

entire

glucagon

molecule

is

required

for

binding to receptor and activation of adenylyl cyclase.

Broadly,

the

receptor

carboxyl

terminus

of

glucagon

is

important

in

binding, while the amino terminus is important in transduction of the receptor occupation signal to N g . amino terminal His residue cyclase activation by 98%.

Removal of even the single

reduces binding by 90% and

adenylyl

Of the various reactive groups on the

glucagon peptide, modification of only particular residues is not detrimental to receptor binding or cyclase activation.

Iodina-

tion of either Tyr 1 0 or Tyr 13 increases the binding affinity for receptor.

Modifications

amino terminus at His

1

of the two reactive amino groups,

the

e

(N°) or the amino group of Lys12 (N ), as

well as of the aromatic ring of Trp 2 5, seem to have minor effects on glucagon action. cations glucagon

of

the

Knowledge of the effects of various modifi-

glucagon

antagonists

as

peptide well

as

has

allowed

the

rational

the

creation

of

construction

of

photoaffinity labels for the glucagon receptor. Factors influencing glucagon binding The most notable and perhaps most important factors the binding tides.

of

glucagon with

its receptor

influencing

are guanine

nucleo-

It was first observed in 1971 that addition of micromolar

concentrations

of

GTP

[l25x]iodoglucagon

by

glucagon receptor

(7).

stimulated decreasing

the

the

dissociation

apparent

of

affinity

bound of

the

That the glucagon receptor exists in two

interconvertible affinity states in the cell was first demonstrated on isolated rat hepatocytes by Sonne et al. (32).

It is now

known that the guanine nucleotide-sensitive high affinity

state

results from the association of the glucagon receptor with the N s protein. Divalent cations have also been shown to be important regulators of glucagon action.

Addition of M g + + reduces binding of glucagon

136 to the glucagon receptor (7), and also increases the stimulatory activity of glucagon for adenylyl cyclase (4). Treatment of liver membranes with trypsin reduces both glucagon binding

and

lipase

A

adenylyl

cyclase

treatment

activities

(6,8).

N-ethyl

(33), as does maleimide,

a

phosphosulhydryl

blocking agent, has no effect on glucagon binding but eliminates adenylyl

cyclase

activity

(34).

Other

chemical

reagents

affecting glucagon binding to the glucagon receptor are listed by Desbuquois (13).

Structure of the Glucagon Receptor Crosslinking Direct come

structural

information

from photoaffinity

about

labeling

glucagon

receptor

has

and crosslinking

the

studies.

In

(35) reported the use of iodinated N e -4-

1977, Bregman and Levy

azido-2-nitrophenyl-Lys 1 2 -glucagon as a photoaffinity the

glucagon

showed

a

Johnson

receptor.

radioactive et

al.

(36)

SDS-PAGE species used

of

the

running

the

label

photolabeled

at

23-25

Kd.

heterobifunctional

for

products In

1981,

crosslinking

agent N-hydroxysuccinimidyl-p-azidobenzoate

(HSAB) to covalently

attach

receptor.

[ 125i]iodoglucagon

revealed

53

Kd

Demoliou-Mason

and

(38)

of

a

the

band

to

which

Epand

glucagon

was

describe

photoaffinity

sulfenyl]-Trp25-gi U cagon

the

specific

and

the preparation

label

SDS-PAGE

GTP-sensitive. (37) and

use

2-[(2-nitro-4-azidophenyl)

(glucagon-NAPS).

Labeling

of

the

receptor with [ 125i]iodoglucagon-NAPS yielded bands in the region 52-70 Kd after SDS-PAGE. phenylamidinoglucagon lactoperoxidase, specific gels. Herberg

as

and GTP

Wright et al. (39) prepared Ne-4-azido-

(APA-glucagon), a photoaffinity

sensitive

labeling

which was label.

iodinated

They

reported

(40)

observed

a

63

Kd

the

of a 55 Kd protein on SDS

Using HSAB and [ 125i-Tyr l O]monoiodoglucagon,

Iwanij and Hur

using

species

after

Iyengar and

SDS-PAGE,

(41) reported the direct UV-induced

while

crosslinking

137 [ 1 2 5x]iodoglucagon

of

into a 62 Kd band on SDS gels.

structural studies have been carried out on the HSAB directly

crosslinked

(41) glucagon-receptor

Detailed

(40,22) and

complexes,

and

will

be described below. In this chapter, the glucagon receptor will be referred to as a 63 Kd species, since that is the size most often seen in our SDSPAGE system.

However, the apparent mobility of the

crosslinked

receptor changes as a function of the acrylamide concentration of the gel, so that the 63 Kd band in a 10% gel runs as 55 Kd on an 8%

gel

(M.

Because

of

Benson this

crosslinked

and

R.

Iyengar,

variability,

the

unpublished

observed

observations).

molecular

weight

of

glucagon receptor and its fragments should be viewed

as approximate.

This may also, in part, explain the differences

between the size of the crosslinked receptor measured by different laboratories. Using HSAB crosslinking to covalently label the glucagon receptor with

[125i-Tyr 1 O]monoiodoglucagon

has,

extensively

(40,22). city

covalent

attachment,

membranes

without

the

hepatic

this

laboratory

glucagon

receptor

A typical crosslinking experiment, showing the specifi-

of

plasma

characterized

([ 1 25i]MIG),

various

were

is shown

incubated

unlabeled

with

peptide

in Figure 1

nM

hormones,

1.

Rat

[125I]MIG unbound

liver

with label

or was

washed away, and the membranes treated with HSAB in the presence of

ultraviolet

light.

Crosslinked

membranes were

subjected

to

electrophoresis in the presence of SDS, and the dried gel exposed to

X-ray

film.

single band and most Since 2%,

at

In

the

63,000 M R

absence

of

(~98%) of the radioactivity

the

the

efficiency

remainder

of HSAB

of

competing

is covalently

with

only

label

of

a

[125I]MIG,

is found at the dye

crosslinking

receptor-bound

peptides,

labeled

[125I]MIG

front. is

is dissociated

only under

the denaturing conditions of electrophoresis and migrates to the bottom of the gel. initial

binding

[125I]MIG

Addition of excess unlabeled glucagon to the

reaction

prevents

the

covalent

attachment

of

into the 63 Kd band, indicating that the labeled pro-

tein specifically and saturably

interacts with glucagon.

Excess

138

CO O *

CO Ts

O

CO

*

7s

Glucagon Insulin 8-AVP ACTH Figure 1; Specificity of crosslinking of [12SI]MIG to the glucagon receptor. HSAB crosslinking of [125I]MIG bound to rat liver membranes yields a major labeled band at 63 Kd on SDS-PAGE (-). Addition of unlabeled glucagon to the binding reaction abolishes labeling of this band, while insulin, [8-arginine] vasopressin (8-AVP), and adrenocorticotropic hormone (ACTH) are without effect. Shown is an autoradiogram after 48 hours of exposure to the dried 10% gel. Note the free [125I]MIG in the dye front, which was membrane bound but not crosslinked. insulin,

vasopressin

and ACTH, unrelated peptide hormones which

do not effect glucagon stimulation of adenylyl cyclase or binding to the glucagon receptor, are without effect on the labeling of the

63

Kd

band.

If the

effects

of

varying

concentrations

of

unlabeled glucagon on labeling of the 63 Kd band are examined, a competition curve between [125I]MIG and glucagon can be constructed.

Such

experiments

show that the I C 5 0

for

[125I]MIG

linking to the 63 Kd band in the presence of unlabeled is

approximately

10

nM,

with the receptor (22).

indicating

high

affinity

cross-

glucagon

interactions

Therefore, labeling of the 63 Kd protein

by [125I]MIG and HSAB is specific for glucagon within the proper concentration range.

139

63K

Additions during Binding

GTP

GL

Figure 2: GTP sensitivity of crosslinking of [125I]MIG to the glucagon receptor. The addition of 100 YM GTP to the binding reaction causes a reduction in the [125I]MIG covalently attached to the 63 Kd band. Note also the reduction in uncrosslinked label in the dye front, reflecting the lowering of the affinity of the glucagon receptor in the presence of GTP. Besides specificity, one of the hallmarks of the glucagon receptor is the reduction of its affinity for glucagon by GTP. In Figure 2 is shown the effect of GTP on the crosslinking of [125I]MIG by HSAB into the 63 Kd band. Addition of GTP to the initial binding reaction transforms the glucagon receptor from its high to low affinity state, so that less [125I]MIG is bound to the membranes. Upon crosslinking with HSAB, less label is available for covalent attachment, so the 63 Kd band is markedly diminished. Additionally, dose responses of both [125I]MIG binding to liver membranes and crosslinking into the 63 Kd band to varying concentrations of GTP exhibit similar ICJQ'S of less than 1 yM (22), well within the physiological range. Taken together, the 63 Kd band seen in SDS-PAGE of crosslinked liver membranes exhibits both the GTP sensitivity and glucagon specificity expected of the glucagon receptor, so the 63 Kd band repre-

140 sents the hormone-binding unit of the glucagon receptor. Since

the

preceding

glucagon-receptor disulfide

electrophoretic

identification

of

the

complex was carried out in the presence of the

reducing

agent

g-mercaptoethanol,

it

can

only

be

claimed that the 63 Kd complex is the hormone-binding subunit of the

glucagon

covalently

receptor.

Other

(by disulfide

subunits,

bridges)

either

attached

ionically

or

to this peptide

and

which do not bind hormone, will be separated during the electrophoresis.

To determine if the 63 Kd receptor complex is associa-

ted with another subunit through disulfide linkages, receptor

samples were treated with varying

mercaptoethanol prior to electrophoresis reducing

agents,

the

crosslinked

concentrations complex migrated

an apparent molecular weight of 60,000, while increasing trations of previously

(3-mercaptoethanol observed.

This

shifted

slight

of

this band

apparent

g-

In the absence of

(22).

receptor

crosslinked

with

concen-

up to 63 Kd

shift

in

as

migration

produced by reducing agents is indicative of intrachain disulfide linkages

being

broken,

and

implies

that

no

other

peptides

are

associated with the 63 Kd glucagon receptor complex by disulfide bonds.

If liver membranes are incubated at 32°C prior to binding

of [125I]MIG,

crosslinking

reveals a minor

labeled band with an

apparent M r of 115,000 which is specific for glucagon and

sensi-

tive to GTP (22). The glucagon-binding unit of the receptor has now been identified in a number al.

(42)

of

species

reported

a

using HSAB

63

receptor from human liver. receptors

from rat,

Kd

size

crosslinking. for

the

Livingston

crosslinked

lack of GTP

glucagon

In Figure 3 are shown the crosslinked

rabbit, chicken, and mouse

liver

All have very similar apparent sizes of from 60-64 Kd. apparent

et

sensitivity

be discussed in a later section.

membranes. Note the

in the rabbit liver; this will

141

Figure 3: Crosslinked glucagon receptors from several species. Liver membranes from the indicated species were incubated in 1 nM [125i]MIG without additions (lane 1), with 100 UM GTP (lane 2), with 10 yg/ml glucagon (lane 3), or with 10 yg/ml insulin (lane 4). Washed membranes were crosslinked with HSAB, and electrophoresed. Protease and endoglycosidase mapping Having established that the 63 Kd band seen after SDS-PAGE of crosslinked membranes has the characteristics expected of the glucagon receptor, this crosslinked receptor complex can be further characterized. Treatment of the crosslinked receptor complex from rat liver membranes with various proteases before electrophoresis causes the appearance of a labeled 33 Kd fragment from both membrane-bound and Lubrol-PX solubilized proteins (40). In Figure 4 is shown a limited elastase treatment experiment. In lane 2, [ 1 2 5 I]MIG was bound to membranes, the membranes treated with elastase, and the specifically-bound [125I]MIG crosslinked with HSAB. Autoradiography of the SDS-PAGE separated membrane proteins reveals the appearance of a 33 Kd labeled fragment in addition to the major 63 Kd receptor band. This 33 Kd band is also observable in longer exposures of untreated crosslinked membranes, unless the membranes have been previously treated with the serine protease inactivator, diisopropylfluorophosphate (DIFP) (40). In lane 3, membranes were treated with elastase

142

1

2

3

3 3 — -

2

4

—

Figure 4: Limited elastase treatment of occupied and vacant rat liver glucagon receptors. In lane 1 is a crosslinking with no additions. Following [125I]MIG binding, the membranes in lane 2 were treated in a limited manner with elastase, which was then inactivated by addition of DIFP. Crosslinking was then carried out as usual. In lane 3, membranes were treated with elastase in a limited manner, and elastase inactivated with DIFP prior to [125I]MIG binding. After binding, membranes were crosslinked as usual and run on a 10% SDS gel. prior

to

before.

[l 2 5 ]MIG

binding,

and

bound

label was

Elastase treatment of thé unoccupied

crosslinked

glucagon

as

receptor

produces a fragment which retains the capability to bind glucagon specifically and in a GTP-sensitive manner

(38), and upon cross-

linking, migrates as a 24 Kd band in SDS-PAGE. ment similarly

retains

Kd fragment (40). these

membrane

its GTP sensitivity,

In view of the washing procedures used during

treatments,

only

remain to be run on the SDS gel. 24

Kd

proteolytic

fragments

well as hormone binding these

fragments

The 33 Kd frag-

and contains the 24

indicate

with the N s protein.

membrane-bound

contain

sites. that

proteins

will

Therefore, both the 33 Kd and membrane-bound

regions

as

The apparent GTP sensitivity of they

also

functionally

interact

All known functional sites of the glucagon

receptor, then, are found on only 1/3 of the labeled complex, and are associated with a membrane-bound region of the molecule.

143

r 6 1K

6 3 K — • • ' P 5 6 K

5 1K 45K

—

0

4

24 Time (hr)

Figure 5: Endoglycosidase F treatment of crosslinked rat liver glucagon receptors. Rat liver membranes crosslinked to [125I]MIG were solubilized in 1% Lubrol-PX. The soluble extract was untreated (0 hour) or was treated for 4 or 24 hours with Endo F and run on a 10% SDS gel. It

has

been

observed

that

the

crosslinked

glucagon

receptor

complex can be specifically adsorbed to wheat germ lectin-Sepharose (WGL-Sepharose) (22) and to concanavalin A-Sepharose Sepharose)

(41),

glycoprotein

indicating

containing

that

the

and mannose and/or glucose residues. a

mixture

of

glycosidase

activities N-linked

Treatment

from

receptor

and/or F

and

Flavobacterium

oligosaccharides

of crosslinked

sialic

is

a

acid,

Endoglycosidase F (Endo F),

endo-g-N-acetylglucoseaminidase

F

deglycosylates (43,44).

glucagon

N-acetylglucosamine

(ConA-

from

glucagon

peptide:N-

meningosepticum, glycoproteins

receptor

complexes

with Endo F for various times gives four discrete bands on SDSPAGE,

as

clearly

shown yields

treatment

have

crosslinked apparent

in Figure four

of

the

Four hours

different

shifted

receptors

size

5.

to for

bands,

the

lower

longer

labeled

band

of

which Mr

periods beyond

Endo

F

after

forms. fail 45

24 hours

of

Treatment

of

to

Kd

treatment

reduce

(40).

the

These

discrete bands are due to the sequential removal of the N-linked glycans

from

the

receptor,

and

they

indicate

that

these

four

144

63

-56

-51 •45 33

Endo F -

+

Figure 6; Endoglycosidase F treatment of elastase treated 33 Kd receptor fragment. Crcsslinked membranes were subjected to limited digestion with elastase, under conditions favoring the formation of 33 Kd receptor shown in Figure 4, and are shown in the (-) lane. A portion of these elastase-treated membranes were then incubated with Endo F, as in the legend to Figure 5. SDSPAGE reveals that while the unproteolyzed 63 Kd fragment remains susceptible to Endo F, the elastase-generated 33 Kd fragment is unaffected by Endo F. glycans

alter

the

Mr

of

the

receptor

by

2,

5,

Similar patterns of Endo F digestion of directly receptor

complexes

has

been

reported

by

5, and

6

Kd.

UV-crosslinked

Iwanij and

Hur

(41).

Endo F treatment of the elastase 33 Kd fragment, shown in Figure 6, implies that the 33 Kd fragment does not contain the N-linked glycans.

Similar experiments with the 24 Kd elastase

from the vacant receptor

fragment

indicate that this fragment also does

not contain any N-linked glycans

(40), which is expected if the

24 Kd fragment is contained within the 33 Kd fragment. With

this

information,

constructed, structure

is

as is as

a map

shown

follows:

of

the

in Figure the

glucagon

7.

receptor

The rationale

completely

linked receptor runs as 45 Kd on SDS-PAGE.

can

deglycosylated This 45 Kd

be

for this crossfragment

represents the complete peptide backbone of the receptor. Four N—

145 45,000 33,000 E.T.V8.S

Cut from the hormone occupied state Cut from the vacant receptor

24,000

Hydrophobic membrane binding region 4 GTP sensitive hormone binding region Figure 7: Proposed structure of the 63 Kd crosslinked hormoneBlack bar binding unit of the rat liver glucagon receptor. represents the peptide chain, open circles the N-linked glycans. Arrows demark points of protease cleavage by trypsin (T), elastase (E), subtilisin A (S), and protease V8 (V8). linked glycans are attached

to this backbone, and increase its

apparent molecular weight by

2, 5, 5, and 6 Kd,

respectively.

The 33 Kd elastase fragment of the occupied receptor is insensitive to deglycosylation and is produced even in limited digestion (implying

only

a single cleavage

is necessary

for its produc-

tion) , so this fragment extends from the non-glycosylated end of the peptide backbone.

The 24 Kd fragment of the vacant receptor

is contained within the 33 Kd fragment, and since it too is produced upon limited digestion and without a 33 Kd intermediate, it also extends from the non-glycosylated end of the peptide.

The

24 Kd fragment is bound to the membrane, interacts with N s functionally,

and

contains

the

glucagon

binding

site.

It

is

important to remember that the measured molecular weight of the crosslinked covalently

receptor

complex

crosslinked

on SDS-PAGE

[125I]MIG

and

the

includes

that of

crosslinkers,

the

so all

sizes shown are, in fact, at least 3 Kd higher than would be the actual

receptor

molecule

itself.

Another

important

point

to

understand is the nature of the pattern of protease susceptibilities.

As mentioned above, several proteases of different site

specificities approximately likely

that

cleave 33 Kd the

33

the

hormone-occupied

crosslinked Kd

receptor

to

fragments on SDS gels.

fragments

are

not

identical,

but

give It is that

occupancy by hormone exposes a small region of the receptor to the action of proteases.

This is supported by the observation

146

that

elastase

produces

no

33

Kd

fragment

from

the

unoccupied

receptor, suggesting ligand-induced conformational change in the receptor molecule.

Such an allosteric shift could play a criti-

cal role in the activation of N g . Physicochemical_analysis Crosslinking of the glucagon receptor using HSAB has been used not

only

as

a

probe

of

the

fine

structure

of

the

receptor, but also to ascertain its physicochemical Once crosslinked,

the receptor complex

glucagon

properties.

can be removed

from the

membrane with detergents without the need to be concerned about maintaining

the

activities

of

the

receptor.

The

hydrodynamic

properties of the crosslinked receptor complex have been measured in this manner

(40).

Liver membranes with crosslinked receptors

have been solubilized in 1% Lubrol-PX and chromatographed on AcA 34 gel

in the presence of 0.5% Lubrol.

Eluted

fractions were

analyzed for internal marker enzyme activities and aliquots were run on SDS-PAGE.

Autoradiograms were scanned for the appearance

of the 63 Kd band, and the peak corresponded to a Stokes' radius of 6.3 nM.

Solubilized crosslinked receptors have been sedimen-

ted in 5-15% sucrose gradients in the presence of 0.5% Lubrol-PX in both H2O and D2O»

an

I 15 20 10

1 30

1 . . . . 1 . 10 15

I

A

i . . . 5

2 0

» I \

I l/*4.3S

20 •

DC

Sucrose Density Gradient Profile | L i v e r N.

0

1

\

GTPTS + Glucagon + 0.4mM Mg2+

AH

M%

V* ' o\

^ :V. 2.5S|

I .... 1• .• • I .... I

5

10

20

15

4 0

Jo

y*

/

GTPTS + 50mM Mg2+

rw

\ V

Â

fcl

2.4SI

Top

25

o\s 0»?

• "ò 8 8 8 o 20

Top

Fraction Number

10

15

F r a c t i o n Number

Figure 14: A: M g T T and glucagon dependent shift in N sedimentation upon activation. Membranes treated as indicated were solubilized in 1% Lubrol-PX, and the extracts separated on 5-20% sucrose gradients. Fractions were assayed for S49 cyc~ reconstiB: M g + + and tuting activity with (o) or without (o) added GTPyS glucagon dependent shift in Nj sedimentation. Fractions of gradient in A were labeled with "T 32 P]NAD + and PT, and labeled a ^ identified in autoradiographs of SDS-PAGE. Plotted are densitometric scans of 40 Kd a^ radioactivity from autoradiograms. both of which sedimented at 2S. units

allowed

their

Cooling of the dissociated sub-

reassociation

to the

"pre-active"

3S form.

It has recently been directly shown that addition of glucagon and guanine nucleotide

to membranes alters N s

form to the active, dissociated

from the inactive

4S

2S form when extracted membrane

157

proteins are analyzed on sucrose gradients (56).

This study also

shows a glucagon- or high Mg ++ -dependent dissociation of N^ from its inactive 4S form to the active 2S form, implying that glucagon

receptors

may

also

unsuspected manner.

interact

with

Nj_

in

To sum, binding of guanine nucleotides to N g high

Mg++

promotes

or

in

interaction with

a conformational

high temperatures

some

previously

These results are shown in Figure 14.

change

an

in the presence of

agonist-occupied

receptor

in the heterotrimer, which

at

(such as those found in a living cell) allows

the dissociation

of

from the active

ag subunit.

In trans-

ferring this biochemical model to the living state in a cell, it is

important

to

remember

that

the

GTP

concentrations

seen by this system are saturating, while it is the M g is limiting.

tration which

sufficient M g + + ,

Without

normally

++

concen-

even with

GTP bound, the N g will not undergo the allosteric change of activation.

Upon addition of agonist, the occupied receptor complex

alters the M g + + requirement for the activation of N s , and allows its activation at the ambient intracellular M g + +

concentration.

For this reason, the glucagon receptor (and other R s 's) has been called a "Mg + + switch" (34), controlling N s activity through its hormone occupancy state. Functional states of the glucagon receptor If the glucagon receptor acts as a "Mg + + switch", increasing the affinity

of N g

essential

to

"switching". observation

for M g + +

understand The that

when occupied by glucagon, the

mechanism

first hint guanine

of

this

at this mechanism

nucleotides

[ 125j]iodoglucagon to liver membranes

reduce (7).

it is then

agonist-induced is the the

initial

binding

of

This guanine nucleo-

tide effect is a reflection of the observation (mentioned previously) that the glucagon receptor exists in two interconvertible forms: one of high affinity.

affinity

for glucagon and the other of

low affinity form of the receptor. of

the

low

Guanine nucleotides promote the transformation to the

high

and

low

affinity

The mechanistic

states

of

receptors

significance coupled

to

158 adenylyl cyclase has been elucidated in studies of the binding of antagonists (57,58).

and

The

partial

agonists

to

the

g-adrenergic

g-adrenergic receptor exhibits guanine

receptor

nucleotide

sensitivity to the binding of agonists, just as does the glucagon receptor. insensitive nucleotide partial

However, the binding of catecholamine antagonists to guanine nucleotides. sensitivity

agonists,

between the intrinsic extent

of

reveals

a

nucleotide

their

et al.

of several

(57) obtained

a

catecholamine

linear

relation

activity of the partial agonists and

guanine

functional

In a study of the guanine

of the binding

Kent

nucleotide

sensitivity.

relationship between

regulation

of

receptor

is

This

agonism and

affinity,

and

the

study

guanine-

implies

that

antagonism is due to the inability of the antagonist to bind the high affinity state of the receptor. of comparing N g

This data, and the results

activation with the known affinity state ratios

(58), suggest that only the high affinity form of the receptor is active in stimulating adenylyl cyclase activity. Since

it is N g

guanine

which

nucleotides

responsible

binds

on

Rs

guanine

nucleotides,

affinity

for the affinity

states

the effect

implies

of the receptor.

that

Ng

of is

In studies with

co-reconstituted preparations of g-adrenergic receptor and N s in varying

degrees

of purity

(59-61),

it has been

shown that

the

receptor exhibits the high affinity state only in the presence of Ns.

Addition

of guanine nucleotides

shift these

reconstituted

receptors to their low affinity form, just as observed in native membranes.

The presence of N s

is necessary and sufficient

receptors to exist in the high affinity state. Ns,

In the absence of

or when GTP is present, receptors are unable to convert

the high affinity form.

for to

The high affinity state of the receptor,

the active state, only occurs if the receptor is coupled to N s . This explains the earlier observation CHAPS solubilized

(see Figure

10) that the

glucagon receptor exhibited only low affinity

binding in the absence of GTP (22), since the soluble receptor is apparently unable to interact with N s -

159 Knowing

that N s

Mg++

that

controls the affinity of R g coupled to it, and

is

an

activity, M g

++

binding

Rg.

to

important

regulator

of

Ns

conformation

and

may be expected to exert some effects on hormone It

has

been

reported

for

the

g-adrenergic

receptor that inclusion of M g + + in the binding reaction increases the

apparent

affinity

of

the

receptor

for

agonists

but

not

antagonists, without altering the total number of binding sites, and that guanine nucleotides abolish this Mg ++ -dependent increase This agrees with the role of M g + + in the acti-

in affinity (62).

vation of N s by R s .

[125I]MIG binding to the glucagon receptor

on rat liver membranes, however, has been shown to be insensitive to M g + + , and more surprisingly, GDP has been shown more effective than

GTP

affinity

in

promoting

for

glucagon

reconciles

this

(64).

the

In

the

receptor

(63).

course

of

Recent in M g + +

discrepancy

shift

studying

from

work

in

effects

the

high

this

to

laboratory

on binding

guanine

low

to

nucleotide-

Rg and

Mg ++ -regulation of [125I]MIG binding to the glucagon receptors of liver membranes from various species, three distinct patterns of regulation were

observed:

1) Mg + + -dependent

guanine

nucleotide

++

sensitivity in chick liver membranes; 2) Mg -independent guanine nucleotide

sensitivity

independent

guanine

membranes. receptors species

activity

revealed

an

that

relative

of

of

liver

membranes;

insensitivity

the

in the

interesting

relative

in

Mg++-

rabbit

liver

levels

liver membranes pattern.

3)

and

The

of

glucagon

of the three

ratio

of N g

to

in rat is nearly twice that found in chicken,

rabbit

ratios

rat

nucleotide

Measurement and N g

glucagon receptor while

in

imply

is

five

that

one

times

that

in

explanation

chicken. for

the

These observed

differences in M g + + and guanine nucleotide regulation of glucagon binding

in these membranes may be that where there

excess of N S ,

is a great

as in rabbit liver, the glucagon receptors always

find enough N s

in the proper

state to couple with to form the

high affinity state, even in the presence of GTP. tial regulation of glucagon receptors also seen within species. liver membranes

This differen-

from different species is

Lafuse and Edidin (65) have shown that

from mice of differing major

histocompatability

haplotypes have differing levels of high and low affinity gluca-

160 gon receptors. membranes

In this laboratory, experiments with mouse liver

(E. Padrell and R. Iyengar, unpublished

observations)

indicate that glucagon binding is GTP sensitive, but that addition of M g + + increases equally the extent of binding and of GTP sensitivity:

glucagon

receptor

regulation

in

mouse

liver

(of

mixed haplotype) is between that seen in rat and chicken liver. This leads to the suspicion that ratios of N g to glucagon receptor in mouse liver vary with histocompatability haplotype, and to the

intriguing

speculation

that

these

ratios

are perhaps

con-

still seem

con-

trolled by the major histocompatibility complex. The effects of N g

and

guanine

fusing: how can receptor

nucleotides may

"find" N s to promote its high affinity

state in rabbit liver membranes even in the presence of saturating guanine nucleotides? guanine nucleotides by glucagon receptor vation) when

It does seem very contradictory

increase the activation of adenylyl

(and are absolutely required for this acti-

guanine

nucleotides

the receptor for glucagon. in

which

the

that

cyclase

system

is

also decrease the affinity

of

This discrepancy is due to the manner studied,

since

the

affinity

of

the

receptor can be measured only at equilibrium or by dissociation. The

glucagon

receptor-N s

system

is

a dynamic

one,

continually

moving through a cycle of activation and deactivation.

If the

receptor is coupled to N s , it has high affinity; if it is not, it has

affinity,

regardless

binding,

low

and

the

receptor

is uncoupled

state.

after

of

allosteric

from N s

and

GTP.

It

is

activation returns

after

of N s ,

to the

hormone that

low

the

affinity

Guanine nucleotides only reduce the receptor affinity by

allowing N g

to proceed in its activation.

It is important here

to keep in mind that there exists large populations of R s , N s and C,

all

concurrently

undergoing

this

cycle

of

activation

and

deactivation. When a receptor couples to N s , even in the presence of GTP,

it exists

in the high affinity

state.

However,

in an

experiment, only the macro properties of the system are visible, and since most receptors are low affinity most of the time when active

(with hormone

affinity

and

sites are seen.

guanine nucleotide present), If in the case of rabbit

only

low

liver mem-

161 branes where larger

there

is much more N g

than

Rs

fraction of the receptor population

(comparatively),

is coupled to N g

a at

any instant, binding experiments will show this as a lack of GTP sensitivity. membranes

It should

be noted

that

even

these rabbit

liver

(which show no GTP effect on glucagon binding) require

added GTP for adenylyl cyclase activity. Many models of agonist-occupied receptor stimulation of adenylyl cyclase have been proposed in the course of study of this system. A portion of one of the more recent of these models (63) will be outlined

here.

Ns

can

be

postulated

domains, one of which interacts with R g which

interacts with C

to

have

two

distinct

(r domain) and the other

In the r + conformation,

(c domain).

Ns

interacts with and stabilizes the receptor in its high affinity form, while the r~ conformer of N s either does not interact with the

receptor

or

interacts

Similarly, when c + , N g it does not.

The

would be r + c~. increased form.

with

only

the

low

affinity

form.

stimulates adenylyl cyclase, but when c~

"resting

state"

of N g

in the

cell

membrane

In the presence of GTP, addition of agonist

intracellular

Mg++)

would

transform

Ng

to

its

(or r+c+

Whether receptor remains coupled to N g during stimulation

of C or not is inconsequential to this discussion, so the r~c + and

r~c~

states will

not be

mentioned

further.

The

glucagon

receptor can exist in two affinity states, high (R^) or low (R-^), and only the high affinity form is associated with N g form).

R^,

the

receptor coupled

to N g ,

further

(in its r +

exists

in two

functionally distinct states, agonist unoccupied (-R^) or agonist occupied while

(+R h ).

The unoccupied receptor-N s complex is -Rj1.r+c~,

the agonist-occupied

the presence of GTP.

receptor-N g

complex

is +R- h .r + c + ,

in

Since in the presence of high M g + + concen-

trations and GTP, N g can convert from r + c~ to r + c + without dissociation (55), the change from r + c - to r + c + state must involve an allosteric

conformational

been proposed previously +

its r c~ state, N s affinity

-R^

change involving M g + + binding, as had (50).

The implication is that while in

stabilizes the glucagon receptor in its high

state,

agonist

change in the receptor to the

binding

induces

a

conformational

state, which in turn stabilizes

162 Ns

in its r + c +

easy

to

see

"activated" conformation.

how

the

occupied

In this model, it is

receptor

could

lower

requirement to stabilize N s in its active conformation equivalent

to increasing

the affinity of N s

Mg++

the

(which is

for Mg + + ).

At the

present time, there is little direct evidence to support such a mechanism.

However, the observation that the glucagon

receptor

exhibits differential protease susceptibilities depending on its occupation

state

suggest

(40, see also Figure receptor

agonist-induced

4).

reconstituted

Future

with

conformational

studies

(and most

of purified probably

changes glucagon

using

site-

directed mutants of each) will be required to define the conformational changes associated with initiation of the signal transduction event).

Regulation of the Glucagon Receptor In

addition

adenylyl

to

its

cyclase

regulation

system,

regulation as a hormone receptors

do

not

the

as

a

glucagon

functional receptor

part

receptor on the cell surface.

comprise

receives signals from afar.

a

static

antenna

of

is under which

the

other

Hormone passively

Just as the secretory cell tightly

regulates its release of hormonal messages, hormonally responsive cells carefully adjust their mileau of surface receptors to conform

to

changing

needs,

in

light

of

other

signals.

Hormone

receptors are in continual motion in the membrane, and in and out of the cell. Internalization Hormone binding to specific receptors produces an effect in the cell. is

But since hormone responsiveness

necessary

received.

to

eliminate

In some

systems,

the as

is a temporal event, it

hormone is the

once

the

case with

message

is

neuromuscular

junction acetylcholinesterase, specific extracellular enzymes are present to destroy the spent hormone.

The very short (less than

5 minute) half-lives of glucagon and insulin in the bloodstream

163 do hint at a very efficient peptide hormone clearing system (see for

example:

mechanism

66).

that

internalized,

hormones

lysosomes, and (67-69). the

However,

it

appears

agonist-occupied are

dissociated

molecules and

general

are

digested

rapidly

in

free receptors are recycled to the cell

acidic surface

And while it has been reported that unbound glucagon in

presence

of

liver

plasma

membranes

receptor bound glucagon is not (6). gon for its receptor ligand

a more

to be

receptor

and

will

is

quickly

degraded,

The high affinity of gluca-

implies that the receptor will retain its

remain

activated

if

no

specific

inactivation

event occurs. Several studies have been undertaken to visualize internalization of

glucagon

receptors.

Since

no

antibody

to

the

glucagon

receptor is available, these studies have relied on either electron

microscopic

localization

of

either

radiolabeled

glucagon

(70,71) or of colloidal gold-glucagon conjugates

(72,73).

of

depending,

these methods

example,

on

may

how

the

give

artifactual

modified

results,

glucagons

interact

receptor, and on background levels of labeling. tion

aside,

these

studies have

provided

Both for

with

the

This qualifica-

a direct

view

of

the

glucagon receptor in action, in the intact mouse liver (71), and isolated (70),

mouse

and

hepatocytes

in human

(73),

peripheral

in

isolated

leukocytes

rat

(72).

hepatocytes

All

papers are supportive of the hypothesis that glucagon are

internalized

upon

occupancy,

although

of

these

receptors

Asada-Kubota

(71) failed to see preferential labeling over lysosomes.

et

al.

Another

related EM study showed that the glucagon receptors of cultured mouse hepatocytes regained their regionalization on the trabacular face of the cells as the cells reformed tight junctions, but that this regionalization was lost upon prolonged culture

(74).

More definitive studies of the movements of the glucagon receptor await the availability of anti-receptor antibodies. More biochemical gon-receptor researchers

evidence for the internalization of the gluca-

complex

comes

reported

that

from in

Canivet isolated

et rat

al.

(75).

hepatocytes,

These the

164 addition

of

agents

chloroquine) ("internal")

known

prevented

to block

lysosomal

degradation

[ 1 2 5i]iodoglucagon.

of

This

earlier study by Rouer et al. (76).

(NH4CL

action

acid-wash

and

resistant

is in agreement with an

More recently, Lipson et al.

(77) have directly measured glucagon binding activity associated with

internal

vesicular

membrane

fractions.

These

internal

glucagon receptors were not associated with adenylyl cyclase or plasma membrane markers; however, determine if these receptors

these workers were unable

represented

internalized

to

receptors

or newly synthesized molecules. Physical removal of the receptor from the cellular surface is not the only possible mechanism for the cessation of hormonal stimulation by

the

transduces

its occupation

occupied

receptor.

Since the glucagon

receptor

signal through the N s protein

to the

catalyst of adenylyl cyclase, it is possible that modification of one or more

of the components

in this pathway

could

force

the

system into an "uncoupled" state, unresponsive to continued hormone occupancy of the receptor.

In particular, a coupling defect

due to modification of the receptor would leave the stimulatory pathway through N g

open to unmodified,

unoccupied

well as to other receptors of the R g type. fication has been observed receptor phosphorylation (78-80), surface occupied

and

in

fact,

receptors

(81).

for the g-adrenergic receptor,

appears to reduce receptor-N s appears This

g-adrenergic receptor

receptor kinase

receptors, as

Such a receptor modi-

to

precede

specific

where

coupling

internalization

phosphorylation

is performed by the

of

of the

g-adrenergic

(82), in a manner highly analogous to the action

of rhodopsin kinase (83).

Agonist-induced increased phosphoryla-

tion has recently been reported for the muscarinic acetylcholine receptor

(an R^-type receptor), as well (84).

not been demonstrated

Although this has

for the glucagon receptor, it seems likely

that it too follows a similar mechanism. Desensitization It is a general observation that prolonged exposure of a system

165

to stimulation

leads to an attenuation

system to further stimulation.

of the

been termed tachyphylaxis, or desensitization. tizations can be subdivided

response of the

Such an adaptation phenomenon has Hormonal desensl-

into two classes: reduction

in the

responsiveness to only the inducing hormone (homologous desensitization), or a generalized loss of stimulability by other effectors, as well as the inducing hormone (heterologous desensitization ) . Uncoupling recycling

of occupied to the

responsiveness

receptors, and their internalization and

cell

of

surface, need

adenylyl

not involve a loss

cyclase

further

to

there exists a large enough pool of internal,

in the

stimulation stored

if

receptors

which move to the cell surface as occupied receptors are removed for cycling, or if cell surface low affinity receptors convert to the

high

affinity

removed.

state

as

occupied,

modified

and in desensitization need not be the same. larities

receptors

are

Therefore, the mechanisms involved in receptor cycling between

the

two processes would

they can be and often are identical.

However, some simi-

tend

to suggest

that

Almost by definition, homo-

logous desensitization must involve either a modification of the receptor which produces

a defect

i n its coupling

removes the receptor from the cell surface. known

as

receptor

"down-regulation" .

to N

and/or

This latter case is

The

difference

between

receptor recycling and receptor down-regulation is in the return of the internalized receptor to the cell surface. short-term receptors number

receptor are

of

available

desensitizations although

removal,

retained

within

cell

appear

modification

the

surface

to be due

of

been reported as well.

while

receptors

in

Recycling is a

down-regulation,

the

thereby

the

cell,

reducing

receptors.

Most

to

down-regulation,

receptor

without

homologous

internalization

has

For a more mathematical model of the role

of receptor modification in desensitization, see the recent work of Knox et al. (85).

While receptor uncoupling from N g may play

an important role in homologous desensitization, uncoupling of N s from

the

receptor,

of

Ns

from

C,

or

of

mechanisms for heterologous desensitization.

C

from

Ns

could

be

166 In the

glucagon-stimulated

desensitization cultured

has

adenylyl

been

rat hepatocytes

cyclase

reported

in

system,

isolated

(87), while homologous

together with heterologous

homologous

(86)

and

in

down-regulation

desensitization has been reported

in

cultured rat hepatocytes (88) and in the MDCK canine kidney cell line

(89).

their

Houslay

homologous

and

collaborators

desensitization

(86) have

is

due

reported

to

a

that

receptor-N s

uncoupling without internalization of receptors, and is complete within

5 minutes.

This uncoupling

is glucagon

but independent of cAMP action (86).

dose-dependent,

In addition, these workers

have found that pertussis toxin treatment of hepatocytes blocks the desensitization by glucagon (90), while the protein kinase inactivating (TPA,

phorbol

also

ester

called

PMA)

12-0-tetradecanoyl reduces

phorbol-13-acetate

glucagon

stimulated

adenylyl

cyclase action (91) apparently by desensitizing the system in the absence

of

activity kinase

glucagon

in

C

(92).

The

desensitization

in

receptor

and

possible the

modification

requirement

possible

indicate

role

that

for

of

N^

protein

the

feedback

mechanisms involved in receptor uncoupling could be very complex. Other workers have observed homologous down-regulation of glucagon

receptors

together with heterologous

desensitization.

The

apparent discrepancy between these studies and those of Houslay is

likely

due

to

the

experimental

treatment

of

the

cells.

Houslay uses freshly isolated hepatocytes, while Noda et al. used primary

cultures

of

cultured cells (89). and

heterologous

cultures

of

hepatocytes

(88)

Rich

et

al.

stable

In addition, similar patterns of homologous

desensitization

chick

and

hepatocytes

manuscripts in preparation).

have (R.

been

Premont

seen and

in R.

primary Iyengar,

In the study of Noda et al. (88),

homologous loss of glucagon receptors and heterologous desensitization Neither

are

complete

after

9

hours

form of desensitization

treatment

with

dibutryl-cAMP,

lyzable analogue of cAMP.

of

exposure

to

glucagon.

can be elicited by 12 hours of a

membrane-permeable,

Labeling of N s

(a s )

b

non-hydro-

Y cholera toxin

catalyzed ADP-ribosylation using [3 2p]NAD+ indicates no change in the amount of N s present

following glucagon treatment, so these

167

researchers probably observe

conclude

is caused complete

following

that the heterologous

by

a modification

desensitization

of

both

2 hours of glucagon treatment.

bromo-cAMP,

another

heterologous

cAMP

component

analogue,

differences

levels of N G receptors.

types

between

most

et al.

in

MDCK

(89) cells

The treatment with 8-

of desensitization,

of N s and N g activity assays

Rich

induces

component appears to be cAMP-independent. no

desensitization

of N s .

in

this

while

system

the

the

homologous

Cholera toxin labeling

(cyc~ membrane reconstitution) show

untreated

and

desensitized

membranes

in

or its ability to couple to C or to other hormone However, pertussis

toxin catalyzed

ADP-ribosylation

of N^ (D^) using [3 2 P ]NAD + indicated a two-fold enhancement in AI labeling

in desensitized

mean that the tion, give

and

amount of

this

lower

over

"extra"

levels

of

untreated

«i present N^

further

membranes.

increases

This

upon

desensitiza-

inhibits adenylyl

stimulation.

However,

could

cyclase

labeling

of

a^

to b

y

pertussis toxin is not a measure of absolute N^ levels.

It is

therefore

which

allow

not

it to

possible

further

to

rule

out

modifications

inhibit adenylyl

cyclase and

of

N^

concomitantly

increase its capacity to be labeled by pertussis toxin.

Further

alterations in gy levels would decrease adenylyl cyclase stimulation and increase the pertussis toxin catalyzed

ADP-ribosylation

of N^ since ai is substrate for pertussis toxin only when associated with gy. Glucagon-induced

desensitization has also been shown to occur in

isolated rat liver plasma membranes

(93) and in chick hepatocyte

membranes (R. Premont and R. Iyengar, manuscript in preparation). In the presence of ATP and GTP, glucagon added to membranes was capable of promoting the apparent uncoupling of glucagon tors

from

adenylyl

cyclase

(i.e.,

homologous

recep-

desensitization).

Since assays for adenylyl cyclase activity contain 1 mM cAMP, the lack of desensitization cates one.

that More

produced

the

in the absence of added

desensitization

importantly,

process

however,

high

is

not

glucagon a

concentrations

identical loss of glucagon-stimulability

indi-

cAMP-mediated of

Mg++

in a GTP-inde-

pendent manner, and desensitization by glucagon was shown to have

168 an absolute

requirement

dependence,

similar

that N s

for divalent

to that required

is important

for N s

(93).

Mg++-

This

activation,

in the desensitization process,

occupation of the receptor. is known of the

cations

implies

as well

Despite these studies, quite

specific mechanisms

underlying

as

little

glucagon-induced

desensitization. Desensitization and

its

of glucagon-stimulated

relationship

to

diseased

adenylyl

states

has

studied, with often conflicting results. common

complication

diabetes mellitus glucagon

into

of

both

(95,96).

normal

rats

renal

in

cyclase been

situ

extensively

Hyperglucagonemia is a

insufficiency

(94,95)

and

The effect of repeated injection of is

the

reduction

of

both

glucagon

receptor number and glucagon-stimulated adenylyl cyclase activity in subsequently ment

with

isolated

studies

liver plasma membranes

on both

(97),

isolated hepatocytes

vitro desensitization of membranes

(93).

unilateral

"desensitized"

nephrectomy

causes

a

in

agree-

(86,88)

and

Uremia resulting state

from

indis-

tinguishable from that due to injection of glucagon in rats Diabetes mellitus modeled in rats by injection of is

characterized

number

and

by

dose-dependent

a parallel

loss

of

loss

adenylyl

of

glucagon in isolated liver plasma membranes activity (heterologous desensitization) ment

of

streptozotocin-induced

receptor

stimulation

(98).

diabetic

rats

with

by

A loss in N s

is also observed.

partially reverse these desensitized states

(97).

streptozotocin

glucagon

cyclase

in

Treat-

insulin

can

(98).

Studies of the regulation of glucagon receptor levels on the cell surface are only in their infancy.

The preparation of antibodies

to the receptor molecule will make it possible to directly

study

the movements of the receptor both in and out of the cell.

Future Directions Utilization

of

the

specific

crosslinking

of

glucagon receptor with the heterobifunctional

[125I]MIG

to

crosslinking

the

agent

169

HSAB has led to a significant increase of our knowledge of the structure of the glucagon receptor. The major focus of research on the glucagon receptor in the near future will be on three interrelated fronts: the purification of the hormone-binding glycoprotein unit of the receptor, the preparation of specific antibodies against the glucagon receptor, and cloning of the gene for the receptor. The data obtained from studies of the crosslinked receptor provides a basis from which to accomplish these objectives. It is important to realize, however, that these goals are not ends unto themselves, but rather tools to be used in the further characterization of signal transduction through adenylyl cyclase systems. All that is known of the activation of N s by the occupied glucagon receptor and of the functional and structural interactions of N g with the receptor has been obtained using native membranes or is assumed by analogy to reconstituted systems of purified N s with (3-adrenergic receptors in defined phospholipid vesicles. Purified preparations of glucagon receptors are necessary to define the mechanisms of receptor interaction with N g . In addition, the nature of the difference between the 63 Kd receptor in SDS gels and the 119 Kd receptor seen in hydrodynamic experiments, either due to a different receptor subunit of unknown function or due to dimerization of hormone-binding units, can be investigated in the purified receptor. Antibodies to the glucagon receptor will be very useful in future studies of the glucagon receptor as much as the crosslinking technique has in the past few years. By allowing the specific labeling of the receptor (at essentially 100%, rather than the 12% seen in crosslinking) in both living and in vitro systems, antibodies will allow direct visualization of glucagon receptor levels, distribution, and movements. Uncertainties about internalization and recycling of occupied receptors, the fate of down-regulated receptors, and the existence of intracellular pools of receptors can be eliminated with antibody probes. The rapid purification of the receptors with the use of antibody

170

affinity

columns

will

allow

through phosphorylations

questions

of

receptor

or other modifications

regulation

to be addressed

as well. Cloning the gene

for the glucagon receptor will require

the

of

purification

the

receptor antibodies.

receptor

or

the

preparation

either

of

anti-

Once cloned, the DNA sequence of the gene

will give great insight into the structure of the receptor protein, and will allow comparison of the glucagon receptor sequence with that of other R s receptors.

Alterations of the gene through

site-directed

allow

domains

mutagenesis

important

will

for hormone binding

the and

direct

probing

of

interaction with

Ng,

while bacterial expression of the gene may provide large quantities of receptors for purification.

Perhaps more fundamentally,

analysis of regulatory regions of the glucagon receptor gene may give insight into its tissue-specific expression, and how expression of receptors by particular monal "address system".

cells creates the complex hor-

The role of receptor transcription and

synthesis and its regulation, as well as that of receptor downregulation in determining the resting and stimulated cell surface receptor number could also be directly examined. There are more general questions concerning hormone action yet to be addressed. any hormone

In a very real sense, understanding

requires

not only

a complete

the role of

understanding

of

its

effects on target cells, but also knowledge of the specific conditions which accelerate or inhibit its release, and the mechanisms underlying them. messenger generating

Adenylyl cyclase is an integrative second

system:

signals received

from many

are transformed into a single rate of cAMP synthesis.

sources

This would

seem to imply that all information about the hormones generating the cAMP signal will be lost.

That this does not seem to be the

case is exemplified by the observation that the maximum extent of stimulation by glucagon hepatocytes.

is greater than that of epinephrine

in

Whether this is due simply to different numbers of

surface receptors, or to an inherent difference in the efficacy of these receptors

remains to be shown.

Within the cell, how-

171

ever, the attained concentrations of cAMP will differ for these hormones.

How

the

levels

of

cAMP

regulate

the

instantaneous

activity of the cAMP-dependent protein kinase, and the possibilities for differential regulation of its substrates, are important questions

if

there

is

a

functional

reason

for

differences

in

stimulation by various hormones. Recent work has raised the possibility that the glucagon receptor system

is

Houslay

not

and

as

straightforward (99) have

co-workers

phosphatidylinositol

breakdown

cAMP-independent manner.

by

as

has

been

reported

described

the

glucagon,

in

here.

stimulation an

of

apparently

These workers have suggested that there

are, in fact, two distinct populations of glucagon receptors: the first coupled to adenylyl cyclase; and another, coupled to phosphatidylinositol-specific for

glucagon

phospholipase

stimulation

of

C.

phospholipase

Another C,

explanation

however,

would

require only a single population of receptors which can couple to two N proteins: N s for stimulation of adenylyl cyclase, and "N p " for the stimulation of phospholipase C.

It is presently thought

that a single class of angiotensin II receptors couple to both N^ (to inhibit adenylyl

cyclase) and to the yet unidentified

"Np"

(to stimulate phospholipase C) (100). That glucagon does stimulate phospholipase C directly in a cAMPindependent manner workers

has

fashion,

is not clear.

indicated

potentiate

hydrolysis

by

that the

Recent work by Exton and co-

glucagon

stimulation

vasopressin

or

can, of

angiotensin

in

a

cAMP-dependent

phosphatidylinositol II

(101).

More

directly, these researchers failed to see a significant increase inositol phosphates following glucagon treatment, but do observe increased phospholipase C activity when NaF is added along with the glucagon

(102).

forskolin or

cAMP

In this case, glucagon can be replaced by

analogues,

and NaF activates

the presumptive

"N_" protein. This discrepancy will require additional work to r resolve, but is clear that glucagon (either directly or through adenylyl

cyclase) does

phospholipase C.

affect

the

phosphatidylinositol-specific

172 Acknowledgements R.I. is an Established Investigator of the American Heart Association. Portions of this work have been supported by grants AM26905 and CA-29808 from the National Institutes of Health. The authors wish to thank Ms. Suzanne Saltalamacchia for expert secretarial assistance. References 1.

Unger, R.H. (1985) Diabetologia 28:574.

2.

Unger, R.H., and L. Orci, eds. (1981) GLUCAGON: Physiology, Pathophysiology, and Morphology of the Pancreatic A-Cells. Elsevier North Holland.

3.

Rail, T.W., 232:1065.

4.

Pohl, S.L., L. Birnbaumer, and M. Rodbell Chem. 246:1849.

(1971) J. Biol.

5.

Birnbaumer, L., S.L. Pohl, and M. Rodbell Chem. 246:1857.

(1971) J. Biol.

6.

Rodbell, M. , H.M.J. Krans, S.L. (1971) J. Biol. Chem. 246:1861.

Pohl,

and

L.

Birnbaumer

7.

Rodbell, M. , H.M.J. Krans, S.-L. Pohl, (1971) J. Biol. Chem. 246:1872.

and

L.

Birnbaumer

8.

Rodbell, M. , L. Birnbaumer, S.L. (1971) J. Biol. Chem. 246:1877.

9.

Abood, M.E., J.B. Hurley, M.C. Pappone, H.R. Bourne, and L. Stryer (1982) J. Biol. Chem. 257:10540.

10.

Litosch, I., C. Wallis, and J.N. Fain (1985) J. Biol. Chem. 260:5464.

11.

Houslay, M.D., A.V. Wallace, M.E. Cooper, N.J. Payne, S.R. Wilson, and C.M. Heyworth (1984) Biochem. Soc. Trans. 12:766.

12.

Saltiel, A.R., J.A. Pox, (1986) Science 233:967.

13.

Desbuquois, B. (1975) in: Polypeptide Hormone Receptors (B. Posner, ed.), Marcel Dekker, pp. 345-417.

14.

Iyengar, R. (1986) in: Hormonal Control of Gluconeogenesis (N. Kraus-Friedmann, ed. ), CRC Press.

and

E.W.

Southerland

(1958)

Pohl,

J.

and

Biol.

H.M.J.

Chem.

Krans

P. Sherline, and P. Cuatrecasas

173 15.

Hunter, W.M.,

and F.C. Greenwood

16.

Birnbaumer, 248:2056.

17.

Bromer, W.W., M.E. Boucher, Biochem. Biophys. Res. Commun.

18.

Desbuquois,

19.

R o j a s , F . J . , T . L . S w a r t z , R. B i r n b a u m e r (1983) E n d o c r i n o l .

20.

Goldfine, I.D., Chem. 247:1211.

J.

21.

Cuatrecasas,

(1972)

22.

H e r b e r g , J . T . , J. C o d i n a , K. A. R i c h , F.R. Iyengar (1984) J. Biol. Chem. 259:9285.

23.

Neville,

24.

Berry,

25.

Bataille, D., P. Endocrinol. 95:713.

26.

Couvineau,

27.

Nguyen, 25:361.

28.

Hossein,

29.

Sasaki, K., S. D o c k e r i l l , D.A. A d a m i a k , B l u n d e l l (1975) N a t u r e 257:751.

30.

Bromer, W.W., L.G. Chem. Soc. 79:2807.

31.

Hruby,

32.

Sonne, O., T. Berg, Chem. 253:3203.

33.

Pohl, S . L . , H . M . J . K r a n s , L. B i r n b a u m e r , (1972) J. B i o l . C h e m . 2 4 7 : 2 2 9 5 .

34.

Iyengar, Sci. USA

35.

Bregman, M.D., and Commun. 78:584.

L.,

B.

N.M.,

A.,

N.M.,

(1982)

Williams,

R. , a n d 79:5179.

Mol.

O.K.

L. D.

Levy

Acta

Gray

L.

Biol.

69:318. and

R.

154:540. 43:506.

J.

(1974) 225:473.

(1986)

Biochem.

178:83.

I.J. T i c k l e ,

Behrens

and

(1957)

J.

T. Am.

44:49.

(1982)

(1977)

J.

Rosselin

Christofferson

Birnbaumer

and

Rojas,

(1984) F E B S Lett.

and

T.

USA

Biochem.

and G.M.

Cell Biochem. and

(1972)

Sci.

G.

(1985)

(1973)

Garber,

(1969) J. C e l l Biol.

a n d M. L a b u r t h e

Sinn,

A.J.

Biophys.

and

Chem.

53:569.

Natl. Acad.

Freychet,

and R.S. Gurd

Biol.

Patterson

L. B i r n b a u m e r

Biochim.

194:495.

J.

and J.M. 53:134.

Iyengar, 113:711.

and

Proc.

(1968)

J.A.

(1973)

Eur. J. Biochem.

Roth,

Jr.

Pohl

and D.S. Friend

T.D.,

V.

S.L.

(1975)

P.

D.M.,

and

(1962) N a t u r e

(1978) and

Proc.

Biochem.

J.

M.

Biol.

Rodbell

Natl. Biophys.

Acad. Res.

174 36.

Johnson, G.( V. MacAndrew, Jr., Natl. Acad. Sci. USA 78:875.

and

37.

Demoliou-Mason,

C.,

a n d R. E p a n d

(1982)

Biochem.

21:1989.

38.

Demoliou-Mason,

C.,

a n d R. E p a n d

(1982)

Biochem.

21:1996.

39.

Wright, D.E., R. B i o c h e m . 1 4 1 : 6 3.

40.

Iyengar, 259:5222.

41.

Iwanij, 82:325.

42.

L i v i n g s t o n , J . N . , K. E i n a r r s o n , L. B e c k m a n , P. A r n e r ( 1 9 8 5 ) J . C l i n . I n v e s t . 7 5 : 3 9 7 .

43.

E l d e r , J . H . , a n d S. A l e x a n d e r USA 79:4540.

44.

Plummer, T.H., A.L. Tarentino

45.

Johnson, G.L., H.R. Kaslow, (1980) Adv. C y c l i c N u c l e o t i d e

46.

Gilman,

47.

B i r n b a u m e r , L., T. S u n y e r , F.J. R o j a s , M . G . C a r o n , R.J. L e f k o w i t z , a n d R. I y e n g a r (1985) in: M o l e c u l a r M e c h a n i s m s of Transmembrane Signalling (Cohen and Houslay, eds.), Elsevier Science Publishers, pp. 131-182.

48.

Iyengar, R., 256:11036.

49.

Iyengar

50.

B i r n b a u m e r , L., Chem. 244:3468.

51.

Northup, J.K., M.D. Smigel, P.C. Sternweis, (1983) J. B i o l . C h e m . 2 5 8 : 1 1 3 6 9 .

52.

Northup, J.K., P.C. Sternweis, Biol. Chem. 258:11361.

53.

Asano, (1984)

T.L., S.E. Pederson, Biochem. 23:5460.

54.

Asano,

T.L.,

55.

Codina, J., J.D. Hildebrandt, L. Birnbaumer, S e k u r a (1984) J. Biol. Chem. 259:11408.

R.,

V.,

R.

and and

A.G.

Horuk,

and

J.T.

K.

Hur

M.

Pilch

Rodbell

Herberg (1985)

P.

(1984)

(1984)

Proc.

(1982)

(1981)

J.

Natl.

Proc.

Proc.

Eur.

Biol.

Acad.

J.

Chem.

Sci.

USA

S.

Ewerth,

and

Natl.

Acad.

Sci.

J . H . E l d e r , S. A l e x a n d e r , A . W . P h e l a n , (1984) J. B i o l . C h e m . 2 5 8 : 1 0 7 0 0 .

(1984)

and

(1981)

Cell

L. J.

S.L.

and E.M.

Z. F a r f e l , Res. 13:1.

and

H.R.

and

Bourne

36:577.

Birnbaumer

Biol. Pohl,

Ross

Chem. and

(1981)

J.

Biol.

Chem.

256:11042. M.

Rodbell

and S.W.

(1984)

A.G.

(1969)

Biochem.

and A.G.

Gilman

Scott,

J.

and

Biol. Gilman

(1983) E.M.

J.

Ross

23:5467. and

R.D.

175 56.

Rich, K.A., G. Floyd, (manuscript submitted).

57.

Kent, R.S., A. Delean, Pharmacol. 17:14.

58.

Delean, A., J.M. Chem. 255:7108.

59.

Citri,

60.

K e l l e h e r , D.J., A. R a s h i d b a i g , A . E . (1983) J. Biol. Chem. 258:12881.

61.

C e r i o n e , R.A., J. C o d i n a , J . L . B e n o v i c , R.J. L e f k o w i t z , B i r n b a u m e r , a n d M.G. C a r o n (1984) B i o c h e m . 23:4519.

62.

Bird, S.J., 253:8826.

63.

Rojas, F.J., 260:7829.

64.

P a d r e l l , E. , J . T . H e r b e r g , B . M o n s a t i r s k y , G . F l o y d , P r e m o n t , a n d R. I y e n g a r ( m a n u s c r i p t s u b m i t t e d ) .

65.

Lafuse,

66.

P o h l , S . L . , H . M . J . K r a n s , L. B i r n b a u m e r , (1972) J. B i o l . C h e m . 2 4 7 : 2 2 9 5 .

67.

Oka, Y., L.M. 260:9435.

68.

Sullivan, 261:3571.

69.

Hunt, R.C., 261:3681.

70.

B a r a z z o n e , P., P. G ö r d e n , J . - L . C a r p e n t i e r , L. O r c i , P. F r e y c h e t , a n d B. C a n i v e t ( 1 9 8 0 ) J . C l i n . I n v e s t . 6 6 : 1 0 8 1 .

71.

A s a d a - K u b o t a , M. , J. W a t a n a b e , S. K a n a m u r a , M. Y o s h i k a w a (1983) Exp. P a t h o l . 2 3 : 9 5 .

72.

Ackerman, Histochem.

73.

W a t a n a b e , J . , S. K a n a m u r a , M . A s a d a - K u b o t a , M. O k a ( 1 9 8 4 ) A n a t . R e e . 2 1 0 : 5 5 7 .

74.

Watanabe, 214:1.

Y.,

W.,

and

Stadel,

a n d M.

M.E.

and

L.

S.J., and

and L.

S.

and

(1985)

A.

Shonbrunn

Czech

Kanamura,

and

and

K.

Johnson L.

Biol.

Chem.

Biol.

Chem.

and J. J.

(1986)

Kanai

Biol.

R.T.

19:49.

(1985)

K.W.

(1980) J.

J.

(1986)

Marshall-Carlson

Mol.

J.

Biochem.

M.P.

(1980)

and G.L.

(1976)

and

Iyengar

287:297.

Ruoho,

Birnbaumer

(1980)

R.

Lefkowitz,

(1980) N a t u r e

G.A., J. Yang, Cytochem. 31:433.

J.,

R.J.

Maguire

and M. E d i d i n

Rozek,

Herberg,

and R.J. Lefkowitz

Schramm

and

J.T.

J.

K.

Wolken

K.

(1986)

M.

Rodbell

Biol.

Chem.

Biol.

Chem.

Biol.

Chem.

Kanai,

and

(1983)

J.

Kanai,

and

Anat.

Ree.

176 75.

Canivet, Freychet

76.

R o u e r , E . , B. D e s b u q u o i s , Cell Endocrinol. 19;143.

77.

Lipson, K., A.A. Kolhatkar, (1986) B i o c h e m . 2 5 : 2 6 1 2 .

78.

S t a d e l , J . M . , P. N a m b i , Caron, and R.J. Lefkowitz 80:3173.

79.

S i b l e y , D . R . , J . R . P e t e r s , P. N a m b i , M . G . L e f k o w i t z (1984) J. Biol. Chem. 2 5 9 : 9 7 4 2 .

80.

Strasser, 260:4561.

81.

Strasser, R.H., D.R. Biochem. 25:1371.

Sibley,

82.

Benovic, Lefkowitz

Strasser, Natl. Acad.

83.

B e n o v i c , J., F. M a y o r , Jr., R.J. L e f k o w i t z (1986) N a t u r e

84.

Kwatra, M.M., 261:12429.

85.

Knox, B.E., P.N. Devreotes, A. Goldbeter, (1986) Proc. Natl. A c a d . Sci. USA 8 3 : 2 3 4 5 .

86.

Heyworth,

C.M.,

87.

Plas,

a n d J. N u n e z

88.

N o d a , C., F. S h i n j y o , and A. I c h i h a r a (1984)

89.

Rich, K.A., J. Codina, G. Floyd, R.D. Sekura, J.D. H i l d e b r a n d t , a n d R. I y e n g a r ( 1 9 8 4 ) J . B i o l . C h e m . 2 5 9 : 7 8 9 3 .

90.

Heyworth, C.M., J. 222:189.

91.

Heyworth, C.M., S.P. Wilson, (1984) FEBS L e t t . 1 7 0 : 3 8 .

92.

Heyworth, C.M., A.D. Whetton, A.R. H o u s l a y (1985) FEBS L e t t . 1 8 7 : 1 9 6 .

93.

I y e n g a r , R. , P . W . M i n t z , T . L . S w a r t z , (1980) J. Biol. C h e m . 2 5 5 : 1 1 8 7 5 .

C.,

B . , P. G o r d e n , J . - L . C a r p e n t i e r , L . (1981) Mol. C e l l E n d o c r i n o l . 23:311.

R.H.,

J.L., (1986)

and

Postel-Vinay Cherskey,

and

Hosey

(1975)

(1980)

and D.B.

Caron,

(1985) R.J.

Mol.

J.

and

Biol.

(1986)

(1983)

J. Biol.

and M.D. D.J.

R.J.

Chem. (1986)

M.G. Caron, and Sci. USA 83:2797. M.G. J.

Biol.

and

Biochem. Chem.

Houslay Gawler,

J.

and Chem.

L.E.

Segal 214:93.

250:5304. Nakamura,

(1984)

Biochem.

and M.D.

Kinsella, and

R.J.

Caron,

A. T o m a m u r a , S. K a t o , T. J. Biol. Chem. 259:7747.

E. H a n s k i ,

P.

Donner

Lefkowitz

R.L. Somers, 321:869.

Houslay

and

Shorr, D.F. Sawyer, M.G. Proc. Natl. Acad. Sci. USA

Lefkowitz

M.M.

and M.D.

B.D.

R.G.L. (1983)

R.J.

R.H. Proc.

and

and M.C.

Orci,

L.

Houslay

and"

M.D.

Birnbaumer

177

94.

Bilbrey, G.L., G.R. Falonna, M.G. White, and J.P. Knöchel (1974) J. Clin. Invest. 53:841.

95.

Kuku, S.F., A. Zeidler, D.S. Emmanouel, A.I. Katz, and A.H. Rubenstein (1976) J. Clin. Endocrinol. Metab. 42:173.

96.

Bhatena, S.J., N.R. Voyles, S. Smith, and L. Recant J. Clin. Invest. 61:1488.

(1978)

97.

Dighe, R.R., F.J. Rojas, L. Birnbaumer, (1984) J. Clin. Invest. 73:1004.

and A.J.

Garber

98.

Dighe, R.R., F.J. Rojas, L. Birnbaumer, (1984) J. Clin. Invest. 73:1013.

and

Garber

99.

Wakelam, M.J.O., G.J. Murphy, V.J. Hruby, and M.D. HcJuslay (1986) Nature 323:68.

100.

Enjalbert, A., F. Sladeczek, G. Guillon, P. Bertrand, C. Shu, J. Epelbaum, A. Garcia-Sainz, S. Jard, C. Lombard, C. Kordon, and J. Bockaert (1986) J. Biol. Chem. 261:4071.

101.

Morgan, N.G., R. Charest, P.F. Blackmore, (1984) Proc. Natl. Acad. Sei. USA 81:4208.

102.

Blackmore, 261:11056.

P.F.,

and

J.H.

Exton

(1986)

A.J.

and J.H. J.

Biol.

Exton Chem.

INSULIN RECEPTOR

Sam J. Bhathena Carbohydrate Nutrition Laboratory Beltsville Human Nutrition Research Center U.S.D.A. Agricultural Research Service, Beltsville, Maryland 20705

Introduction, History and Definition Assay Properties Regulation Isolation, Purification and Characterization Structure Visualization Microheterogeneity Antibodies to Insulin Receptors Mechanism of Action Clinical Applications Future Trends

Introduction

Among the receptors for peptide hormones, the insulin receptor is one of the most widely studied.

Since several excellent reviews

have been written on several aspects of the insulin receptor, this chapter will summarize the salient features and for greater in depth analysis of each point, the reader will be referred to specific reviews of the subject. The binding of insulin to its specific receptor is a first step in insulin action.

This complex then activates several biochemical

and physiological processes at the membrane and/or

Peptide Hormone Receptors © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

intracellularly.

180 The a c t i v a t i o n is g e n e r a l l y m e d i a t e d t h r o u g h a s e c o n d U n f o r t u n a t e l y , such a m e s s e n g e r or a m e d i a t o r

messenger.

for i n s u l i n

action

is not c l e a r l y i d e n t i f i e d a n d c o u l d be m o r e t h a n o n e m a t e r i a l . The m a g n i t u d e of the b i o l o g i c a l e f f e c t of i n s u l i n d e p e n d s o n three f a c t o r s : receptor

i) the n a t u r e of insulin,

and iii) p o s t receptor e v e n t s .

r e s t r i c t e d to the l a s t two

T h i s r e v i e w w i l l be

aspects.

It is n e c e s s a r y to d i f f e r e n t i a t e a receptor B o t h m a y b i n d to the h o r m o n e

from an

«

s p e c i f i c and m a y or m a y n o t be d i s s o c i a b l e .

receptor-

B i n d i n g to a

is s p e c i f i c , w h i l e binding to the a c c e p t o r

of the latter are b i n d i n g

acceptor.

(e.g., i n s u l i n ) , b u t o n l y the

hormone complex can produce biological effects. receptor

mainly

ii) b i n d i n g to its

is u s u a l l y

S o m e of the

examples

to g l a s s , p l a s t i c , t a l c and o t h e r

ials f r e q u e n t l y used in the s t u d y of r e c e p t o r s . c a n be in p a r t i c u l a t e or s o l u b l e

The

non-

mater-

receptor

form.

History

The p r e s e n c e of in 1 9 4 9 - 1 9 5 2

i n s u l i n r e c e p t o r s was s u g g e s t e d by S t a d i e et al.

(1,2).

T h a t the b i n d i n g of i n s u l i n to its

receptor

is r e q u i r e d for b i o l o g i c a l a c t i v i t y w a s p r o p o s e d by H e c h t e r and R e i s e r

(4).

H o w e v e r , it was n o t u n t i l 1970 that the

for i n s u l i n w a s f i r s t e x p e r i m e n t a l l y

(5) using

(6,7), he d i d d e m o n s t r a t e

of i n s u l i n r e c e p t o r s using r a d i o a c t i v e l y used s e v e r a l y e a r s earlier

interactions.

of i n s u l i n r e c e p t o r

and

presence

technique

(14,15)

and

This technique with only minor

m o d i f i c a t i o n is still used today for i n s u l i n and o t h e r hormone-receptor

absorbed

the

(8-13), a

in d e m o n s t r a t i n g A C T H

receptors.

on

iodinated insulin and

d i s p l a c e m e n t of its b i n d i n g by n a t i v e i n s u l i n (16,17)

insulin

T h o u g h the m e t h o d was later c r i t i c i z e d

the r e s u l t s w e r e c h a l l e n g e d

angiotensin

receptor

(directly) d e m o n s t r a t e d

fat c e l l s and m e m b r a n e s by C u a t r e c a s a s on sepharose beads.

(3)

peptide

The e a r l y s e v e n t i e s saw a n

s t u d i e s o n the k i n e t i c s of b i n d i n g ,

explosion

identifica-

181

tion of insulin receptors on a variety of tissues, on intracellular organelles, and on receptor regulation in diabetes and obesity. However, the structure of the insulin receptor was not studied until the late seventies when newer affinity techniques became available.

Early eighties saw a proliferation of reports of

the possible second messenger(s) generated by interactions.

insulin-receptor

Only recently, the amino acid sequence of the

insulin receptor has been indirectly determined and the gene for the insulin receptor in human tissue has been assigned.

Definition

It is difficult to define an insulin receptor. aspects to be considered:

There are two

i) recognition of a hormone

(insulin)

(binding unit) and ii) initiation of a biological event(s)

attri-

butable to that hormone either by activation or induction of some factor(s) known as mediator or second messenger

(effector unit).

It is, however, not necessary that the effector unit be present within the receptor

structure.

Earlier studies defined insulin receptors based only on the recognition aspect

(18,19).

Thus, any molecule or chemical structure

present in any animal tissue which could specifically bind

(recog-

nize) a hormone and from which a bound hormone can be displaced was classified as a receptor.

By this definition, a molecular

structure isolated from a cell in soluble form which binds insulin is a receptor. The requirement of effector function to define a receptor poses problems for insulin and for other hormones. recognized) to almost all animal tissues.

Insulin binds

(i.e.,

However, in some tissues

no known biological effect has been demonstrated.

It is certainly

possible that insulin does exert some biological effect in these tissues which has not yet been demonstrated or recognized by the present status of our knowledge

(e.g., growth promoting effect

182

of insulin in cells in culture has only recently been recognized). Thus, using this dual requirement, a chemical structure which recognizes insulin in one tissue where there is a demonstrable effect of insulin is a receptor but in other tissue where the effects of insulin are not yet demonstrated may not be considered a receptor.

This also applies to a soluble receptor which is

isolated from a target tissue but has now no opportunity to express its biological effect. At present we know enough about the structure of the insulin receptor.

As described later in this chapter, insulin receptor

is a heterotetramer with 2 a and 2 6 subunits joined by disulfide linkages. It has been demonstrated that a subunit(s) bind insulin (i.e., have recognition sites) while 6 subunit has tyrosine kinase activity, a possible site for mediation of insulin action effector unit?).

(putative

Since disulfide bonds holding 2 halves of the

receptors are not required for either binding of insulin or for the biological activity, a structure with 1 a subunit joined to 1 3 subunit will also function as a receptor since it can bind as well as initiate a chain of events leading to a demonstrable effect.

Thus, by the rigid definition this molecular

insulin

arrangement

is a receptor, even though in all tissues where receptor

structure

has been identified, insulin receptor is a heterotetramer.

Further,

since only the a subunit is responsible for binding of insulin, then based on the recognition requirement, an isolated single a subunit may be considered as an insulin receptor. A more precise definition of insulin receptor will have to wait until some biological effects of insulin are recognized in all tissues that bind insulin.

Also, when the putative second messen-

ger (s) for insulin is more fully characterized and when it is demonstrated with some certainty whether tyrosine kinase on B subunit can itself act as a mediator of insulin effect, a better definition will emerge.

Based on our present knowledge,

insulin

receptor is a molecule or chemical structure that can bind insulin and when not dissociated from the tissue or a cell or its component (e.g., plasma membrane), can initiate the production of a biological effect or an event leading to it.

Such an event may not have yet

183 been described as an effect of insulin.

This definition though

not precise will at least include solubilized molecules having most of the components of a structure that can function.

This

would exclude the isolated a subunit since it can only bind but possibly lacks effector

function.

Assay

Insulin binds to almost all the tissues of animals.

Insulin

receptors have been demonstrated on primary as well as established cell lines from various tissues grown in culture.

Since the

receptors for insulin (and for other peptide hormones) reside on the membrane, insulin binding has been measured from isolated membranes of several tissues as well as on isolated intact cells. It is not possible to describe insulin binding to all the tissues and plasma membranes from these tissues.

This review is therefore

restricted to insulin binding to hepatocytes, erythrocytes and adipocytes.

These three cells are selected, because, they require

different techniques to separate bound insulin from free insulin. Insulin binding to circulating lymphocytes in culture has been described in great detail by Roth (20) .

Fortunately,

insulin

binding to different tissues has very similar binding characteristics and therefore the same method with minor alterations can be employed to measure insulin-receptor interactions in other systems. In essence, cells or membranes are incubated with a tracer quantity (50-100 pg/ml) of mono-iodinated insulin in the presence of varying concentrations

(0-10 yg/ml) of native insulin for 30 min to 18 hr

at 4° to 37°C. several factors.

The selection of time and temperature depend on Many tissues have enzymes which degrade either

insulin or the receptor and hence a lower temperature with a prolonged incubation time is used.

Membranes on the other hand

have very little degrading activity and hence binding is measured at 30-37° for short periods of time, usually 30 min to 1 hr.

Many

184 cells, e.g., granulocytes

(21), d e g r a d e

insulin significantly

hence it is e s s e n t i a l to use v a r i o u s p r o t e a s e to m e a s u r e b i n d i n g .

inhibitors

Some c e l l s r a p i d l y i n t e r n a l i z e

i n s u l i n w h i c h is either d e g r a d e d by i n t r a c e l l u l a r r e b i n d s to r e c e p t o r s o n i n t r a c e l l u l a r o r g a n e l l e s ribosomes, etc.).

bound

e n z y m e s or (nucleus,

S i n c e i n t e r n a l i z a t i o n is t e m p e r a t u r e

(22,23), it is n e c e s s a r y to m e a s u r e b i n d i n g at lower A longer

the

golgi,

dependent

temperatures.

time of i n c u b a t i o n is r e q u i r e d w i t h these c e l l s ,

overnight, since binding reaches equilibrium slowly.

usually

Insulin

b i n d i n g to v a r i o u s t i s s u e s also d e p e n d s o n p H a n d i o n i c It is t h e r e f o r e n e c e s s a r y

environment.

to e s t a b l i s h the r e q u i r e m e n t of time, pH,

t e m p e r a t u r e , i o n i c m i l l i e u and p o s s i b l e r e q u i r e m e n t for

protease

i n h i b i t o r s b e f o r e b i n d i n g m e a s u r e m e n t s to a new type of c e l l is c a r r i e d

system

out.

S l i g h t l y d i f f e r e n t t e c h n i q u e s are r e q u i r e d to s e p a r a t e h o r m o n e f r o m free h o r m o n e

in d i f f e r e n t t i s s u e s .

bound

Since most

and 1 m e m b r a n e s s e d i m e n t e a s i l y a n d form a t i g h t p e l l e t u p o n f u g a t i o n , the m e t h o d d e s c r i b e d here for h e p a t o c y t e s is used.

and,

in o r d e r

A t the e n d of

tissues centri-

routinely

i n c u b a t i o n , the tubes are g e n t l y s h a k e n

make c e r t a i n that the c e l l s are h o m o g e n e o u s l y

s u s p e n d e d and

to 200

y l d u p l i c a t e a l i q u o t s are l a y e r e d o v e r 200 y l of c h i l l e d b u f f e r microfuge

tubes.

a n d the s u p e r n a t a n t is r e m o v e d by s u c t i o n . that p e l l e t is n o t t o u c h e d .

C a r e is t a k e n to see

Two h u n d r e d m i c r o l i t e r s of 10%

sucrose

is l a y e r e d g e n t l y over

the c e l l p e l l e t and the tubes are

a g a i n in the m i c r o f u g e

for 1 m i n .

c a r e f u l l y by s u c t i o n .

The tubes are c u t just above the p e l l e t

the tip c o n t a i n i n g

in

The tubes are spun for 1-2 m i n in a m i c r o f u g e

spun

The s u p e r n a t a n t is a g a i n

the p e l l e t is c o u n t e d in a g a m m a

removed and

counter.

For e r y t h r o c y t e s , a t the end of i n c u b a t i o n , d u p l i c a t e a l i q u o t s of 200 y l are l a y e r e d over phthalate)

200 y 1 of n - b u t y l p h t h a l a t e

a n d 200 y l of buffer

in c h i l l e d m i c r o f u g e

(dibutyl tubes.

(The

v o l u m e s of p h t h a l a t e and buffer c a n be r e d u c e d to 100 y l e a c h to use 400 y l m i c r o f u g e c o m p a r e d to larger

tubes w h i c h are m o r e c o n v e n i e n t to use as

v o l u m e tubes) .

The tubes are s p u n for 1 m i n

in a m i c r o f u g e as d e s c r i b e d for h e p a t o c y t e s .

The upper

c o n s i s t i n g of buffer and p a r t of the p h t h a l a t e layer

layer

is r e m o v e d

185 by s u c t i o n .

The tubes are c u t t h r o u g h the p h t h a l a t e layer

just

a b o v e the e r y t h r o c y t e p e l l e t and the p e l l e t is c o u n t e d in a g a m m a counter. of

It is n o t n e c e s s a r y to w a s h the p e l l e t as in the c a s e

hepatocytes.

For a d i p o c y t e s , w h i c h f l o a t o n c e n t r i f u g a t i o n ,

200 y l

duplicate

a l i q u o t s are l a y e r e d over

200 y l of d i n o n y l p h t h a l a t e a n d

in m i c r o f u g e

The c e l l s f l o a t to the top.

for 15 sec.

spun

The

tubes

are c u t t h r o u g h the p h t h a l a t e layer j u s t b e l o w the c e l l

pellet

and the upper p a r t c o n t a i n i n g

in a

gamma

the a d i p o c y t e s is c o u n t e d

counter.

In all c a s e s , the total r a d i o a c t i v i t y p r e s e n t in the m e d i u m is a s s e s s e d by p o o l i n g

incubation

the r e s i d u a l m e d i u m from

t u b e s a n d c o u n t i n g d u p l i c a t e 200 y l

several

aliquots.

I n s t e a d of c e n t r i f u g a t i o n , an a l t e r n a t i v e m e t h o d i n v o l v i n g tion is a l s o c o m m o n l y u s e d . (8).

T h i s was f i r s t d e s c r i b e d for

The f i l t e r s m u s t be of p o r e size s m a l l e r

t h a n the

filtraadipocytes

cells

and m e m b r a n e s w h i c h are being used and the f i l t r a t i o n m u s t be rapid.

A l i q u o t s of the i n c u b a t i o n m i x t u r e are r a p i d l y p o u r e d o n

the w e t t e d m e m b r a n e s a t t a c h e d to the M i l l i p o r e f i l t r a t i o n box. filters are w a s h e d w i t h large v o l u m e s of buffer f i l t e r s are d r i e d and c o u n t e d in the g a m m a Insulin binding

(5-10 m l ) .

counter.

to c e l l s is e x p r e s s e d per c e l l n u m b e r ,

is o b t a i n e d by c o u n t i n g a p p r o p r i a t e l y d i l u t e d c e l l containing

t r y p a n b l u e in a N e u b a u e r

which

suspensions

hemocytometer.

The

of the c e l l s is judge by the e x c l u s i o n of t r y p a n b l u e . ity s h o u l d be g r e a t e r cells

than 90%.

The

The

S i n c e d e a d c e l l s and

viability The

viabil-

broken

(which take up t r y p a n blue) r e l e a s e c y t o s o l i c and o t h e r

intracellular

p r o t e a s e s , w h i c h either d e g r a d e

receptor, decreasing

v i a b i l i t y to lower

m e a s u r e m e n t s are u s u a l l y n o t r e l i a b l e . p h y s i o l o g i c or p a t h o l o g i c s t a t e s the c e l l

(e.g., a d i p o c y t e s )

i n s u l i n or

Occasionally

binding

in c e r t a i n

(obesity for example)

is a l t e r e d .

the

than 90%, i n s u l i n

the size of

I n s u l i n binding

fat c e l l s c o r r e l a t e s w e l l w i t h insulin s e n s i t i v i t y w h e n

to h u m a n binding

is e x p r e s s e d per unit c e l l s u r f a c e t h a n w h e n e x p r e s s e d per

cell

186 number

(24,25).

Hence, insulin binding is also expressed per cell

volume instead of cell number.

Insulin binding to membranes is

usually expressed on a protein basis which is measured by the method of Lowry et al. (26). Recently, Whitcomb et al. (27,28) have described an in vivo method for studying insulin receptors on several tissues simultaneously. The method relies on several assumptions.

Experimentally the

procedure involves the measurement of labelled insulin distribution between plasma and interstitial space in the absence and presence The method can be applied to study

of excess unlabelled insulin.

other peptide hormone receptors also.

However, the method has not

been tested in other laboratories.

Properties of Insulin Receptor

When insulin binds to its receptor, the reaction is governed by several factors.

The receptor has certain characteristics which

separate it from acceptors.

In the latter instance, the attachment

of insulin is only a physical attachment. Specificity - The binding of insulin to its receptor is highly specific, i.e., the receptors which bind insulin do not bind unrelated peptides or other hormones and insulin will not bind to receptors of other hormones.

However, the insulin receptor will

bind closely related peptides such as proinsulin, several growth factors such as insulin-like growth factor I, non-suppressible insulin-like activity, somatomedins and multiple stimulating activity, as well as insulin from different species.

However,

binding is not as efficient as with true insulin of the appropriate species.

The biological activity will in general be proportional

to the binding.

Similarly, insulin will bind to specific receptors

for these growth factors but with much lower affinity and will produce relatively small biological (growth promotion) effects. Binding of several insulin derivatives to insulin receptors from

187 liver and fat c e l l s have b e e n s t u d i e d

(29-31).

The b i n d i n g

of these d e r i v a t i v e s c l o s e l y p a r a l l e l s the b i o l o g i c a l e x e r t e d by them in those tissues as d e m o n s t r a t e d Affinity - Affinity

in F i g u r e

1.

is the a v i d i t y w i t h w h i c h i n s u l i n b i n d s to a n d

d i s s o c i a t e s f r o m its r e c e p t o r s . tissue to t i s s u e .

affinity

activity

T h u s , a f f i n i t y w i l l vary

from

G e n e r a l l y , c l a s s i c a l t i s s u e s w h e r e i n s u l i n has a

m o r e p r o n o u n c e d e f f e c t w i l l h a v e a m u c h higher a f f i n i t y than t i s s u e s w h e r e the e f f e c t is o n l y m a r g i n a l .

Several

mathematical

m o d e l s h a v e b e e n d e v i s e d to a s s e s s a f f i n i t y and have b e e n by R o d b a r d

(32,33)

and o t h e r s

(34).

those

reviewed

A l l of them a s s u m e that a

steady s t a t e of b i n d i n g has b e e n r e a c h e d .

Affinity depends on

the rate c o n s t a n t s for a s s o c i a t i o n as w e l l as for

dissociation,

and is e x p e r i m e n t a l l y d e r i v e d from S c a t c h a r d p l o t s

(35).

However,

in m o s t i n s t a n c e s of i n s u l i n b i n d i n g , S c a t c h a r d p l o t s are

curvi-

linear and h e n c e a f f i n i t y c a n be c a l c u l a t e d m o r e a c c u r a t e l y

from

c o m p e t i t i o n - i n h i b i t i o n p l o t s as c o m p a r e d to S c a t c h a r d p l o t s

(36-

39).

Before constructing

s e c t i o n o n assay)

the p l o t s , n o n - s p e c i f i c b i n d i n g

is s u b t r a c t e d from all tubes and the

(see

maximum

b i n d i n g m e a s u r e d o n l y in the p r e s e n c e of a tracer q u a n t i t y of i n s u l i n is t a k e n as 100%

(Fig. 2).

from v a r i o u s s o u r c e s b e c a u s e : adipose, liver, muscle)

I125-

T h i s w i l l n o r m a l i z e the d a t a

i) b i n d i n g

is m u c h higher

to t a r g e t tissues

than to n o n - t a r g e t

(e.g.,

tissues,

ii) d i f f e r e n t n u m b e r s of c e l l s or c o n c e n t r a t i o n s of m e m b r a n e

are

used for v a r i o u s tissues and iii) c h a n g e s in m a x i m u m b i n d i n g

may

be o b s e r v e d in the same tissue under d i f f e r e n t p h y s i o l o g i c or pathologic conditions

(36-38).

as the a m o u n t of n a t i v e tracer

insulin.

The a f f i n i t y c a n then be d e t e r m i n e d

insulin r e q u i r e d to d i s p l a c e

50% of

The a f f i n i t y c a n thus be m e a s u r e d w h e t h e r

bound binding

is to w h o l e c e l l s or to i s o l a t e d cell c o m p o n e n t s such as m e m b r a n e s . N u m b e r - M o s t of the r e c e p t o r s for i n s u l i n are o n the cell and since s u r f a c e a r e a is f i n i t e , the n u m b e r of r e c e p t o r s

surface are

f i n i t e , e v e n t h o u g h the number of r e c e p t o r s c a n increase or

decrease

d e p e n d i n g o n the c o n d i t i o n s in w h i c h r e c e p t o r s are a s s e s s e d

(see

s e c t i o n o n r e g u l a t i o n of receptors) . of r e c e p t o r s

Experimentally,

the number

is a s s e s s e d from the i n t e r c e p t of S c a t c h a r d

E v e n t h o u g h S c a t c h a r d p l o t s for insulin are u s u a l l y

plots.

curvilinear,

188

e o -o

«

•iH c 4J • m > •H

U -H U

4) 4J J3 •o 1000 values from

452

The calculated inhibition constants are shown in Table 1.

Of

particular interest is the K^ for unlabeled AVP (0.8 nM), which is identical to the K^ for [ 3 H] AVP (0.8 nM) determined from saturation analysis in the same membrane preparation. This 3 finding supports the use of [ H] AVP in radioligand assays. Hormone Analogue Selection by Radioreceptor Assay g Since the synthesis of arginine -vasopressin (49) nearly thirty years ago, the development of synthetic analogues with altered bioactivity has been an area of active research.

Initially,

primary attention was focused upon the development of agonists which were either selective antidiuretics or pressor agents. Later, antagonists of the physiological actions of AVP were developed.

Excellent reviews on the development of these com-

pounds have been written (50,51). It should be noted that the synthesis of AVP analogues for the most part preceded the development of radioligand assays for vasopressin receptors.

Therefore, early workers relied upon

bioassays to characterize the specificity of synthetic analogues. Recently, synthetic vasopressin analogues have been used to validate radioligand assays for vasopressin receptors through comparison of inhibition constants in radioligand assays with published data from bioassays.

In addition, vasopressin

analogues have been used to confirm the presence of vasopressin receptor subtypes. Synthetic vasopressin analogues can be grouped into three classes. In the first category are the agonists and antagonists of the antidiuretic action of AVP. produced deamino AVP (dAVP).

Initially, deamination of AVP

This compound was shown to possess

453

enhanced antidiuretic activity (52).

In addition, substitution

of D-arginine in the number eight position of AVP was shown to g produce a peptide, D-arginine -vasopressin (DAVP), with an antidiuretic/pressor ratio of approximately 240 (52). 2

Deamina8

tion of DAVP results in a peptide, deamino -D-arginine -vasopressin (dDAVP), with an antidiuretic/pressor ratio of approximately 3000 (52) .

The first antidiuretic antagonists were developed

from a parent compound 4 1-(8-mercapto-S,B-cyclopentamethylene8 propionic acid)-valine ,-D-arginine -vasopressin (d(CH2)j- VDAVP), which rn vivo was not an antidiuretic antagonist; but, rather a weak antidiuretic agonist and a potent vasopressor antagonist (53).

Interestingly, d(CH2),-VDAVP was shown to be a potent

inhibitor of [^H]-LVP binding to membranes prepared from rat renal medullary membranes (54).

Substitution of either O-methyl-

tyrosine (Tyr(Me)) or O-ethyl-tyrosine (Tyr(Et)) into the number two position of d(CH 2 ) 5 VDAVP resulted in d(CH 2 ) 5 Tyr(Me)VDAVP and d(CH2)j- Tyr (Et) VDAVP, analogues, that when injected into rats produced transient antidiuresis followed by reversible antagonism of the antidiuretic effect of AVP (55).

However, both analogues

retained antivasopressor activity (56).

More recently, relati-

vely selective antidiuretic antagonists with diminished antivasopressor activity have been reported with substitutions of either D-Phe or D-Ile in position two (27).

Finally, deletion of Gly

(NH2) from 1-(6-mercapto-B,g-cyclopentamethylenepropionic acid) 2 4 8 -D-phenylalanine ,-isoleucine ,argimne -vasopressin (d(CH2),-[D2 4 Phe ,Ile ]AVP) resulted in an analogue that retained its antagonist properties (57).

The synthesis of a series of analogues

with substitution in position 9 have resulted in the most potent

454 antidiuretic antagonist, d(CH 2 ) 5 [D-Phe 2 ,He 4 ,Ala(NH 2 ) 2 9 AVP, to date (57). The second category of synthetic vasopressin analogues includes agonists and antagonists of the pressor action of AVP. Phenyla2 3 8 lanine -isoleucine -ornithine -vasopressin with a pressor/antidiuretic ratio of approximately 225 (58) is the most selective pressor analogue known. However, more attention has been devoted to developing antagonists of the pressor action of AVP. 8 4

The

first pressor antagonist was deaminovaline -D-arginine -vasopressin (dVDAVP) (59) .

Subsequently, substitution of either a

penicillamine group into position one giving 1-deaminopenicil4 8 lamine, valine ,D-arginine -vasopressin (dPVDAVP) (59) or a cyclopentamethylene group on the 6 carbon in position one giving 1-(6-mercapto-B,6-cyclopentamethylenepriopionic acid) valine4 , g -D-arginine -vasopressin (d (CH2) ,-VDAVP) (53) led to enhancement of antipressor activity.

Modifications of AVP have also produced

potent vasopressor antagonists.

Addition of either a pencilla-

mine group on a cyclopentamethylene group in position one in combination with substitution of O-methyltyrosine in position two have resulted in two of the commonly used pressor antagonists in radioligand assays. These compounds, 1-(6-mercapto-B,B-cyclopentamethylenepropionic acid), O-methyltyrosine 2 , arginine 8 vasopressin (d (CH2) j-Tyr (Me) AVP) (60) and deamino pencillamine*, 2

8

O-methyltyrosine ,arginine -vasopressin (dPTyr (Me)AVP) (61), are commercially available. The third category includes synthetic analogues which modulate learning and memory processes.

It should be noted that these

compounds have not been extensively tested in pressor and anti-

455

9 diuretic assays.

8

Initially, desglycinamide -lysine -vasopressin

(DG-LVP) was shown to be as potent as AVP in restoring passive avoidance behavior in homozygous Brattelboro rats (62).

DG-LVP

was also shown to be devoid of activity (63). 9 antidiuretic 8 Subsequently, desglycinamide -arginine -vasopressin (DG-AVP) has been shown to have many of the same central effects as AVP.

More

recently, a metabolite of vasopressin (AVP^_g) in the brain of rats has been shown to facilitate memory consolidation in a passive avoidance situation (64) . Relationship Between Receptor Binding and Biological Activity AVP affects a wide variety of physiological events in a number of target organs.

Thus far, in tissues in which AVP actions are

well characterized, AVP through interaction with V2 receptors has* been shown to alter intracellular cyclic AMP levels and through interaction with V^ receptors increase cytosolic calcium concentration.

In other tissues, particularly the central nervous

system, the relationship between binding of AVP to its receptor and biological activity has not been established.

A summary of

the vasopressin receptor subtypes, representative tissues and cells in which they have been identified and intracellular second messengers are listed in Table 2. Table 2.

Vasopressin Receptor Subtypes Tissue

Intracellular Second Messenger

V.

Hepatocytes, vascular smooth muscle

+ Cytoplasmic Calcium

V2

Renal tubular epithelium

+ Cyclic AMP

Other

CNS, Adenohypophysis

not established

456 V 2 receptors The following evidence supports the hypothesis that binding sites for vasopressin in kidney membrane fractions are involved in adenylate cyclase activation.

First, a large number of synthetic

vasopressin analogues, both agonist and antagonists, have been shown to have similar rank orders of potency in either receptor assays or adenylate cyclase assays (29-32,35).

Second, in the

rat kidney the ontogenical development of the vasopressin receptor parallels the development of vasopressin-sensitive adenylate cyclase activity

(33) .

Third, in the rat kidney the distribution

of vasopressin receptors (65) is similar to the distribution of vasopressin-sensitive adenylate cyclase activity

(66).

It is likely that the molecular mechanism by which vasopressin elevates cyclic AMP levels in tubular epithelial cells is identical to that of other hormone receptors coupled to adenylate cyclase.

Detailed discussions of this topic have been published

(67,68).

In such systems, the hormone receptor is coupled to the

catalytic subunit (C) via a heterotrimeric protein termed the guanine nucleotide regulatory subunit (Ng).

N g belongs to a

family of related guanine nucleotide binding proteins; N g , N^ and transducin

(67).

Purified N

s

consists of an a subunit (M = r

45,000); a B subunit (Mr=35,000) and a y subunit (Mr=5,000).

The

a subunit of N g binds guanine nucleotides and possesses GTPase activity.

The 6 subunit of N g is apparently identical to the 6

subunit of N^ and furthermore is similiar to the 6 subunit of transducin. at this time.

The precise function of the y subunit is not clear

457 Through interaction with the a subunit of N g , guanine nucleotides have been shown to play two important regulatory roles in hormone-mediated activation of adenylate cyclase (69).

First,

guanine nucleotides appear to be obligatory for hormonal stimulation of adenylate cyclase.

This requirement has been demonstra-

ted for the V^ receptor in rat renal medullary membranes (42) . Second, guanine nucleotides appear to reduce the affinity of 3 hormones for their receptors.

The binding of [ H] LVP to human

(35) and rat (70) renal medullary membranes appears to be modulated by guanine nucleotides.

In human renal medullary membranes,

the addition of 10 uM guanylyl imidodiphosphate (Gpp(NH)p), a non hydrolyzable guanine 3 nucleotide, increased the apparent dissociation constant of [ H] LVP by a factor of eight (35).

In rat

renal medullary membranes, both guanosine-5'-triphosphate and Gpp(NH)p decreased binding of [^H] LVP with the same potency (70).

This effect was specific for guanine nucleotides since

adenosine-5'-triphosphate reduced [^H] LVP at concentrations approximately 1000-fold higher than that required for either of the guanine nucleotides (70) . Similarly, the role of guanine nucleotides in the binding of AVP to rat renal medullary membranes has been examined (71) .

Equili-

brium binding studies analyzed by the iterative curve fitting 3 program LIGAND (48) revealed an interaction of [ H] AVP with a single class of binding sites with a dissociation constant of 1.4+0.2 nM and a binding site concentration of 201±37 fmol/mg protein.

With the addition of 100 yM Gpp(NH)p the binding site

concentration decreased to 151+36 fmol/mg protein with no change in receptor affinity. Furthermore, Gpp(NH)p accelerated 3 dissociation of [ H] AVP from the receptor.

458

Despite the suggestive evidence cited above, the definitive elucidation of the mechanism by which AVP increases cyclic AMP levels will require purification of each of the membrane components and reconstitution of the purified components into phospholipid vesicles. Studies in which

receptor occupancy has been correlated with

adenylate cyclase activity indicate that spare in the renal medulla.

receptors exist

Half maximal adenylate cyclase activation

by vasopressin is obtained in porcine (72) , bovine (31) , rat (34) and human (35) renal medulla with less than half receptor occupancy.

The presence of spare

receptors may explain why

synthetic vasopressin analogues which act as partial agonists in adenylate cyclase assays, act as full agonists in antidiuretic assays (34). V^ receptors The physiological actions which result from the interaction of AVP with the Vj receptor do not appear to be mediated by cyclic AMP.

AVP does not increase cyclic AMP levels in either liver

(18) or rat aorta (73), tissues with demonstrable V^ receptors. In addition, AVP activation of phosphorylase in the liver is not associated with increased activity of cyclic AMP-dependent protein kinase (21,74).

Thus far, in hepatocytes, vascular

smooth muscle and adrenal glomerulosa cells binding of AVP to V^ receptors has been associated with mobilization of intracellular calcium. The early evidence which linked increased cytoplasmic calcium levels with AVP stimulation of hepatocytes has been discussed (75).

An area of intense interest has been investigation of the

459 coupling mechanisms which link AVP-V^ receptor complex to intracellular calcium mobilization.

Kirk et al. (76) first demon32

strated that incorporation of [

P] into phosphatidylinositol was Earlier, it was suggested that

stimulated by AVP in hepatocytes.

changes in phosphatidylinositol metabolism were involved in hormonally-mediated mobilization of intracellular calcium

(77).

Currently, it is known that AVP stimulates breakdown of D-myoinositol-4,5-bisphosphate

(PIP2) by a phosphodiesterase producing

the biologically active metabolites, D-myoinositol-1,4,5-trisphosphate

(IP3) and 1,2-diacylglycerol

(DG) (78-80).

IP 3 has

been shown to stimulate release of calcium from saponin-permeabilized hepatocytes (81,82).

Thus IP^ may be the intracellular

mediator of AVP-induced calcium mobilization in hepatocytes.

DG

has been shown to activate protein kinase C (83,84), an action also produced by the phorbol esters (83,85).

The role of protein

kinase C in AVP stimulated calcium mobilization remains to be completely elucidated.

Fain et al. (86) have shown that DG and

calcium synergistically increase phosphorylase activity in hepatocytes.

However, Cooper et al. (87) have shown that pre-

treatment of hepatocytes with either synthetic DG or 4 6-phorbol12-myristate-13-acetate results in reduced calcium mobilization induced by AVP. For some time, intracellular mobilization of calcium has been recognized as an essential event for vascular smooth muscle contraction

(88) .

Contraction of vascular smooth muscle is

reduced in calcium free medium (89,90).

Elevated cytoplasmic

+2 calcium is thought to activate Ca

-calmodulm-dependent myosin

light chain kinase which ultimately results in smooth muscle

460

contraction (91).

Furthermore, as is the case in hepatocytes,

AVP has been shown to stimulate incorporation of [32P] into phosphatidylinositol in rat thoracic aorta (92) . More recently, +2 using the fluorescent Ca

indicator Quin2, AVP was shown to

elevate cytosolic calicum levels in smooth muscle cells obtained from rat aorta (93).

In addition, AVP caused the accumulation of

inositol phosphates, although the concentration of IP^ was not significantly increased by concentrations of AVP up to 100 nM (93) . ACTH and angiotensin II are the major regulators of aldosterone secretion by the zona glomerulosa of the adrenal cortex. However, AVP has been shown to increase circulating levels of aldosterone (94), potentiate the effect of low doses of ACTH on aldosterone secretion (95) and stimulate mitotic activity of rat glomerulosa cells in culture' (96).

Using radioligand assays, the

V^ receptor subtype has been identified in rat adrenal glomerulosa cells (97).

Recently, the mechanism by which AVP stimulates

aldosterone secretion from glomerulosa cells has been investigated.

AVP stimulated the formation of IP3/ inositol-1,4-bis-

phosphate manner (97,98).

an
c 01 01 c Ol O " O s - 0J iO í - - E o í ai a » 4-1 O o "

O

crt

u'r— o ai o. (/>

tA >>

•o ai c/> IO ai 4o ai a

XJ i10 .c U 4-1 10 c_> C/I

il a

IO c IO ai o> c •0 .E o

ai ai 00 IO ai su ai a

o z

704 concentration although both castration and administration of an estrogenic agent appear to down-regulate the receptors. Thus, the multiple factors that may affect the regulation of-Angll receptors make an understanding of this process difficult.

Further complicating an under-

standing is the potential interaction of two or more of the factors in the regulation.

It is clear that additional study is required to understand the factors

and processes involved in the regulation of Angll receptors in the various organs as well as their roles in the physiological processes initiated and maintained by these organs.

References 1.

Skeggs, L.T., J.R. Kahn, N.P. Shunway.

2.

Elliot, D.F., W.S. Peart.

3.

Page, I.H., F.M. Bumpus.

4.

Peart, W.S.

5.

De Bono, E., G. Lee, F.R. Mottram, G.W. Pickering, J.J. Brown, H. Keen, W.S. Peart, P. Sanderson. 1963. Clinical Sci. 25, 123.

6.

Haddy, F.J., J.I. Molnar, C.W. Borden, E.C. Texter. 25, 239.

7.

Feldberg, W., G.P. Lewis.

8.

Zimmerman, B.G., S.K. Gomez, J.C. Liao.

9.

Boadle, M.C., J. Hughes, R.H. Roth.

10.

Laragh, J.H., M. Angers, W.G. Kelly, S. Lieberman. Assoc. 174, 234.

11.

Bonjour, J.P., R.L. Malvin.

12.

Munday, K.A., B.J. Parsons, J.A. Poat.

13.

Ganten, D., J.S. Hutchinson, P. Schelling, U. Ganten, H. Fischer. Clin. Exp. Pharmacol. Physiol. 1°3.

14.

Phillips, M.I.

15.

Ganten, D., K. Hermann, C. Bayer, T. Unger, R.E. Lang. (Wash., DC) 22!, 8 6 9 •

16.

Ganong, W.F.

1965.

1961.

J. Exp. Med. 103, 301.

Nature (Lond.) 177, 527. Physiol. Rev. 41, 331.

Pharmacol. Rev. 17, 145.

1978.

1984.

1956.

1956.

1964.

1970.

1962.

Circulation

J. Physiol. 171_, 98. 1972.

1979.

Fed. Proc. 31, 1344.

Nature (Lond.) 222, 987. 1960.

J. Am. Med.

Am. J. Physiol. 218, 791. 1972.

J. Physiol. 224, 195. 1971.

Neuroendocrinology 25, 354.

Ann. Rev. Physiol. 46, 17.

1982.

Science

705 17.

Bickerton, R.K., J . P . Buckley. 834.

1961.

18.

Epstein, A.N., J . T . Fitzsimons, B.J. R o l l s . 210, 457.

19.

Buggy, J . , A.E. F i s h e r .

20.

Mouw, D., J . Bonjour, R.L. Malvin, A. Vander. 239.

21.

Ganong, W.F., J . Shinsako, I . Reid, L.C. K e i l , D.L. Hoffman. N. Y. Acad. S e i . 394, 619.

22.

Ganten, D., K. Fuxe, M . I . P h i l l i p s , J . F . E . Mann. 1978. In: Frontiers in Neuroendocrine ogy 5 (W.F. Ganong and L. M a r t i n i , L . , e d s . ) . Raven P r e s s , New York, p. 61.

23.

Lin, S . - Y . , T.L. Goodfriend.

24.

Douglas, J . G . , G.P. Brown, C. White.

25.

Catt, K . J . , F.A.0. Mendelsohn, M.A. M i l l a n , G. Aguilera. vasc. Pharmacol. 5575.

26.

Baudouin, M., P. Meyer, M. Worcel. 42, 434.

27.

Mukherjee, A . , P.V. K u l k a r n i , Z. Haghani, J . L . Sutko. Biophys. Res. Commun. 105, 575.

28.

Mendelsohn, F.A.0.

29.

Bennett, J . P . , S.H. Snyder.

30.

S i r e t t , N.E., A.S. McLean, J . J . Bray, J . I . Hubbard. 122, 299.

31.

Harding, J.W., L.P. Stone, J.W. Wright.

32.

Khosla, M.C., R.R. Smeby, F.M. Bumpus. 1974. In: Handbook of Experimental Pharmacology 37 ( I . H . Page and F.M. Bumpus, e d s . ) . Springer-Verlag, New York, p. 126.

33.

Mendelsohn, F . A . 0 . , R. Q u i r i o n , J.M. Saavedra, G. Aguilera, K.J. Catt. 1984. Proc. Natl. Acad. S e i . U.S.A. 81, 1575.

34.

Mendelsohn, F.A.0., G. A g u i l e r a , J.M. Saavedra, R. Q u i r i o n , K.J. Catt. 1983. C l i n . Exp. Hypertension [A] 5, 1081.

35.

Healy, D.P., M.P. P r i n t z .

36.

Gehlert, D.R., R.C. Speth, J.K. Wamsley. 145.

37.

Glossman, H., A. Baukal, G. Aguilera, K.J. Catt. Enzymology 109, 110.

1974.

1985.

Proc. Soc. Exp. B i o l . N. Y. 106, 1969.

J. Physiol.

(Lond.)

Nature (Lond.) 250, 733.

1970.

1971.

An. J . P h y s i o l . 220, 1982.

Ann.

Am. J . P h y s i o l . 218, 1319. 1984.

1971.

Metabolism 33, 685. 1984.

J . Cardio-

Biochem. Biophys. Res. Commun. 1982.

Biochem.

J . Hypertension 3, 307. 1976.

1985.

J . B i o l . Chem. 251,

1981.

74

1977.

23. Brain Res.

Brain Res. 205, 265.

Fed. Proc. 4 4 ( 3 ) , 1808. 1984.

Europ. J . Pharmacol. 98, 1985.

Methods in

706 38.

Devynck, M.A., P. Meyer.

1976.

Am. J. Med. 61, 758.

39.

Glossman, H., A. Baukal, K.J. Catt.

40.

Gunther, S., M.A. Gimbrone, R.W. Alexander. 278.

41.

Douglas, J.G., P. Bartley, T. Kondo, K.J. Catt. 1921.

42.

Campanile, C.P., J.K. Crane, M.J. Peach, J.C. Garrison. Chen. 257, 4951.

43.

Glossman, H., A. Baukal, K.J. Catt.

1974.

J. Biol. Chem. 249, 664.

44.

Glossman, H., A. Baukal, K.J. Catt.

1974.

Science 185, 281.

45.

Douglas, J., G. Aguilera, T. Kondo, K.J. Catt. 685.

46.

Douglas, J., S. Saltman, P. Fredlund, T. Kondo, K.J. Catt. tion Res. 38, 11-108.

47.

Le Morvan, P., D. Palaic.

48.

Penit, J., M. Faure, S. Jard.

49.

Fregly, M.J., N.E. Rowland, C. Sumners, D.B. Gordon. 338, 115.

50.

Wilson, K.M., C. Sumners, S. Hathaway, M.J. Fregly. (in press).

51.

Raizada, M.K., J.W. Yang, M.I. Phillips, R.E. Fellows. 297, 343.

52.

Sumners, C., M.K. Raizada. C502.

53.

Raizada, M.K., T.F. Muther, C. Sumners. Physiol. 16), C354.

54.

Cox, H.M., K.A. Munday, J.A. Poat.

1984.

Brit. J. Pharmacol. 82, 891.

55.

Cox, H.M., J.A. Poat, K.A. Munday.

1983.

Biochem. Pharmacol. 32, 3601.

56.

Sernia, C., L. Sinton, W.G. Thomas, W. Pascoe. 103.

1985.

57.

Schiffrin, E.L., F.S. Thome, J. Genest.

Hypertension 6_, 682.

58.

Speth, R.C., S.I. Harik.

59.

Weyhenmeyer, J.A., C.-J. Hwang.

60.

Chen, F.-C. M., M.P. Printz.

1975.

1974.

1980.

Circulation Res. £7,

1978.

1978.

Endocrinology 102, 1982.

J. Biol.

Endocrinology 102, 1976.

Circula-

J. Pharmacol. Exp. Ther. 195, 167.

1983.

1984.

1985.

J. Biol. Chem. 249, 825.

Am. J. Physiol. 244» £72. 1985. 1986.

Brain Res. Brain Res.

1981.

Brain Res.

Am. J. Physiol. 246 (Cell Physiol. 15), 1984.

1984.

Am. J. Physiol. 247 (Cell

J. Endocrinol. 106,

Proc. Natl. Acad. Sci. U.S.A. 82, 6340. 1985.

1983.

Brain Res. Bulletin 14, 409.

Endocrinology 113, 1503.

707 61.

Capponi, A.M., C.A. Favrod-Coune, R.C. G a i H a r d , A.F. Müller. Endocrinology 110, 1043.

62.

Moore, T.J., G.H. Williams.

63.

Rouzaire-Dubois, B., M.A. Devynck, E. Chevillotte, P. Meyer. F.E.B.S. Letters 55, 168.

64.

Capponi, A., K.J. Catt.

65.

Healy, D.P., M.P. Printz.

66.

Israel, A., F.M. Correa, M. Niwa, J.M. Saavedra. Hypertens. [A] 6(10-11), 1761.

67.

Heidrich, H.G., R. Kinne, E. Kinne-Saffran, K. Hannig. Biol. 54, 232.

68.

Wei, J.W., R.A. Janis, E.E. Daniel.

69.

Harik, S.J., G.H. Doull, A.P.K. Dick. 5, 156.

70.

Brecher, P.I., H.Y. Pyun, A.V. Chobanian.

71.

Devynck, M.A., M.G. Pernollet, P. Meyer, S. Ferraandjian, P. Fromageoat, F.M. Bumpus. 1974. Nature (Lond.) 249, 67.

72.

Forget, G., S. Heisler.

73.

Capponi, A.M., K.J. Catt.

74.

Paglin, S., J.D. Jamieson.

1982.

Proc. Natl. Acad. Sei. U.S.A. 79, 3739.

75.

Chang, R.S.L., V.J. Lotti.

1981.

Life Sei. 29, 613.

76.

Birabeau, M.A., A.M. Capponi, M.B. Valloton. crinol . 37, 181.

77.

Imai, N., F. Masugi, T. Ogihara, Y. Kumahara, S. Hirose, F. Akiyama, K. Murakami. 1984. Biochem. Biophys. Res. Commun. 123, 452.

78.

Sen, I., K.F. Jim, R.L. Soffer.

79.

Sen, I., H.G. Bull, R.L. Soffer. 1679.

80.

Beaufils, M., J. Sraer, C. Lepreux, R. Ardaillou. 230, 1187.

81.

Aguilera, G., K. Catt.

82.

Reid, W.D., J.H. Laragh.

83.

Hollenberg, N.K., H.S. Solomon, D.F. Adams, H.L. Adams, J.P. Merrill. 1972. Circ. Res. 31, 750.

1981.

1979.

1979.

1975.

Neuroscience Lett. 44, 167.

1976.

1984.

Clin. Exp. 1972.

J. Cell.

Blood Vessels 13, 279.

1985.

J. Cereb. Blood Flow Metab.

1974.

Endocrinology 95, 1026.

Experientia 35, 125.

1980.

1965.

Circulation Res. 51, 314.

J. Biol. Chem. 254, 5120.

1984.

1981.

1982.

J. Biol. Chem. 225, 12081.

1983. 1984.

1984.

Molec. Cell. Endo-

Europ. J. Biochem. 136, 41. Proc. Natl. Acad. Sei. U.S.A. 81, 1976.

Am. J. Physiol.

Circ. Res. 49, 751. Proc. Soc. Exp. Biol. Med. 120, 26.

708 84.

Kaplan, N.M., J.G. Silah.

1964.

85.

Healy, J.K., J.B. Suszkiw, V.W. Dennis, G.E. Schreiner. 329.

86.

Swales, J.D., J.D. Tange, H. Thurston.

87.

Schiffrin, E.L., F.S. Thome., J. Genest. V16.

1983.

Hypertension ^(Suppl. 5),

88.

Schiffrin, E.L., D.J. Franks, J. Genest.

1983.

Clin. Res. 31^, 688A.

89.

Berecek, K.H., D.F. Bohr.

90.

Marks, A.D., D.B. Marks, Y.N. Kim, J. Moctezuma, E.V. Adlin, B.J. Channick. 1978. Circ. Res. 42, 864.

91.

Campanile, C.P., T.L. Goodfriend.

92.

Schiffrin, E.L., J. Gutkowska, J. Genest. H608.

93.

Schiffrin, E.L., D.J. Franks.

94.

Carriere, P.D., A. DeLean, J. Gutkowska, E.L. Schiffrin, J. Genest, M. Cantin. 1984. Fed. Proc. 43, 1038.

95.

Williams, G.H., N.K. Hollenberg, L.M. Braley. 1343.

96.

Samwer, K.F., M. Schreiber, M. Molzahn, W. Oelkers. Arch. 346, 307.

97.

Barraclough, M.A., N.F. Jones, C.D. Marsden, B.C. Bradford. Experientia 23, 553.

98.

Wright, G.B., R.W. Alexander, L.S. Ekstein, M.A. Gimbrone, Jr. Circ. Res. 50, 462.

99.

Dawson-Hughes, B.F., J.T. Moore, R.G. Dluhy, N.K. Hollenberg, G.H. Williams. 1981. Clin. Sei. 61_, 527.

1978.

J. Clin. Invest. 43, 659.

1975.

1969.

Nephron 3,

Circ. Res. 37., 96.

Circ. Res. 42, 764.

1981.

1984.

Steroids 37, 681. 1984.

Am. J. Physiol. 246,

Fed. Proc. 43, 1037.

100. Meyer, P., A. Papadimitriou, M. Worcel.

1974.

1976.

Endocrinology 98, 1974.

Pfluger's 1967. 1982.

Br. J. Pharmacol. 51, 435.

101. Bean, B.L., J.J. Brown, J. Casals-Stenzel, R. Fräser, A.F. Lever, J.A. Miliar, J.J. Morton, B. Petch, A.J.G. Reigger, J.I.S. Robertson, M. Tree. 1979. Circ. Res. 44, 452. 102. Johnson, C.I., A.D. Jose.

1963.

J. Clin. Invest. 42, 1411.

103. Deheneffe, J., M. Cuesta, J.D. Briggs, J.J. Brown, R. Fräser, A.F. Lever, J.J. Morton, J.I.S. Robertson, M. Tree. 1976. Circ. Res. 39, 183. 104. Cowley, A.W., T.E. Lohmeier.

1978.

Circ. Res. 42, 503.

105. Speth, R.C., R. Singh, R.R. Smeby, C.M. Ferrario, A. Husain. Neuroendocrinology 38, 387.

1984.

106. Cole, R . E . , H.L. Blakesley, K.A. Graci, E.D. F r ö h l i c h , A.A. MacPhee. Brain Res. 190, 272.

1980

107. S i r e t t , N.E., V . l . Hubbard, J . J . Bray. 1982. In: The Renin Angiotensin System in the Brain (D. Ganten, M. P r i n t z , M . I . P h i l l i p s and B.S. Scholkens, e d s . ) . Springer-Verlag, B e r l i n , pp. 233. 108. Mann, J . F . E . , E.L. S c h i f f r i n , P.W. S c h i l l e r , W. Rascher, R. Boucher, J . Genest. 1980. Hypertension 2, 437. 109. Ashida, T . , Y. Ohuchi, T. S a i t o , Y. Yazaki.

1982.

110. Kapsha, J.M., L.C. K e i l , P.A. Klase, W.B. Severs. 25.

Jap. C i r c . J . 46, 1328 1979.

Pharmacology 18,

111. Davis, J . O . , P.M. Hartroft, E.0. T i t u s , C.C.J. Carpenter, C.R. Ayers, H.E. Spiegel. 1962. J . C l i n . I n v e s t . 41_, 378. 112. Singh, R., A. Husain, C.M. F e r r a r i o , R.C. Speth. 25, 297. 113. Slack, B . L . , J.M. Ledingham.

1976.

114. Mizuno, K., S. Hata, S. Fukuchi.

1982.

The P h y s i o l o g i s t

C l i n . S e i . Mol. Med. 50, 285.

1981.

C l i n . S e i . 61_, 249.

115. Bianchi, G., J . J . Brown, A.F. Lever, J . I . S . Robertson, N. Roth. C l i n . S e i . 34, 303. 116. Blair-West, J . R . , J . S . McKenzie. 117. Kinson, G.A., B. Singer.

1968.

1966.

1968.

Experientia 22, 291.

Endocrinology 83, 1108.

118. Mendelsohn, F.A.O., C . I . Johnston, A.E. Doyle, B.A. Scoggins, D.A. Denton, J . P . Coghlan. 1972. C i r c . Res. 31, 728. 119. S c h i f f r i n , E . L . , D.J. Franks, J . Gutkowska. macol . 63, 1522. 120. Douglas, J . G . , J . P . Brown. 121. Douglas, J . G . , K.J. Catt.

1982. 1976.

1985.

Can. J . P h y s i o l . Phar-

Endocrinology 111, 988. J. C l i n . I n v e s t . 58, 834.

122. Strewler, G.J., K.J. H i n r i c h s , L.R. Guiod, N.K. Hollenberg. Res. 31, 758. 123. S i r e t t , N.E., S.N. Thornton, J . I . Hubbard.

1979.

1972.

Circ.

Brain Res. 166, 139.

124. Van Houten, M., E.L. S c h i f f r i n , J . F . E . Mann, B . I . Posner, R. Boucher. 1980. Brain Res. 186, 480. 125. Molteni, A . , K.B. M u l l i s , R.M. Zakheim, L. M a t t i o l i . 35, 569.

1976.

126. Kurihara, H., A. Gregory, P. Dewailly, R. Boucher, J . Genest. Tokohu J . Exp. Med. 109, 19. 127. Healy, D.P., M.P. P r i n t z .

1984.

Fed. Proc. 43, 653.

Lab. Invest. 1973.

710 128. Gipe, D., S. Slwka, S. H a r r i s , P. Hornsby, M. P r i n t z . 43, 653.

1984.

Fed. Proc.

129. Mann, J . F . E . , P.W. S c h i l l e r , E.L. S c h i f f r i n , R. Boucher, J . Genest. Am. J . P h y s i o l . 241, R124. 130. Van Houten, M., M.L. Mangiapane, I . A . Reid, W.F. Ganong. science 10, 1421. 131. Huang, B . , R.L. Malvin, R.J. Grekin. 132. Healy, D.P., M.P. P r i n t z .

1984.

133. Bennett, J . P . , J r . , S.H. Snyder.

1984.

1983.

1981.

Neuro-

Fed. Proc. 43, 721.

Hypertension 6(Suppl. I ) , 1130. 1980.

134. A g u i l e r a , G., R.L. Hauger, K.J. Catt. 75, 975.

Europ. J . Pharmacol. 1978.

11.

Proc. Natl. Acad. S c i . USA

135. Catt, K . J . , G. Aguilera, A. Capponi, K. F u j i t a , A. S c h i r a r , J . Fakunding. 1979. J . Endocr. 81, 37P. 136. Hauger, R . L . , G. A g u i l e r a , K.J. Catt.

1978.

Nature 271_, 176.

137. Hollenberg, N.K., W.R. Chenitz, D.F. Adams, G.H. Williams. I n v e s t . 54, 34.

1974.

J. Clin.

138. Oelkers, W., J . J . Brown, R. Fraser, A.F. Lever, J . J . Morton, J . I . S . Robertson. 1974. C i r c . Res. 34, 69. 139. Mizuno, K . , S. Shigetomi, J . Matsui, S. Fukuchi. 839.

1981.

Jap. Heart J . 22,

140. Mendelsohn, F.A.O., J . Csicsmann, J . S . Hutchinson, R. Di Nicolantonio, Y. Takata. 1982. Hypertension 4 , 590. 141. Saltman, S . , A Baukal, S. Waters, F.M. Bumpus, K.J. Catt. ology 97^, 275. 142. A g u i l e r a , G., A. S c h i r a r , A. Baukal, K.J. Catt. (Suppl. I ) , 1118.

1980.

143. Dluhy, R.D., L. Axelrod, R.H. Underwood, G.H. Williams. I n v e s t . 51, 1950. 144. McCaa, R . E . , C.S. McCaa, A.C. Guyton.

1975.

147. Vander, A.J.

1979. 1970.

Endocrin-

C i r c . Res. 46 1972.

J. Clin.

C i r c . Res. 36(Suppl. I ) , 57.

145. Sealey, J . E . , I . Clark, M.B. B u l l , J.H. Laragh. 49, 2119. 146. Douglas, J.G.

1975.

1970.

J. Clin.

Invest.

Am. J . P h y s i o l . 237, E519. Am. J . P h y s i o l . 219, 455.

148. Douglas, J.G., J . K . L i t o w i t z .

1978.

C l i n . Res. 26, 304A.

149. Hollenberg, N.K., G. Williams, B. Burger, I . Hooshmond. Mol. Med. 49, 527.

1975.

Clin.

Sci.

711 150. Fredlund, P . , S. Saltman, T. Kondo, J . Douglas, K.J. Catt. Endocrinology 100, 481. 151. Deane, H.W., J.H. Shaw, R.O. Greep. 152. Jonklaas, J . , J . Buggy.

1985.

1948.

Endocrinology 43, 133.

Brain Res. 326, 239.

153. Chen, F.M., R. Hawkins, M.P. P r i n t z .

1982.

Exp. Brain Res. Suppl. 4 , 157.

154. P r i n t z , M.P., R.L. Hawkins, C.J. W a l l i s , F.M. Chen. 155. Thomas, W.G., C. Sernia.

1977.

1985.

1983.

Chest 83.

3

°8.

Brain Res. 34E[, 54.

156. Monney, M., P.A. Schlegel, H.R. Brunner. and Pract. A5, 1735.

1983.

C l i n . Exper. Hyper.-Theory

157. Blair-West, J . R . , J . P . Coghlan, D.A. Denton, J . R . Goding, J . A . Munro, R.D. Wright. 1963. Aust. J . Exp. B i o l . Med. S c i . 41, 369. 158. P a l l e r , M.S., J.G. Douglas, S . L . Linas.

1984.

159. Rosenmann, R.H., S.C. Freed, M. Friedman.

J . C l i n . Invest. 73, 79.

1952.

160. Galvez, O.G., W.H. Bay, B.W. Roberts, T.F. F e r r i s . (Suppl. I ) , 11. 161. Campbell, W.B., J.M. Schmitz.

1978.

C i r c u l a t i o n 5^, 412. 1977.

C i r c . Res. 40

Endocrinology 103, 2098.

162. Adamick, R . , M.E. Gold, S. Hayes, R. Coleman, J.T. McCreary, S. S a b a t i n i , A.L. Arruda, N.A. Kurtzman. 1981. C i r c . Res. 49, 932. 163. B l a i r - W e s t , J . R . , J . P . Coghlan, D.A. Denton, J.R. Goding, M. Wintour, R.D. Wright. 1965. C i r c . Res. 17, 386. 164. Laragh, J . H . , P . J . Cannon, R.P. Ames.

1964.

Can. Med. Assoc. J . 90, 248.

165. Ames, R . P . , A.J. Borkowski, A.M. S i c i n s k i , J.M. Laragh. I n v e s t . 44, 1171.

1965.

J. Clin.

166. Oelkers, W., M. Schoneshofer, G. Schultze, J . J . Brown, R. Fraser, J . J . Morton, A.F. Lever, J . I . S . Robertson. 1975. C i r c . Res. (Suppl. I) 36, 37, 149. 167. Peach, M.J.

1971.

C i r c . Res. (Suppl. I I ) 28, 29, I I 1 0 7 .

168. Douglas, J . G . , M. Michailov, M.C. Khosla, F.M. Bumpus. ology 106, 120. 169. F r e g l y , M.J., W.G. Luttge. 1982. Test, E l s e v i e r , New York, p. 131. 170. Peach, M.J.

1977.

1980.

Endocrin-

Human Endocrinology, An I n t e r a c t i v e

P h y s i o l . Rev. 57, 313.

171. Saltman, S . , P. Fredlund, K.J. Catt.

1976.

Endocrinology 98, 894.

172. Douglas, J . G . , M. M i c h a i l o v , M.C. Khosla, F.M. Bumpus. ogy 104, 71.

1979.

Endocrinol-

173. Douglas, J.G., M.C. Khosla, F.M. Bumpus.

1985.

Endocrinology 11JL, 1598.

174. Brecher, P., M. Tabacchi, N.Y. Pyun, A.V. Chobanian. Biophys. Res. Comm. 54, 1511. 175. Aguilera, G., C.L. Hyde, K.J. Catt.

1982.

1973.

Endocrinology l j U , 1045.

176. Hauger, R.L., G. Aguilera, A. Baukai, K.J. Catt. crinol . 25, 203.

1982.

Mol. C e l l . Endo-

177. Mukherjee, A., P. Kulkarni, S.M. McCann, A. Negro-Vilar. ology 110, 665. 178. Ganong, W.F., A.T. Boryczka. 1230.

1967.

1985.

180. Sraer, J.D., J . Sraer, R* Ardaillou, 0. Mimoune. 241.

1974.

Kidney Int. 6.,

1980.

Endocrinology

Endocrinology 111, 1830.

183. Brown, C.A., R.M. Zusman, E. Haber.

1980.

Circ. Res. 46, 802.

184. Levens, N.R., M.J. Peach, V.E. Darracot, J r . , R.M. Carey. ology 108, 318. 185. Carroll, J . E . , T.L. Goodrfriend.

1984.

186. Sumners, C., L.L. Watkins, M.K. Raizada:

1981.

J . Neurochem. (in press).

188. Gehlert, D.R., R.C. Speth, D.P. Healy, J.K. Wamsley. 1565.

1980. 1984.

189. Devynck, M.A., B. Rouzaire-Dubois, E. Chevillotte, P. Meyer. J . Pharmacol. 40, 27. 190. Moore, A.F., M.M. Hall, P.A. Khairallah. 101.

192. Moore, G.J.

1980.

1980.

Endocrin-

Science 224, 1009.

187. Baxter, C.R., J.S. Horvath, G.G. Duggin, D.J. T i l l e r . 106, 995.

191. Moore, G.J., E.M. Ko.

Endocrin-

Endocrinology 116,

181. Brown, G.P., J.G. Douglas, J.K. Krontiris-Litowitz. 106, 1923. 1982.

1982.

Proc. Soc. Exper. B i o l . Med. 124,

179. Healy, D.P., A.R. Maciejewski, M.P. Printz. 1221.

182. Brown, G.P., J.G. Douglas.

Biochem.

1976.

Endocrinology Life Sei. 34, 1976.

Eur.

Eur. J . Pharmacol. 39,

Pharmacology 20, 36.

Can. J . Physiol. Pharmacol. 58, 1373.

193. Gabelman, E.H., P.A. Rondell. 194. Strouder, D.A., R.L. Wathen.

1966. 1972.

Circ. Res. 18, 705. Proc. Soc. Exper. Biol. Med. 141, 548.

195. Devynck, M.A., M.G. Pernollet, P.G. Matthews, G.J. MacDonald, R.S. Raisman, P. Meyer. 1979. J . Cardiovasc. Pharmacol. 163.

713 196. Perno!1 et, M.G., M.A. Devynck, P.G. Matthews, P. Meyer. Pharmacol. 43, 361.

1977.

Eur. J .

197. Devynck, M.A., M.G. P e r n o l l e t , G.J. MacDonald, P.G. Matthews, R.S. Raisman, P. Meyer. 1978. C l i n . S c i . Mol. Med. 55, 1715. 198. Douglas, J.G.

1982.

J . C l i n . Invest. Ctf, 1171.

199. Douglas, J.G.

1980.

Am. J . P h y s i o l . 239, E317.

200. S c h i r a r , A., A. Capponi, K.J. Catt.

1980.

Endocrinology 106, 5.

201. K h a i r a l l a h , P.A. 1971. In: Kidney Hormones (J.W. F i s h e r , ed.). Academic Press, New York, p. 129. 202. Douglas, J . , J . Hansen, K.J. Catt.

1978.

Endocrinology 103, 60.

Author Index

R. A. ADLER

63

p. B. KAPLOWITZ

519

A. ALECOZAY

615

J. L. KIRKLAND

335

S. J. BHATHENA

179

R. F. KLEIN

481

J. M. BURNS

615

W. W. LEAVITT

615

T. T. CHEN

561

T. H. LIN

335

S. D. CHERNAUSEK

519

R. B. LINGHAM

335

L. E. CORNETT

437

R. K. MISHRA

385

M. A. MORENCY

385

J. DAVE

63

R. W. DOWNS, JR.

639

V. R. MUKKU

335

M. FREGLY

663

P. A. NISSENSON

481

N. GALLO-PAYET

287

R. T. PREMONT

129

D. D. HATMAKER

561

G. M. STANCEL

335

C. SUMNERS

663

J. R. HUBBARD R. IYENGAR

1 129

R. J. WITORSCH

63

Subject

ACTH

Index

receptors adenylate cyclase

308

antibody

322

autoradiography

316

binding assay

292

binding parameters

297

calcium channels

314

desensitization

315

electrophysiological photoaffinity A n g i o t e n s i n II

studies

labelling

313 320

receptor

assays

666

modulation

685

purification

680

Calcitonin

receptors

autoradiography

654

defination

646

internalization

652

purification

654

structure

655

Cholecystokinin

receptors

agonist/antagonist

selection

399

brain

398,407

clinical application

422

fractionation

412

gastro intestinal

406

modulation

417

pancreatic

400

phylogeny

409

radioreceptor assays

396

visualization

410

718 Epidermal growth factor receptor affinity labelling

344

antibodies

363

autophosphorylation

349

binding measurements

340

internalization and degradation

357

primary structure

364

purification

361

regulation

367

synthesis

357

tyrosine kinase activity

346

Glucagon receptor adenyl cyclase system

151

binding assays

133

desensitization

164

functional states

157

internalization

162

purification

148

regulation

162

structure

136

Growth hormone receptors binding assays

8

characterization

34

chemical modulators

24

clinical aspects

27

In vivo measurements

10

measurement

4

monoclonal antibodies

52

purification

28

receptor assays regulation

5 14

719 Insulin receptors assay

183

anti-insulin receptor antibodies

220

clinical applications

236

defination

181

micro heterogeneity

214

properties

186

purification

201

regulation

191

spare receptors

190

structure

206

visualization

212

LH/hCG receptors antagonists

572

biochemical approaches

579

clinical application

601

during development

590

during reproduction

591

induction, receptor

581

localization

573

monoclonal antibodies

600

regulation

584

Oxytocin receptors assays

624

modulation

624

physiological consideration

632

properties

621

Prolactin receptors antibodies

79

assay methodology

74

binding assay

75

internalization

97

kidney

82

liver

85

mammary gland

80

modulation

86

purification

78

720 PTH receptors adenyl cyclase system

491

assays, radioreceptor

501

binding assays

493

functional properties

483

purification

492

structural properties

495

Somatomedin receptors binding characteristics

524

down regulation, type I

547

function, type II receptor

5A4

in cultured fibroblasts

528

in lymphocytes

530

phosphorylation

541

primary structure

539

post-translation modification

540

purification

537

receptor subtypes

526

structure

535

Vasopressin receptors autoradiography

465

biophysical characterization

462

modulation

466

purification

460

solubilization

462

V

receptors

458

V

receptors

456

w DE

G

v. K. Moudgii (Editor)

Walter de Gruyter Berlin • New York Molecular Mechanism of Steroid Hormone Action Recent Advances 1985.17x24 cm. XII, 824 pages. Numerous illustrations. Hardcover. ISBN 3110101181 The chapters in this book represent invited contributions from the leading investigators engaged in research in the general area of steroid hormone receptors. The effort of comprising this volume has brought together the most significant and current work of prominent, world renowned scientists. It provides a firsthand interaction with the various approaches and directions adopted by the authors in their quest of knowledge of the molecular mechanism of steroid hormone action.

K. Fotherby S.B.Pal (Editors)

Exercise Endocrinology 1985.17x24 cm. XII, 300 pages. Numerous illustrations. Hardcover. ISBN 311009557 2 There has been a marked increase in the amount of interest taken in exercise physiology during the past few years. This is partly due to the higher standards demanded in most sports and the most efficient use of strength, energy and stamina required of the sports-person, which can have a significant effect on the final result. This book will be of importance to both "trained" and "untrained" subjects in helping them to understand the physiological changes which take place during exercise.

K. Fotherby S. B. Pal (Editors)

The Role of Drugs and Electrolytes in Hormonogenesis 1984.17x24 cm. XII, 360 pages. Numerous illustrations. Hardcover. ISBN 311008463 5

w

Walter de Gruyter Berlin • New York

K. Fotherby S.B.Pal

Steroid Converting Enzymes and Diseases

DE

G

(Editors)

M.K.Agarwai (Edltlor)

M. K. Agarwai M.Yoshida (Editors)

1984.17 x 24 cm. XII, 261 pages. Numerous illustrations. Hardcover. ISBN 311009556 4

Adrenal Steroid Antagonism Proceedings • Satellite Workshop of the VII. International Congress of Endocrinology. Quebec, Canada, July 7,1984. 1984.17x24 cm. VIII, 399 pages. With numerous illustrations. Hardcover ISBN 3110086131

Immunopharmacology of Endotoxicosis Proceedings of the 5th International Conference of Immunology. Satellite Workshop. Kyoto, Japan, August 27,1983. 1984.17x24 cm. XIV, 376 pages. Numerous illustrations. Hardcover. ISBN 311009887 3

M. K. Agarwai