PACAP and VIP Receptors

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PACAP and VIP Receptors Edward J. Goetzl*, Julia K. Voice and Glenn Dorsam Immunology and Allergy, University of California, San Francisco, Room UB8B, Box 0711, 533 Parnassus Avenue, San Francisco, CA 94143-0711, USA * corresponding author tel: 415-476-5339, fax: 415-476-6915, e-mail: [email protected] DOI: 10.1006/rwcy.2000.23009.

SUMMARY

BACKGROUND

This subfamily of G protein-coupled receptors consists of PAC1, which binds PACAP alone, and VPAC1 and VPAC2, which bind PACAP and VIP with equal affinity. These homologous receptors all have a long N-terminal extracellular sequence with five conserved cysteines, as well as conserved other cysteines and basic amino acids in the first and second extracellular loops and third intracellular loop and cytoplasmic tail. There are numerous splice variants of PAC1, but not of the VPAC receptors. As for the corresponding ligands, these receptors are distributed preferentially in the nervous system, endocrine and immune organs, intestines and lungs. Signaling through two or more G proteins is mediated principally by increases in [cAMP]i and [Ca2‡ ]i. Genetic manipulation of expression of the receptors is now in progress.

Discovery Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are constituents of one structural superfamily of neuroendocrine hormones, which also includes secretin, glucagon, glucagon-like peptide, and growth hormone-releasing hormone or factor. VIP and PACAP are linked by some functional similarities and by their sharing of two of the three receptors in one subfamily of G protein-coupled cellular receptors (GPCRs) (Table 1) (Harmar et al., 1998). The three VIP/PACAP receptors were cloned initially by PCRbased and GPCR hybridization techniques.

Table 1 Structures and encoding genes of the PACAP and VIP receptors Receptor subtype (IUPHAR nomenclature)

Gene (HUGO)

Chromosomal location Human

Other

Size (aa)

PAC1

ADCYAP1R1

7p14

4 (rat)

495 (rat)

VPAC1

VIPR1

3p22

8 (rat)

457 (human)

9 (mouse)

437 (mouse)

4 (rat)

437 (human)

VPAC2

VIPR2

7q36.3

12 (F2 mouse)

2250 Edward J. Goetzl et al.

Alternative names

Chromosome location and linkages

PAC1 was formerly designated the type I PACAP receptor. VPAC1 had been termed the type II PACAP receptor and the type I VIP receptor. VPAC2 had been termed the type III PACAP receptor and type II VIP receptor.

The gene encoding rat PAC1 has been isolated and characterized, demonstrating a single copy and a complex structure similar to those of genes for other members of this subfamily of GPCRs. Rat PAC1 gene spans 40 kb and contains 15 exons (Chatterjee et al., 1997). Introns are 320 bp to 10.5 kbp and exhibit splice phasing of types 0, 1, and 2. All splice acceptor± donor sequences conform to the GT/AG consensus rule. The sizes and organization of exons and introns, and the intron±exon boundaries resemble those of genes encoding GPCRs for secretin, glucagon, and parathyroid hormone. Unlike VPAC1 and VPAC2, for which no splice variants have been identified, PAC1 shows extensive alternative splicing in several domains (Pisegna and Wank, 1996; Pantaloni et al., 1996; Chatterjee et al., 1996; 1997). Two alternative exons in the region encoding the third intracellular loop have been termed hip and hop, and the latter exists as hop1 and hop2 due to different splice acceptor sites. The hip-hop variants differ in signaling properties. Alternative splicing of exons equivalent to hip and hop1 in human PAC1 leads to four variants designated null, SV-1, SV-2, and SV-3, that also differ in transductional abilities. Four other variants arise from differential use of alternative exons in the 50 untranslated region of the rat PAC1 gene. A splice variation that leads to a 21 amino acid deletion in the extracellular N-terminus and another with sequence differences in transmembrane domains II and IV appear to represent one type of mechanism responsible for different binding affinities and signaling potencies of PACAP-38 and PACAP-27. There is one copy each of the VPAC1 and VPAC2 genes, and no alternatively spliced forms have been identified for either subtype. Human VPAC1 spans 22 kbp and is composed of 13 exons of 42 bp to 1400 bp and 12 introns of 0.3± 6.1 kbp (Sreedharan et al., 1995). The structure of the gene encoding VPAC2 has not been delineated completely, but chromosomal localization is known (Mackay et al., 1996). Although it seems reasonable to assume that expression of each receptor is regulated transcriptionally

Structure PAC1, VPAC1, and VPAC2 GPCRs show a high level of homology to each other and share structural features, such as a long N-terminus and short loops.

Main activities and pathophysiological roles PAC1 has been cloned from rat, mouse, bovine, and human cells (Pisegna and Wank, 1993; Harmar et al., 1998). PAC1 of each species binds PACAP-27 and PACAP-38 (IC50 = 1 nM) with 1000-fold higher affinity than VIP, and exhibits even lower affinities for PHI (peptide histidine isoleucine), PHV (peptide histidine valine), secretin, and growth hormone-releasing factor (GRF) (Ohtaki et al., 1998). VPAC1 has been cloned from rat, human, and mouse cells (Ishihara et al., 1992; Harmar et al., 1998). VPAC1 of each species binds PACAP-27, PACAP-38, and VIP with equal affinity (IC50 = 0.2±1 nM), but differences exist among species in the rank-order of affinity of binding of other closely related peptides. VPAC2 has been cloned from rat, mouse, and human cells (Lutz et al., 1993; Inagaki et al. 1994; Harmar et al., 1998). VPAC2 of each species binds PACAP-38 and VIP with equal affinity (IC50 = 2± 4 nM), but has slightly lower affinity for PACAP-27.

GENE

Accession numbers See Table 2.

Table 2 Accession numbers for PAC1, VPAC1, and VPAC2 PAC1

VAPC1

Human

D17516

U11079, U11080, U11081, U11082, U11083, U11084, U11085, U11086, U11087

Rat

D14908, D14909

AF059678

U09631, Z25885

S82970

S82966

Mouse

VPAC2

PACAP and VIP Receptors 2251 and with tissue specificity, few definitive studies have addressed these questions. One notable contribution is the demonstration that corticosteroids suppress levels of mRNA encoding VPAC1 in cultured lung cells and reduce signals reported by VPAC1 50 -flanking sequence luciferase constructs introduced by transfection (Pei, 1996). The 50 -glucocorticoid negative response element was that mapped to a 126 bp sequence containing a glucocorticoid receptor-binding site between ÿ36 and ÿ21 bp from the transcription start site. Promoters of each gene are now being defined fully with respect to other functional elements.

PROTEIN

Description of protein PAC1, VPAC1, and VPAC2 are related seven transmembrane domain GPCRs. Within each species, amino acid sequence identity among the three receptors is 49% to 51%, and the similarity of each with the GPCRs for secretin, glucagon, glucagon-like peptide I, and growth hormone-releasing hormone ranges from 34% to 47%. As for most subfamilies of GPCRs, the greatest degree of homology is within the transmembrane domains and least in the N- and Cterminal segments. Receptors of this subfamily share other structural features, which as yet have not been related to receptor function, including long Nterminal extracellular sequences with five conserved cysteines, two additional conserved cysteines located one each in the first and second extracellular loops, and conserved basic amino acids in the third intracellular loop and cytoplasmic tail, that may facilitate coupling to Gs.

Affinity for ligand(s) A few aspects of the structural determinants of specific ligand binding have been elucidated for the VPAC1 receptor. A series of chimeras of the rat VPAC1 and secretin receptors were constructed involving exchanges of the N-terminus, first extracellular loop or both (Holtmann et al., 1995). The native receptors bound their respective ligands with 2 nM Kd values and crossbound the other ligand with Kd values three orders of magnitude higher (Table 3). When the VPAC1 N-terminus was substituted for that of the secretin receptor, ligand binding and transduction of biological responses typical of VPAC1 were observed. For reciprocal conversion of VPAC1 to a secretin receptor, however, the chimera required both the N-terminus and the first extracellular loop of the

secretin receptor. Dissociation of ligand-binding affinity from ligand potency in transduction of responses for some chimeras emphasized the likely complexity of composition of complete receptor units. Another series of chimeras of nonidentical components of human and rat VPAC1, and selected sitedirected mutants of both, revealed that the difference in rat VPAC1 high-affinity binding and human VPAC1 low-affinity binding of PHI is attributable to three amino acids in the first extracellular loop and adjacent third transmembrane domain (Couvineau et al., 1996).

Cell types and tissues expressing the receptor The distinctive patterns of tissue distribution of each PACAP/VIP receptor to date have been mapped principally by radioligand binding and semiquantification or probe detection of encoding mRNA (Table 3). The attendant problems of any one approach and the possibility of dissociations between the amounts of mRNA and protein limit confidence in the initial results. Nonetheless, it is clear that VPAC1 and VPAC2 are often expressed with reciprocal densities in one type of cell, tissue representation may be complementary as in the rat CNS (Usdin et al., 1994), and both show high levels of inducibility and repressibility. One such example is in a cell line model for thymocytes, where nearly exclusive expression of VPAC1 changes to much higher expression of VPAC2 and lower levels of VPAC1 within hours of exposure to antigen and antigen-presenting cells (Pankhaniya et al., 1998). Antibodies specific for each receptor in the subfamily now have become available, which should permit definitive analyses of tissue-specific expression and the determinants of regulation of such expression.

SIGNAL TRANSDUCTION PAC1, VPAC1, and PVAC2 each couple to multiple G proteins, resulting in concurrent initiation of diverse signaling pathways (Table 3). Gs is presumed to mediate stimulation of adenylate cyclase and an increase in [cAMP]i, that is the hallmark of cellular effects of VIP and PACAP. In addition to evidence for a physical association between Gs and VPAC1, specific suppression of Gs by transfection of Gs  chain antisense plasmids with a hygromycin-resistance element into VPAC1-expressing epithelial cells, followed by hygromycin selection, blunted VIPinduced increases in [cAMP]i (Goetzl et al., 1993).

2252 Edward J. Goetzl et al. Table 3 Binding, signaling, tissue distribution, and pharmacology of PAC1, VPAC1, and VPAC2 Receptor

PAC1

Kd (nM)

1

Responses (EC50, nM) of [cAMP]i

[Ca2‡ ]i

0.1±1.6

10±50

Predominant tissue expression

CNS (olfactory bulb, thalamus, hypothalamus, hippocampus, cerebellum)

Selective Agonists

Antagonists

Maxadilan

PACAP6-38

VIP1,7/GRF VIP4-28

VIP3,7/GRF

Ro25-1553 Ro25-1392

VIP4-28

Adrenal medulla Macrophages VPAC1

0.2±1

0.3±1

2.5

CNS (cerebral cortex, hippocampus) Lung Gastrointestinal tract Liver Prostate Macrophages and lymphocytes

VPAC2

3±10

10±30

27

CNS (thalamus, suprachiasmatic nucleus, hippocampus, brainstem, spinal cord, dorsal root ganglia) Skeletal and cardiac muscle Gastrointestinal tract Kidney Adipose tissue Testes Macrophages and lymphocytes

VIP1,7/GRF, [K15 R16 L27 ]VIP(1-7)GRF(8-25)-NH2; VIP3,7/GRF, [Ac-His1 D-Phe2 Lys15 Arg16 ]VIP(3-7)GRF(8-27)-NH2; Ro25-1553, Ac-His1 -[Glu8 Lys12 Nle17 Ala19 Asp25 Leu26 Lys27;28 Gly29;30 Thr31 ]-NH2 VIP(cyclo21-25); and Ro25-1392, Ac-His1 [Glu8 OCH3-Tyr10 Lys12 Nle17 Ala19 Asp25 Leu26 Lys27;28 ]VIP (cyclo21-25).

That similar antisense suppression of Gi2 led to marked enhancement of VIP-induced increases in [cAMP]i, suggested that Gi2 has a negative regulatory effect on VIP enhancement of adenylate cyclase. Each receptor also activates phosphoinositide-specific phospholipase C (PLC), with resultant elevation of [Ca2‡ ]i. The greater potency of PACAP-38 than PACAP-27 in PAC1-mediated activation of PLC and elevation of [Ca2‡ ]i has been attributed to a 21 amino acid segment of the N-terminal extracellular domain, based on results of studies of a series of splice variants (Pantaloni et al., 1996). The Gq/11 protein is assumed to couple the receptors to PLC, based on data from other systems, but this has not been demonstrated directly. One transmembrane domain IV splice variant of PAC1 failed to activate either adenylate cyclase or PLC, but stimulated Ca2‡ influx by

activation of L-type Ca2‡ channels (Chatterjee et al., 1996).

DOWNSTREAM GENE ACTIVATION There are several reports of VIP or PACAP enhancing or suppressing proliferation or functional responses of normal or neoplastic cells in vitro (Ogasawara et al., 1997; Maruno et al., 1998). It is assumed that part of the capacity of VIP and PACAP to influence growth is attributable to alterations in transcription of immediate early genes critical for proliferative responses. However, no studies to date have focused on this important question.

PACAP and VIP Receptors 2253

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY PAC1, VPAC1, and VPAC2 are presumed to transduce all of the effects of PACAP and VIP but often act in tissues responding to multiple other mediators and cytokines. The absence of inactivating genetic anomalies of any of the receptors and the lack of potent and selective pharmacological agents precludes specific assignment of each neuropeptide effect to one type of receptor. However, detailed analyses of gene knockout models now in development may permit assessment of the biological and pathophysiological roles of each receptor.

References Chatterjee, T. K., Sharma, R. V., and Fisher, R. A. (1996). Molecular cloning of a novel variant of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor that stimulates calcium influx by activation of L-type calcium channels. J. Biol. Chem. 271, 32226±32232. Chatterjee, T. K., Liu, X., Davisson, R. L., and Fisher, R. A. (1997). Genomic organization of the rat pituitary adenylate cyclase-activating polypeptide receptor gene. J. Biol. Chem. 272, 12122±12131. Couvineau, A., Rouyer-Fesard, C., Maoret, J.-J., Gaudin, P., Nicole, P., and Laburthe, M. (1996). Vasoactive intestinal peptide (VIP)1 receptor. J. Biol. Chem. 271, 12795±12800. Goetzl, E. J., Kishiyama, J. L., Shames, R. S., Liu, Y.-F., Albert, P. R., An, S., Birke, F. W., Yang, J., and Sreedharan, S. P. (1993). Specific inhibition of receptor-dependent human cellular responses by antisense mRNA depletion of individual G proteins. Trans. Assoc. Am. Physicians 56, 69±76. Harmar, A. J., Arimura, A., Gozes, I., Journot, L., Laburthe, M., Pisegna, J. R., Rawlings, S. R., Robberecht, P., Said, S. I., Sreedharan, S. P., Wank, S. A., and Waschek, J. A. (1998). International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol. Rev. 50, 265±270. Holtmann, M. H., Hadac, E. M., and Miller, L. J. (1995). Critical contributions of amino-terminal extracellular domains in agonist binding and activation of secretin and vasoactive intestinal polypeptide receptors. Studies of chimeric receptors. J. Biol. Chem. 270, 14394±14398. Inagaki, N., Yoshida, H., Mizuta, M., Mizuno, N., Fujii, Y., Gonoi, T., Miyazaki, J., and Seino, S. (1994). Cloning and functional characterization of a third pituitary adenylate cyclase-activating polypeptide receptor subtype expressed

in insulin-secreting cells. Proc. Natl Acad. Sci. USA 91, 2679±2683. Ishihara, T., Shigemoto, R., Mori, K., Takahashi, K., and Nagata, S. (1992). Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide. Neuron 8, 811±819. Lutz, E. M., Sheward, W. J., West, K. M., Morrow, J. A., Fink, G., and Harmar, A. J. (1993). The VIP2 receptor: molecular characterization of a cDNA encoding a novel receptor for vasoactive intestinal peptide. FEBS Lett. 334, 3±8. Mackay, M., Fantes, J., Scherer, S., Boyle, S., West, K., Tsui, L.-C., Belloni, E., Lutz, E., Van Heyningen, V., and Harmar, A. J. (1996). Chromosomal localization in mouse and human of the vasoactive intestinal peptide receptor type 2 gene: a possible contributor to the holoprosencephaly 3 phenotype. Genomics 37, 345±352. Maruno, K., Absood, A., and Said, S. I. (1998). Vasoactive intestinal peptide inhibits human small-cell lung cancer proliferation in vitro and in vivo. Proc. Natl Acad. Sci. USA 95, 14373± 14378. Ogasawara, M., Murata, J., Ayukawa, K., and Saimi, I. (1997). Differential effect of intestinal neuropeptides on invasion and migration of colon carcinoma cells in vitro. Cancer Lett. 116, 111±116. Ohtaki, T., Ogi, K., Masuda, Y., Mitsuoka, K., Fujiyoshi, Y., Kitada, C., Sawada, H., Onda, H., and Fujino, M. (1998). Expression, purification and reconstitution of receptor for pituitary adenylate cyclase-activating polypeptide. J. Biol. Chem. 273, 15464±15473. Pankhaniya, R., Jabrane-Ferrat, N., Gaufo, G. O., Sreedharan, S. P., Dazin, P., Kaye, J., and Goetzl, E. J. (1998). Vasoactive intestinal peptide enhancement of antigeninduced differentiation of a cultured line of mouse thymocytes. FASEB J. 12, 119±127. Pantaloni, C., Brabet, P., Bilanges, B., Dumuis, A., Houssami, S. S., Spengler, D., Bockaert, J., and Journot, L. (1996). Alternative splicing in the N-terminal extracellular domain of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor modulates receptor selectivity and relative potencies of PACAP-27 and PACAP-38 in phospholipase C activation. J. Biol. Chem. 271, 22146±22151. Pei, L. (1996). Identification of a negative glucocorticoid response element in the rat type 1 vasoactive intestinal polypeptide receptor gene. J. Biol. Chem. 271, 20879±20884. Pisegna, J. R., and Wank, S. A. (1993). Molecular cloning and functional expression of the pituitary adenylate cyclase-activating polypeptide type I receptor. Proc. Natl Acad. Sci. USA 90, 6345±6349. Pisegna, J. R., and Wank, S. A. (1996). Cloning and characterization of the signal transduction of four splice variants of the human pituitary adenylate cyclase-activating polypeptide receptor. J. Biol. Chem. 271, 17267±17274. Sreedharan, S. P., Huang, J.-X., Cheung, M.-C., and Goetzl, E. J. (1995). Structure, expression, and chromosomal localization of the type I human vasoactive intestinal peptide receptor gene. Proc. Natl Acad. Sci. USA 92, 2939±2943. Usdin, T. B., Bonner, T. I., and Mezey, E. (1994). Two receptors for vasoactive intestinal polypeptide with similar specificity and complementary distributions. Endocrinology 135, 2662±2680.