RON Receptor

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RON Receptor Alla Danilkovitch-Miagkova* and Edward J. Leonard Section of Immunopathology, Laboratory of Immunobiology, Division of Basic Sciences, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702, USA * corresponding author tel: (301) 846-1560, fax: (301) 846-6145, e-mail: [email protected] DOI: 10.1006/rwcy.2001.1907. Chapter posted 5 November 2001

SUMMARY

BACKGROUND

RON (Receptuer d'Origine Nantaise) is a transmembrane receptor tyrosine kinase (RTK) that belongs to the MET receptor tyrosine kinase protein family. RON is expressed in various cell types including macrophages, osteoclasts, epithelial cells, and hematopoietic cells. The RON ligand is macrophagestimulating protein (MSP). MSP is a member of the kringle-domain plasminogen-related protein family; its sequence is similar to that of the MET ligand hepatocyte growth factor (HGF). MSP binding to RON activates a number of intracellular signaling pathways that mediate MSP biological activities, including its effects on adhesion and motility, growth, differentiation, and survival. MSP/RON-induced cellular responses suggest an important role of RON in the regulation of normal cell functions and possible involvement in various pathological conditions. Addition of MSP to macrophages expressing RON induces shape changes, chemotaxis, macropinocytosis, and phagocytosis. RON activation inhibits generation of nitric oxide (NO) by endotoxin and thus suppresses endotoxin lethality. RON promotes adhesion and motility, growth and survival of epithelial cells. Normal RON is overexpressed by a variety of tumors and transfection of mutated forms of RON results in oncogenic transformation. Lethality of RON knockout mice reflects the importance of RON in embryonal development. This review provides a complete RON reference guide showing key points for future directions in RON investigations.

Discovery

Cytokine Reference

Human RON DNA was cloned in 1993 as a result of screening of cDNA libraries prepared from human tumors and foreskin keratinocytes (Ronsin et al., 1993). Analysis of RON DNA revealed that the gene encodes a transmembrane receptor tyrosine kinase structurally similar to the MET receptor kinase (Ronsin et al., 1993). A year later the mouse RON receptor was cloned from purified murine hematopoietic stem cells (Iwama et al., 1994). The similarity between RON and MET receptors, and between macrophage-stimulating protein (MSP) and hepatocyte growth factor (HGF), the MET ligand, suggested that MSP might be a RON ligand. This hypothesis was proved within 2 years, when it was shown that MSP is indeed the ligand for human and mouse RON (Wang et al., 1994b, 1995; Gaudino et al., 1994).

Alternative names Macrophage-stimulating protein receptor (MSP receptor) (Del Gatto et al., 1995). The mouse RON receptor is known as STK (stem cell-derived tyrosine kinase) (Iwama et al., 1994). According to the CD nomenclature: CD136 antigen (SwissProt). Official nomenclature for human RON gene: MST1R (macrophage stimulating 1 receptor)

Copyright # 2001 Academic Press

Alla Danilkovitch-Miagkova and Edward J. Leonard

Structure RON is a transmembrane RTK belonging to the MET RTK protein family. The RON gene encodes a 190 kDa protein, which is expressed on the cell surface as a disulfide-linked heterodimer comprising 40 kDa  and 150 kDa chains (Ronsin et al., 1993; Iwama et al., 1994; Gaudino et al., 1994). The singlechain 190 kDa pro-RON undergoes intracellular proteolytic cleavage at a basic amino acid site which converts pro-RON into the mature two-chain heterodimeric receptor (Gaudino et al., 1994). The  chain of RON is located entirely in the extracellular part of the receptor, whereas the chain consists of an extracellular domain, transmembrane and cytoplasmic domains (Figure 1).

Main activities and pathophysiological roles The ligand for RON is macrophage-stimulating protein (MSP) (Yoshimura et al., 1993), also known as hepatocyte growth factor-like protein (HGFL) (Han et al., 1991). MSP/RON interaction activates a number of intracellular signaling pathways that mediate MSP biological activities. The main targets and biological activities of MSP/RON are summarized in Table 1.

Figure 1 Schematic representation of immature (intracellular) and mature (expressed on cell membrane) RON. RON is synthesized as a 190 kDa single-chain precursor. It has a proteolytic cleavage site location in the extracellular part. To be expressed on the cell surface the RON precursor undergoes proteolytic cleavage by enzymes of the endoplasmic reticulum. The cleavage converts single-chain immature RON into the  disulfide-linked mature RON heterodimer. α chain 40 kDa Proteolytic cleavage site

SS

Intracellular proteolytic cleavage

Transmembrance domain

Extracellular part

(http://www.ncbi.nlm.nih.gov/LocusLink/list.cgi; Angeloni and Lerman, 2001).

chain 150 kDA Cytoplasmic part

2

Kinase domain

Intracellular single chain 190 kDa immature RON

Disulfide-linked heterodimeric mature RON

Chromosome location and linkages GENE

Accession numbers Human RON cDNA: GenBank X70040 (Ronsin et al., 1993) Mouse RON cDNA: GenBank X74736 (Iwama et al., 1994) Intronic sequences of the human RON: GenBank AF 164633±AF164654 (Angeloni et al., 2000) Complete coding sequence of the mouse RON: GenBank U65949 (Waltz et al., 1998).

Sequence The RON gene sequence is available at GenBank (http://www.ncbi.nlm.nih.gov).

The human RON gene is located on chromosome 3 (region 3p21) (Ronsin et al., 1993). The mouse RON gene is located on chromosome 9 (R-positive F1 band) (Iwama et al., 1994).

PROTEIN

Accession numbers Human RON: SwissProt Q04912

Sequence The RON amino acid sequence is available at GenBank and Protein Data Bank (http:// www.ncbi.nlm.nih.gov).

RON Receptor 3 Table 1 RON targets and biological activities Targets

Biological activity

References

Macrophages

Shape changes

Leonard and Skeel, 1976; Iwama et al., 1995; Wang et al., 1997; Waltz et al., 1997; Correll et al., 1997; Nanney et al., 1998;

Chemotaxis in response to C5a

Leonard and Skeel, 1976; Skeel and Leonard, 1991, 1994

Chemotaxis

Skeel and Leonard, 1980, 1991

Ingestion of EigMC3bi

Skeel et al., 1991, 1994; Iwama et al., 1995

Inhibition of iNOS

Wang et al., 1994a, 2000a; Correll et al., 1997; Chen et al., 1998; Liu et al., 1999

Secretion of IL-6

Leonard and Skeel, unpublished

Macropinocytosis

Leonard, unpublished

Adhesion

Willett et al., 1997; Danilkovitch et al., 1999b

Movement

Gaudino et al., 1994; Wang et al., 1995, 1996a, 1996b, 2000b; Maggiora et al., 1998; Montero-Julian et al., 1998; Danilkovitch et al., 1999b; Chen et al., 2000

Cell scattering

Waltz et al., 1997

Ciliary motility

Sakamoto et al., 1997

Growth induction

Gaudino et al., 1995; Wang et al., 1996a; Waltz et al., 1997

Growth inhibition

Willett et al., 1997

Protection from apoptosis

Danilkovitch et al., 2000; Chen et al., 2000.

Apoptosis

Willett et al., 1997

Vascular endothelial cells

Angiogenesis

Cao and Leonard, unpublished

Osteoclasts

Bone resorption

Kurihara et al., 1996

Growth induction

Gaudino et al., 1995

Shape changes

Mera et al., 1999

Growth induction

Iwama et al., 1996; Mera et al., 1999

Growth inhibition

Iwama et al., 1996; Broxmeyer et al., 1996

Cytokine production (IL-6)

Banu et al., 1996

Epithelial cells

Hematopoietic cells

Promotion of differentiation

Banu et al., 1996

Inhibition of differentiation

Broxmeyer et al., 1996

Apoptosis

Iwama et al., 1996

Description of protein Full-length human RON consists of 1400 amino acids (Ronsin et al., 1993); the murine protein comprises 1378 amino acids (Iwama et al., 1994). Analysis of the RON amino acid sequence revealed several domains (Figure 2). Because of a high similarity between human and mouse RON receptors, only the human RON domains are described in detail.

The transmembrane domain (amino acid residues 958±982) divides RON into extracellular (amino acid residues 1±957) and intracellular (amino acid residues 993±1400) regions. In the extracellular part of RON there are one SEMA (residues 58±507 in human RON), one plexin or PSI (residues 526±568) and four IPT (residues 569±671, 684±767, 770±855, and 874±886) domains (Bork et al., 1999; Angeloni et al., 2000). The SEMA domain was initially

4

Alla Danilkovitch-Miagkova and Edward J. Leonard Figure 2

Structural domains of the human RON receptor. Y1238 Y1239 Y1353 Y1360

1

2

3

4

SEMA domain (58-507 amino acid residues)

PSI (plexin) domin (526-568 amino acid residues)

IPT domain (1st: 569-671 amino acid residues; 2nd: 684-767 amino acid residues; 3d: 770-855 amino acid residues; 4th: 874-886 amino acid residues)

Transmembrane domain (?)

IPT domain (?)

Proteolytic cleavage site KRRRR (305-309 amino acid residues)

Y1238 Y1239

Tyrosine residues representing major autophosphorylation site

Y1353 Y1360

Tyrosine residues representing C-terminal multifunctional docking site

identified in the semaphorin protein family (Kolodkin et al., 1993; Yu and Kolodkin, 1999). This domain consists of a highly conserved stretch of about 500 amino acids, and is characterized by 15 conserved cysteines, one conserved potential Nglycosylation site, and several blocks of conserved residues throughout the domain (Kolodkin et al., 1993; Yu and Kolodkin, 1999). In addition to semaphorins and RON, the SEMA domain occurs in MET (Bork et al., 1999; Comoglio et al., 1999), neurophilins (Tamagnone et al., 1999), and the orphan receptors of the SEX family (Maestrini et al., 1996). One of the proposed functions of the SEMA domain is to mediate protein±protein interactions (Bork et al., 1999). The plexin or PSI (plexins, semaphorins, and integrins) domain is approximately 50 residues in length and usually contains eight

cysteine residues (Bork et al., 1999). The IPT domain (for immunoglobulin-like fold shared by plexins and transcriptional factors) has been found in plexins, in proteins belonging to the MET family (including RON) and in the VESPR (the virus-encoded semaphoring receptor) (Bork et al., 1999). It seems that the IPT domain plays a role in neuronal development and immunological functions (Bork et al., 1999). The PSI and IPT domains might be involved in receptor adhesion through a homophilic binding mechanism. The observation that RON can be expressed on the cell surface as a noncovalently linked dimer (Follenzi et al., 2000) raises the possibility that this ligand-independent RON association might be mediated by homophilic interactions between SEMA, PSI, or IPT domains of RON.

RON Receptor 5 The intracellular part of RON (983±1400 amino acids) has a catalytic domain (amino acid residues 1073±1335) (Ronsin et al., 1993). Tyrosine residues at positions 1238 and 1239 represent a RON kinase autophosphorylation site that is essential for upregulation of RON catalytic activity (Gaudino et al., 1994; Longati et al., 1994; Ponzetto et al., 1994). Tyrosines 1353 and 1360 represent a multifunctional docking site that plays an important role in RON-mediated signaling (Gaudino et al., 1994; Iwama et al., 1996). A schematic representation of human RON domain structure is shown in Figure 2. A short form of murine RON containing amino acid residues 900±1378 was described by Iwama et al. (1994). In some human tissues and cell lines a short form of RON has been detected at the level of mRNA (Ronsin et al., 1993; Gaudino et al., 1994; Angeloni et al., 2000). It seems that this RON form is an alternatively spliced protein (Ronsin et al., 1993). Mouse cells have an alternative internal promoter that can drive expression of the short RON protein (Persons et al., 1999). A second promoter in the human RON gene has not yet been found, but since this region is highly conserved in both mouse and human genes, it is likely that the alternative internal promoter occurs in the human RON gene.

Relevant homologies and species differences The sequences of human and mouse RON were published in 1993 and 1994 respectively (Ronsin et al., 1993; Iwama et al., 1994). The amino acid homology between human and mouse Ron is 73.6% in total and 88.6% in the kinase domain (Iwama et al., 1994). The homology of mouse RON with the mouse MET protein and the chicken Sea protein is 31.6% and 45% in total, and 65.8% and 70% in the kinase domain, respectively (Iwama et al., 1994). The homology of human RON with the human MET protein is 63% in the kinase domain (Ronsin et al., 1993). Both human and mouse RON bind and can be activated by human MSP (Wang et al., 1994b, 1995; Leonard and Danilkovitch, 2000). Chicken Sea might represent the ovian RON ortholog but definitive data have not yet been obtained to support this hypothesis (Huff et al., 1993; Wahl et al., 1999). A RON ortholog has been identified in Xenopus (Nakamura et al., 1996). RON has domain structure similarities with other members of the MET RTK family, with plexins, VESPR and proteins belonging to the SEX family (Bork et al., 1999).

Affinity for ligand(s) MSP, the RON ligand, is a heterodimeric protein consisting of disulfide-linked  and chains (Han et al., 1991; Yoshimura et al., 1993). The calculated Kd is 0.6±0.8 nM for the MSP  heterodimer and 1.4 nM for its chain (Wang et al., 1997). Despite high affinity binding of the chain to RON, functional studies indicated that only the MSP  chain heterodimer induced biological responses. Detection of  chain binding to RON (Danilkovitch et al., 1999b) suggests that MSP has two independent binding sites with high and low affinities located in the and  chain respectively. The calculated EC50 values are 0.25 and 16.9 nM for the and  chain, respectively (Danilkovitch et al., 1999a). Ligand± receptor interactions are discussed in the chapter on macrophage-stimulating protein.

Cell types and tissues expressing the receptor RON expression has been found in a number of human and rodent cell lines of different origin and in various human and mouse tissues. Data about RON expression in cell lines and tissues are summarized in Tables 2, 3, 4, 5 and 6 (Only positive data are included. Analysis of data during preparation of these tables revealed some discrepancies in RON expression by cell lines published by different authors. These discrepancies can be explained by detection method limitations and/or by heterogeneity in cell lines cultured in different laboratories.) RON is also expressed in mouse embryos, which suggests RON as a regulator of embryonal development. In situ hybridization analysis demonstrated that RON is expressed in the trophoectoderm representing extraembryonic tissues at embryonic day E3.5 (Muraoka et al., 1999). RON expression in embryonic tissues is detected at E12.5 in the liver and in the central nervous system (Quantin et al., 1995; Gaudino et al., 1995). At E14.5 RON is detected in the digestive tract epithelium, skin keratinocytes and developing bones (Quantin et al., 1995; Gaudino et al., 1995).

Regulation of receptor expression The presence of a number of potential regulatory elements in the RON promoter (Del Gatto et al., 1995; Waltz et al., 1998) suggests that RON gene expression may be positively or negatively regulated

6

Alla Danilkovitch-Miagkova and Edward J. Leonard

Table 2 Human cell lines that express RON Cell line

Origin

Detection methods

References

Osteoclast-like

RT-PCR, FA

Gaudino et al., 1995

Bone GTC-51

Bone marrow/hematopoietic HL-60

Promyelocytic leukemia

N

Gaudino et al., 1994

K562

Erythroleukemia

N

Gaudino et al., 1994

W

Danilkovitch et al., unpubl.

HEL92

Erythroleukemia

W

Danilkovitch et al., unpubl.

TF-1

Erythroleukemia

W

Danilkovitch et al., unpubl.

DAMI

Megakaryocytic

RT-PCR, FA

Banu et al., 1996

CMK

Megakaryocytic

RT-PCR, FC, FA

Banu et al., 1996

Mo7e

Megakaryocytic

RT-PCR, FC

Banu et al., 1996

FA

Broxmeyer et al., 1996

CTS

Megakaryocytic

RT-PCR, FC

Banu et al., 1996

Mammary carcinoma

N,W

Gaudino et al., 1994

Breast T47D

W, FA

Collesi et al., 1996

W,FA

Maggiora et al., 1998

RT-PCR, FA

Gaudino et al., 1995

Breast carcinoma

W, FA

Maggiora et al., 1998

BET-1A

Bronchial epithelial

W, FA,

Sakamoto et al., 1997

BEAS-2B

Bronchial epithelial

W, FA,

Sakamoto et al., 1997

Cervix carcinoma

W

Collesi et al., 1996

SW620

Adenocarcinoma

FC

Montero-Julian et al., 1998

W, FA

Wang et al., 2000b

DLD-1

Adenocarcinoma

W, FA

Wang et al., 2000b

HCT116

Adenocarcinoma

W, FA

Wang et al., 2000b

Colo 201

Adenocarcinoma

FC

Montero-Julian et al., 1998

ZR75.1 Bronchus

Cervix HeLa Colon

RT-PCR, W

Chen et al., 2000

W, FA

Wang et al., 2000b

HT-29-D4

Adenocarcinoma

FC, FA

Montero-Julian et al., 1998

HT-29

Adenocarcinoma

RT-PCR, W

Chen et al., 2000

W, FA

Wang et al., 2000b

W, FA

Wang et al., 1997

FA

Danilkovitch et al., 1999b

Kidney HEK293

Embryonal kidney epithelial

RON Receptor 7 Table 2 (Continued) Cell line

Origin

Detection methods

References

Hepatocarcinoma

FC

Montero-Julian et al., 1998

RT-PCR, W,

Chen et al., 1997

Liver HepG2

Lung H596

Lung carcinoma

RT-PCR

Willett et al., 1997

H60,

Small cell lung cancer

RT-PCR

Willett et al., 1997

H187

Small cell lung cancer

RT-PCR

Willett et al., 1997

H249

Small cell lung cancer

RT-PCR, FA

Willett et al., 1997

H835, H679

Carcinoid cells

RT-PCR, FA

Willett et al., 1997

H1184,

Small cell lung cancer

N

Angeloni et al., 2000

H2081,

Small cell lung cancer

(RON expression in cell lines H1184±H157 was detected by Angeloni et al., 2000 using N)

H2227

Small cell lung cancer

H1086

Small cell lung cancer

H841

Small cell lung cancer

H69

Small cell lung cancer

H1820

Small cell lung cancer

H660

Small cell lung cancer

H1688

Small cell lung cancer

H446

Small cell lung cancer

H740

Small cell lung cancer

H1373

Non-small cell lung cancer

H1264

Non-small cell lung cancer

H1693

Non-small cell lung cancer

H1944

Non-small cell lung cancer

H838

Non-small cell lung cancer

H1299

Non-small cell lung cancer

H727

Non-small cell lung cancer

H157

Non-small cell lung cancer

H460

Non-small cell lung cancer

N RT-PCR

Willett et al., 1998

H1466

Non-small cell lung cancer

N

Angeloni et al., 2000

RT-PCR,

Willett et al., 1998

RT-PCR, WB,

Willett et al., 1998

H661

Non-small lung cancer

Angeloni et al., 2000

H322

Non-small lung cancer

RT-PCR

Willett et al., 1998

H23

Non-small lung cancer

RT-PCR

Willett et al., 1998

CaLu1

Non-small lung cancer

RT-PCR

Willett et al., 1998

ChaGoK1

Non-small lung cancer

RT-PCR

Willett et al., 1998

EPL65H

Non-small lung cancer

RT-PCR

Willett et al., 1998

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Alla Danilkovitch-Miagkova and Edward J. Leonard

Table 2 (Continued) Cell line

Origin

Detection methods

References

H125

Non-small lung cancer

RT-PCR

Willett et al., 1998

H596

Non-small lung cancer

RT-PCR, W, FA

Willett et al., 1998

SKOV3

Adenocarcinoma

W

Collesi et al., 1996

NIH:OVCAR3

Adenocarcinoma

W

Collesi et al., 1996

PT45

Carcinoma

W

Gaudino et al., 1994

W

Collesi et al., 1996

SUIT2

Carcinoma

W

Gaudino et al., 1994

Adenocarcinoma

W, FA

Wang et al., 2000b

SVK14

Keratinocytes

FC

Montero-Julian et al., 1998

HaCat

Keratinocytes

FA

Danilkovitch et al., 2000

RHEK

Keratinocytes

BA, WB, FA

Wang et al., 1996b

SCC-9

Keratinocytes

BA, WB, FA

Wang et al., 1996b

HK-NOC

Keratinocytes

BA, WB, FA

Wang et al., 1996a,1996b

FA

Danilkovitch et al., 2000

Ovary

Pancreas

Rectum SW837 Skin

Stomach MKN-28

Carcinoma

N

Gaudino et al., 1994

GTL-16

Carcinoma

N, W

Gaudino et al., 1994

W

Collesi et al., 1996

KATO III

carcinoma

N, W

Gaudino et al., 1994

W, FA

Collesi et al., 1996

W, FA

Danilkovitch et al., unpubl.

Umbilical cord HUVEC

endothelial

BA, binding assay; FA, functional assays; FC, flow cytometry; N, Northern blotting; RT-PCR, reverse transcriptase polymerase chain reaction; W, Western blotting.

by a variety of cytokines and steroid hormones. Using human hepatocellular carcinoma cell lines as a model system, it has been shown that HGF, EGF, IL-1, IL-6, TNF, serum and PMA can upregulate RON expression (Chen et al., 1997). Upregulation of RON expression has been detected in epidermal burn wound and accessory structures (Nanney et al., 1998). This may be mediated via cytokines and growth factors present at high concentrations in the wound. Investigation of RON expression in macrophages

showed that RON is downregulated during inflammation by lipopolysaccharide (LPS) or TNF plus IFN through induction of NO production (Wang et al., 2000a). Regulatory Sites and Corresponding Transcriptional Factors of the RON Gene Promoter Two regions of the mouse RON gene promoter encompassing nucleotides ÿ585 to ÿ465 and from

RON Receptor 9 Table 3 Rodent cell lines that express RON Cell line

Origin

Species

Detection methods

References

L8057

Megakaryoblastic

mouse

N

Iwama et al., 1994

MEL

Erythroid

mouse

N

Iwama et al., 1994

mouse

RT-PCR

Waltz et al., 1998

DA-1

Il-3-dependent immature

mouse

N

Iwama et al., 1994

BK-1

Keratinocytes

mouse

BA, W, FA

Wang et al., 1996a

BA, FA

Wang et al., 1997

FA

Wang et al., 1996a

BA, FA

Wang et al., 1997

MK308

Keratinocytes

mouse

SP-1

Keratinocytes

mouse

FA

Wang et al., 1996a

PAM212

Keratinocytes

mouse

FA

Wang et al., 1996a

FA

Danilkovitch et al., 2000

FA

Waltz et al., 1997

RT-PCR

Waltz et al., 1998

CMT-93

Rectum carcinoma

mouse

YAMC

Young adult mouse colon

mouse

RT-PCR

Waltz et al., 1998

XB-2

Teratoma

mouse

RT-PCR

Waltz et al., 1998

NIH3T3

Embryonic fibroblasts

mouse

RT-PCR

Waltz et al., 1998

PC-12

Pheochromocytoma

rat

RT-PCR, FA

Gaudino et al., 1995

BA, binding assay; FA, functional assays; N, Northern blotting; RT-PCR, reverse transcriptase polymerase chain reaction; W, Western blotting.

ÿ465 to ÿ285 are important for expression of the RON transcript in CMT-93 cells (Waltz et al., 1998). The minimal promoter to drive RON expression in CMT-93 cells is located in the region containing nucleotides from ÿ465 to ‡1. Positive regulatory elements are located between ÿ465 and ÿ285 nucleotides. Cell-specific negative regulatory elements may be located between nucleotides ÿ585 to ÿ465. Analysis of the positive regulatory region identified four copies of the Ets-1 binding sequences, binding sites for SP-1, AP-1 and AP-2. Ets-1 putative binding sites are also located in the negative regulatory region. In addition to the above-mentioned regulatory elements the 50 -flanking region of the mouse RON gene contains IFN- and IFN -responsive elements, estrogen, and NFB (Waltz et al., 1998). These data suggest that RON expression may be regulated by a variety of cytokines and steroid hormones. Analysis of the human RON gene promoter sequence revealed several possible binding sites for transcriptional factor, including SP-1, AP-2, retinoblastoma control elements (RCE), IL-6responsive elements (IL-6RE) and GATA-1 (Del Gatto et al., 1995). Thus, some potential transcriptional factor binding sites for SP-1 and AP-2 were identified in both mouse and human RON promoters,

whereas several other identified regulatory elements are specific for the mouse or human RON promoter. In addition to the main RON promoter located in the 50 -flanking region of the gene, a strain-specific mouse alternative (internal) RON promoter has been identified (Persons et al., 1999). This internal promoter is located between exons 10 and 11 of the mouse RON gene, and it contains several consensus transcription factor binding sites including SP-1, Ets, Myb, and GATA-1. The internal promoter drives expression of truncated mouse RON, the amino acid sequence of which was published previously (Iwama et al., 1994). Although the human RON alternative promoter has not yet been described, the extremely high homology of this region between the human and the mouse RON gene suggests the existence of the alternative promoter in the human RON gene. Posttranslational Modification The main RON posttranslational modifications include proteolytic cleavage and glycosylation. Both murine and human RON contain eight potential N-linked glycosylation sites (Ronsin et al., 1993; Iwama et al., 1994; Gaudino et al., 1994). Ron is

10 Alla Danilkovitch-Miagkova and Edward J. Leonard Table 4 Human tissues that express RON Tissue

Detection methods

References

Lung

N

Ronsin et al., 1993

N

Gaudino et al., 1994

N

Sakamoto et al., 1997

N

Gaudino et al., 1994

IH

Montero-Julian et al., 1998

N

Gaudino et al., 1994

N

Sakamoto et al., 1997

IH

Montero-Julian et al., 1998

Colon mucosa

IH, RT-PCR

Okino et al., 1999

Small intestine

N

Sakamoto et al., 1997

Skin Colon

IH

Montero-Julian et al., 1998

Thymus

N

Sakamoto et al, 1997

Testis

N

Sakamoto et al., 1997

Prostate

N

Sakamoto et al., 1997

Monocytes

N

Gaudino et al., 1994

PMN

N

Gaudino et al., 1994

Bone marrow

N

Gaudinoet al., 1994

Bronchial epithelium

IH

Sakamoto et al., 1997

Ciliated epithelium of nasal mucosa and oviduct

IH

Sakamoto et al., 1997

Liver

RT-PCR, W

Chen et al., 1997

Tonsil

IH

Montero-Julian et al., 1998

Skin macrophages

IF, IH

Nanney et al., 1998

Keratinocytes

IH, ISH

Nanney et al., 1998

Bone marrow megakaryocytes (CD34‡)

RT-PCR, FC, FA

Banu et al., 1996

FA

Broxmeyer et al., 1996

FA

Kurihara et al., 1996

Osteoclasts

FA, functional assays; FC, flow cytometry; IH, immunohistochemistry; ISH, in situ hybridization; N, northern blotting; RT, PCR, reverse transcriptase polymerase chain reaction; W, western blotting.

synthesized as a 190 kDa single-chain precursor, which is converted into mature form by proteolytic cleavage in the endoplasmic reticulum (Gaudino et al., 1994). A conserved site for furin-like proteases (Barr, 1991; Mark et al., 1992) is present between residues 305±309 and 307±311 in human and murine RON precursors respectively (Ronsin et al., 1993; Gaudino et al., 1994; Iwama et al., 1994). In cells expressing physiological amounts of the receptor, only mature RON is exposed at the cell surface. In cells overexpressing transfected RON cDNA, a small amount of uncleaved protein was found at the cell surface (Gaudino et al., 1994).

Release of soluble receptors At present, data concerning release of soluble RON are unavailable.

SIGNAL TRANSDUCTION

Associated or intrinsic kinases Interaction of MSP with RON causes receptor tyrosine phosphorylation and activation (Gaudino

RON Receptor Table 5

Human patient tumor samples that express RON

Tissue

Detection methods References

Colon carcinomas IH, RT-PCR RT-PCR,

Okino et al., 1999 Chen et al., 1999

Breast carcinomas IH, W, FA

Maggiora et al., 1998

Hepatocellular carcinomas

Chen et al., 1997

RT-PCR, W, FA

Small lung cancer PCR

Angeloni et al., 2000

Non-small lung cancer

Angeloni et al., 2000

PCR

FA, functional assays; IH, immunohistochemistry; N, northern blotting; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction; W, western blotting.

Table 6 Murine tissues that express RON Tissue

Detection methods

References

Hematopoietic stem cells

PCR, RT-PCR

Iwama et al., 1994

Colon

RT-PCR, ISH

Waltz et al., 1998

N, ISH

Quantin et al., 1995

N

Gaudino et al., 1995

N, ISH

Quantin et al., 1995

N

Gaudino et al., 1995

Rectum

N, ISH

Quantin et al., 1995

Brain

RT-PCR,

Waltz et al., 1998

Stomach

RT-PCR

Waltz et al., 1998

N, ISH

Quantin et al., 1995

N

Gaudino et al., 1995

Keratinocytes

FA

Wang et al., 1996a

Adrenal

N

Gaudino et al., 1995

Brain

N

Gaudino et al., 1995

Small intestine

Muscle

N

Gaudino et al., 1995

Lung

N

Gaudino et al., 1995

Testis

N

Gaudino et al., 1995

Liver

N

Gaudino et al., 1995

Kidney

N

Gaudino et al., 1995

11

et al., 1994), a key event in initiation of signal transduction pathways mediating cellular responses to the ligand. RON is a receptor tyrosine kinase (Ronsin et al., 1993; Iwama et al., 1994; Gaudino et al., 1994), and MSP-induced upregulation of RON catalytic activity is mediated by autophosphorylation of two tyrosine residues (Y1238/1239 in human and Y1215/1216 in mouse RON) located within the RON kinase domain (Gaudino et al., 1994; Longati et al., 1994; Ponzetto et al., 1994). Once activated, RON kinase can phosphorylate tyrosines Y1353/ 1360 in human or Y1330/1337 in mouse receptors representing the RON C-terminal multifunctional docking site for downstream signaling molecules (Gaudino et al., 1994; Iwama et al., 1996). This tyrosine residue doublet can directly interact with PLC , the p85 subunit of PI-3 kinase, Shc, and Grb2 proteins (Iwama et al., 1996). Mutations of these tyrosines abolished MSP-mediated biological effects despite an unchanged kinase activity (Iwama et al., 1996). Data about molecules involved in MSP/ RON-activated signaling pathways and their biological role are summarized in Table 7. From Table 7 it is clear that not only intracellular molecules, but also several transmembrane receptors such as EPO receptor (Herley et al., 1999), MET (Follenzi et al., 2000) and the IL-3R c chain (Mera et al., 1999) are involved in MSP/RON-mediated signaling. RON is spatially and functionally associated with other transmembrane receptors including adhesion, cytokine, and growth factor receptors (DanilkovitchMiagkova et al., 2000; Danilkovitch-Miagkova and Leonard, 2001). The receptor collaboration extends throughout the spectrum of cellular responses generated by MSP/RON.

Cytoplasmic signaling cascades Ligand-stimulated RON triggers activation of multiple signaling pathways including the PI-3 kinase/ AKT, MAP kinase, and JNK pathways. These pathways mediate RON-induced biological effects. A schematic representation of RON-activated intracellular signaling cascades is shown in Figure 3.

DOWNSTREAM GENE ACTIVATION

Transcriptional factors activated FA, functional assays; ISH, in situ hybridization; N, northern blotting; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction.

At present, there are no data concerning transcriptional factors activated by MSP/RON interactions.

Table 7 Molecules involved in RON-mediated signaling Molecule

Cell type

Biochemical effects

Biological effects

References

JNK

Hematopoietic

Kinase activation

Apoptosis

Iwama et al., 1996

Epithelial

Kinase

ND

Santoro et al., 1998

Epithelial

Activation

ND

Danilkovitch et al., 2000

FAK

Epithelial

Kinase activation

Unidentified

Danilkovitch et al., 2000

c-Src

Cell-free system

Activation

ND

Ponzetto et al., 1994

Osteoclasts

Redistribution

Bone resorption

Kurihara et al., 1997

Epithelial

Activation

Unidentified

Danilkovitch et al., 2000

Epithelial

Activation

Proliferation

Santoro et al., 1996

Hematopoietic

Activation

ND

Iwama et al., 1996

Epithelial

Activation

ND

Santoro et al., 1998

Epithelial

Activation, and nucleal translocation

Survival

Danilkovitch et al., 2000

AKT

Epithelial

Activation; PY

Survival

Danilkovitch et al., 2000

PI-3 kinase

Macrophages

ND

Shape changes

Wang et al., 1996b

Macrophages

ND

Inhibition of iNOS

Chen et al., 1998

Hematopoietic

Association with RON

ND

Iwama et al., 1996

Epithelial

Association with RON; PY

Migration

Wang et al., 1996b; Xiao et al., 2000

Epithelial

ND

Adhesion

Danilkovitch et al., 1999b

Epithelial

ND

Survival

Danilkovitch et al., 2000

Ras

Epithelial

Translocation; activation

ND

Li et al., 1995

Sos

Epithelial

Association with RON

ND

Li et al., 1995

PLC

Hematopoietic

Association with RON

ND

Iwama et al., 1996

IL-3R c

Hematopoietic

Association with RON; PY

Shape changes

Mera et al., 1999

p90

Hematopoietic

PY

Shape changes

Mera et al., 1999

EPO receptor

Epithelial

PY

ND

MET

Epithelial

PY, activation

Sustained activation of signaling pathways

Follensi et al., 2000

Integrin

Epithelial

RON association

ND

Danilkovitch et al., 1999a

1

Epithelial

RON PY

ND

Danilkovitch-Miagkova et al., 2000

MAPK

Shc Grb2 p61/p65

ND, not determined; PY, tyrosine phosphorylation

RON Receptor

13

Figure 3 RON activated signaling pathways. Interaction of MSP with RON causes receptor tyrosine phosphorylation and activation. Ones, activated, RON phosphorylates tyrosines representing the C-terminal docking site for downstream molecules. These C-terminal phosphorylated tyrosine residues can directly interact with PLC , p85 subunit of PI-3 kinase, Src, and Grb2. Question marks indicate unidentified components of RON-induced signaling pathways.

?

PLCγ

?

P

P13k

? Shape change, adhesion, motility, inhibition of iNOS

RON

RON

MSP

FAK

?

P Grb2

Src

JNK

Mitogenic effects?

Sos AKT

Survival

?

Ras

?

Apoptosis

Bone resorbtion MAPK

Survival mitogenic effects

Genes induced Little is known about RON downstream gene targets. RON-induced secretion of IL-6 by macrophages (Skeel and Leonard, unpublished data) and megakaryocytes (Banu et al., 1996) suggest that the IL-6 gene is one RON target, but transcriptional factors involved in RON-mediated IL-6 gene activation and the molecular mechanism leading to IL-6 gene transcription remain to be investigated. MSP/RON-mediated biological activities include stimulation of cell proliferation and protection from apoptosis. On the basis of these data it is possible to suggest that the list of genes activated by RON may include genes involved in cell cycle regulation, such as cyclins and cyclin-dependent kinases and phosphatases and genes with anti-apoptotic activity such as proteins from the Bcl family.

Promoter regions involved At present, data concerning RON-regulated promoter regions involved in downstream gene activation are unavailable.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique Biological effects of activating the receptors RON Activity on Macrophages Expression of RON on murine macrophages is restricted to highly differentiated subpopulations expressing the F4/80 marker of macrophage maturation, and expression correlates with macrophage functional responses to MSP (Iwama et al., 1995). In humans, RON is detectable in all resident macrophages of the normal human dermis (Nanney et al., 1998). MSP activities on macrophages can be classified into two groups: activities affecting cytoplasmic membrane movement, and activities related to mediator production. Activation of RON by its ligand induces mouse resident peritoneal macrophages to become responsive to the chemoattractant C5a component of complement and to ingest C3bicoated erythrocytes (Leonard and Skeel, 1976; Skeel

14 Alla Danilkovitch-Miagkova and Edward J. Leonard et al., 1991; Skeel and Leonard, 1994). Later it was found that MSP can function alone as a chemoattractant for resident peritoneal macrophages (Skeel and Leonard, 1994). Additional MSP activities on macrophages affecting membrane movements are a macrophage shape change (Leonard and Skeel, 1976) and macropinocytosis (Leonard, unpublished data). The functional importance of the effects of MSP on macrophage motility is not clear, but it might facilitate wound debridement by MSP-stimulated macrophages. The other MSP actions on macrophages are related to mediator production. Endotoxin and/or combinations of proinflammatory cytokines lead to expression of inducible nitric oxide synthase (iNOS) and NO secretion by murine macrophages, which can be inhibited by MSP (Wang et al., 1994a). In vitro inhibition by MSP of endotoxin-induced NO production has also been shown to parallel in vivo effects. The concentration of nitrate in serum of mice that received intravenous endotoxin was higher in RON‡/ÿ than in serum of endotoxin-treated RON‡/‡ mice (Correll et al., 1997; Muraoka et al., 1999). This correlated with a higher lethality of endotoxin in the RON-deficient animals. IL-6 is another biologically active mediator, secretion of which is regulated by MSP (Skeel and Leonard, unpublished). It is possible that inflammatory reactions and immune responses via induction of IL-6 synthesis by macrophages can be modulated by MSP secretion. RON Activities on Epithelial Cells Cloning of human RON from a keratinocyte cDNA library (Ronsin et al., 1993) suggested that RON can be expressed on the surface of epithelial cells, and that in addition to macrophages epithelial cells can be also targets for MSP. Investigations of murine primary keratinocytes and keratinocyte cell lines showed that MSP specifically binds to RON on the surface of murine keratinocytes, causes RON phosphorylation, and induces chemotactic responses and proliferation (Wang et al., 1996a, 1996b). Investigations of the mechanisms of MSP-induced epithelial cell motility showed that MSP affects integrin-dependent cell adhesion (Danilkovitch et al., 1999b). RON was also detected in nasal and bronchial epithelium and in normal oviduct, and MSP was found to increase the ciliary beat frequency of nasal mucosal cells (Sakamoto et al., 1997). RON also was detected in epithelium of epididymis, which suggested a possible role of MSP in sperm motility and in mucociliary transport (Ohshiro et al., 1996). Functional assays of murine and human keratinocyte cell lines showed that MSP can protect

RON-expressing epithelial cells from apoptosis (Danilkovitch et al., 2000). In contrast to the antiapoptotic activity of MSP on keratinocytes, MSP inhibited the growth and induced apoptosis in neuroendocrine cell lines (Willett et al., 1997). Thus, MSP enhances adhesion and motility of epithelial cells, whereas its effects on epithelial cell growth and survival can be positive or negative, possibly depending on cell type and/or a stage of differentiation. RON Activities on Osteoclasts Transient RON expression was detected in foci of bone formation in mouse embryos (Quantin et al., 1995; Gaudino et al., 1995) and in multinucleated osteoclasts isolated from femurs of 14-day-old mice (Kurihara et al., 1996). These data suggested a possible role of MSP and RON in remodeling of developing bone. However MSP/RON effects are restricted to multinuclear osteoclast-like cells derived from cultured bone marrow (Kurihara et al., 1996). RON Activities on Vascular Endothelial Cells RON expression was detected by immunostaining on vascular endothelial cells in human dermis (Nanney et al., 1998). RON also has been found on the surface of a HUVEC cell line, and addition of MSP to these cells induces RON tyrosine phosphorylation (Danilkovitch et al., unpublished observations). MSP has angiogenic activity in the mouse cornea assay (Cao and Leonard, unpublished data). RON Activities on Hematopoietic Cells Mouse RON was cloned from bone marrow cells (Iwama et al., 1994) indicating that hematopoietic cells are MSP targets. MSP has dual effects on differentiation of bone marrow CD34‡ cells: inhibition of granulocyte±macrophage colony formation and stimulation of megakaryocytic differentiation. When isolated bone marrow CD34‡ cells were stimulated by combinations of cytokines such as CSF and SCF or VEGF, addition of MSP inhibited formation of granulocyte±macrophage colonies (Broxmeyer et al., 1996). Addition of MSP to human megakaryocytic cell lines as well as primary bone marrow megakaryocytes stimulated maturation of these cells (Banu et al., 1996). This MSP effect on megakaryocyte differentiation is mediated by MSPinduced secretion of IL-6 (Banu et al., 1996). MSP effects on erythroid and myeloid cells include growth stimulation or induction of apoptosis, depending on the cell type (Iwama et al., 1996).

RON Receptor

Possible pathophysiological role At present there are no data directly showing that abnormalities in RON or RON-mediated signaling lead to development of human disease, but accumulated data about RON expression and functional activities suggest possible involvement of RON in cancer, inflammation, and wound healing. Cancer Three independent sets of data indicate RON involvement in cancer development and progression. These are: (1) RON overexpression and ligandindependent activation in primary tumors and tumor cell lines; (2) experimental mutations in the RON kinase domain that result in oncogenic cell transformation; and (3) RON-mediated susceptibility to Friend virus-induced erythroleukemia in mice. RON analysis in tumor cell lines and samples from cancer patients showed that RON is overexpressed in some primary carcinomas and tumor cell lines (Chen et al., 1997; Maggiora et al., 1998; Willett et al., 1998). In a number of cases RON overexpression is accompanied by ligand-independent activation of RON (Maggiora et al., 1998). For example, RON is expressed at abnormally high levels in about 50% of primary breast carcinomas, but RON is only barely detected in normal mammary gland epithelium (Maggiora et al., 1998). Overexpressed RON is constitutively tyrosine phosphorylated, and in breast cells grown in vitro overexpression of RON resulted in proliferation, migration and invasion through reconstituted basement membrane. A tyrosine kinase expression profile in samples from patients with colon carcinomas contains 17 receptor tyrosine kinases including RON (Chen et al., 1999). RON is highly expressed and constitutively activated in several human colorectal carcinoma cell lines (Chen et al., 2000). Moreover, a novel constitutively active RON variant with molecular weight 160 kDa was identified in the HT-29 cell line, expression of which in human colorectal epithelial cell line CoTr increases cell motility and protects cells from apoptosis (Wang et al., 2000b). Expression of a splicing variant of RON transcript was detected in both malignant and nonmalignant human colonic mucosa (Okino et al., 1999). This splicing variant of RON is identical to the constitutively active RON found in human gastric cell line KATO-III (Collesi et al., 1996). In addition to breast and colon cancers, elevated RON expression has been detected in small and nonsmall lung cancer cell lines and tumor samples (Willett et al., 1998; Angeloni et al., 2000). Simultaneous expression of MSP and its receptor

15

RON by non-small cell carcinomas, and MSPincreased motility of a RON-positive adenosquamous carcinoma cell line support the hypothesis that MSP and RON might represent an autocrine/paracrine system involved in the pathogenesis of human lung cancer (Willett et al., 1998). The anti-apoptotic activity of MSP on epithelial cells suggest that activation of RON by overexpression or autocrine/ paracrine stimulation with ligand may lead to anchorage independence and tumorigenicity. In summary, these data suggest that RON may play a role in progression of human lung, breast and colon cancers, apoptosis resistance and an invasive-metastatic phenotype. Activating mutations in growth factor RTKs may result in oncogenic cell transformation and tumor development. A number of human neoplastic syndromes are associated with activating point mutations in a highly conserved region of the tyrosine kinase domains of Kit, Ret, and Met receptors (Schmidt et al., 1997). Mutation of aspartic acid at position 816 (D816V) in the Kit receptor causes mast cell leukemia and mastocytosis (Nagata et al., 1995; Tsujimura, 1996). Substitution of threonine for methionine in Ret (M918T) and in Met (M1268T) receptors is associated with multiple endocrine neoplasia (Santoro et al., 1995) and renal papillary carcinoma (Schmidt et al., 1997) respectively. Although activating mutations in RON associated with a particular type of cancer has not yet been identified, experimental mutations in the conserved region of the RON kinase domain at the positions that are homologous to the Kit, Ret, and Met oncogenic mutations result in a tumorigenic phenotype (Santoro et al., 1998; Williams et al., 1999). The tumorigenic potential of RON mutants has been determined by an in vitro focus formation assay and in vivo by tumor formation and growth in nude mice (Santoro et al., 1998). Infection of selected strains of mice with Friend virus causes erythroleukemia. A number of host genes affect susceptibility to Friend disease (Best et al., 1996; Kumar et al., 1978; Shibuya and Mak, 1982). Recently it has been shown that the FV2 (Friend virus susceptibility 2) locus, which mediates susceptibility to Friend virus-induced erythroleukemia in mice, encodes a truncated form of RON (Persons et al., 1999). Thus, a truncated form of RON might be involved in erythroleukemia development and/or progression. Because of the high similarity between mouse and human RON receptors, truncated RON might also play a role in human erythroleukemia. Preliminary results show that human erythroleukemic cell lines express truncated RON, which is constitutively tyrosine phosphorylated and activated

16 Alla Danilkovitch-Miagkova and Edward J. Leonard (Danilkovitch et al., unpublished data). This supports the hypothesis that RON is involved in development of human erythroleukemia. The role of IL-6 in multiple myeloma development was discovered when it was shown that IL-6 can induce in vitro growth of myeloma cells freshly isolated from patients (Ludwig et al., 1999). Bone marrow (stromal, monocytoid, and myeloid) cells secrete IL-6, which then can act as a paracrine factor. An autocrine loop of IL-6 stimulation of myeloma cells has also been described. IL-6 supports expansion of myeloma cells by stimulation of cell division and protection from apoptosis. High serum IL-6 has been predominantly found in patients with aggressive disease, and is correlated with a poor prognosis (Ludwig et al., 1999). MSP, the RON ligand, is an inducer of IL-6 synthesis by human bone marrow megakaryocytes and megakaryocytic cell lines (Banu et al., 1996) and by mouse peritoneal macrophages (Skeel and Leonard, unpublished data). These data suggest MSP as a potential regulator of IL-6-dependent cell functions, including proliferation and maturation of normal cells, as well as myeloma progression. Inflammation Macrophages, the first identified MSP targets, are important regulators of inflammation. Early studies showed that MSP stimulates macrophage cell shape changes, chemotactic migration to C5a and phagocytosis (Leonard and Skeel, 1976; Skeel and Leonard, 1994; Skeel et al., 1991). These data suggest that MSP/RON might enhance inflammation by stimulating macrophage motility and phagocytic activity. On the other hand, MSP exerts inhibitory effects on macrophages: addition of MSP to macrophages inhibits LPS and inflammatory cytokine-induced synthesis of iNOS in vitro (Chen et al., 1998; Wang et al., 1994a; Liu et al., 1999). Excessive production of NO by macrophages has cytototoxic effects on cells and tissues. In vitro data correlate with data obtained in vivo. RON‡/ÿ mice (which express half the amount of RON in comparison to RON‡/‡ mice) have enhanced reactions to endotoxin challenge and delayed-type hypersensitivity (DTH) challenge (Correll et al., 1997). RON‡/ÿ mice have higher nitrate concentrations in serum after injections of endotoxin, and at critical endotoxin dose only 20% of the RON‡/ÿ mice survived, compared with 80% survival for normal mice (Correll et al., 1997). DTH responses to oxazalone, measured as ear swelling, were more intense in RON‡/ÿ mice than in normal mice (Correll et al., 1997). In view of a proposed role of NO in inflammation (Grigoriadis et al., 1994), deficiency of MSP/RON-mediated inhibition of iNOS

synthesis and NO production by macrophages might contribute to septic shock and other pathophysiological conditions. Wound Healing In response to human wounds, RON is upregulated in keratinocytes and in accessory epithelial structures including sweat ducts and hair follicles (Nanney et al., 1998). At the same time active MSP and pro-MSP convertase activity were detected in wound fluid (Nanney et al., 1998). These findings suggest a possible role of MSP and RON in wound healing. Both macrophages and keratinocytes are MSP targets (Danilkovitch and Leonard, 1999; Leonard and Danilkovitch, 2000). The ability of MSP to stimulate macrophage motility responses and phagocytosis (Leonard and Danilkovitch, 2000) can enhance wound debridement by MSP-stimulated macrophages. MSP effects on keratinocytes also may have a beneficial role in the host response to tissue injury. In normal skin, detachment of the proliferating layer of keratinocytes from the basement membrane initiates a sequence of maturation steps that terminates in apoptosis. The hypertrophic layer in healing wounds might reflect inhibition of apoptosis by MSP, which is present in the wound fluid (Nanney et al., 1998). In addition to MSP/RON effects on macrophages, other contributions of MSP/RON to wound healing may include inhibition of apoptosis and stimulation of keratinocyte proliferation and migration. Although MSP knockout mice healed an incisional wound as rapidly as normal mice (Bezerra et al., 1998), this does not exclude the contribution of MSP/RON to the healing process.

Phenotypes of receptor knockouts and receptor overexpression mice Complete loss of RON leads to early embryonic death. RONÿ/ÿ embryos are viable through the blastocyst stage of development but fail to survive past the periimplantation period (Muraoka et al., 1999). Hemizygous (RON‡/ÿ) embryos develop normally. These mice can grow to adulthood, but they are highly susceptible to endotoxic shock (Correll et al., 1997; Muraoka et al., 1999). RON conditional knockout is a promising approach that can be used to study the role of RON in embryonic and adult tissues.

Human abnormalities At present, data concerning human abnormalities caused by RON are unavailable.

RON Receptor

THERAPEUTIC UTILITY At present, RON and anti-RON antibodies are not used for therapeutic purposes.

ACKNOWLEDGEMENTS The authors thank Dr J. Oppenheim for critical reading and discussion of this manuscript.

References Angeloni, D., and Lerman, M. (2001). Macrophage Stimulating 1 Receptor (MST1R). ABL. Atlas Genet Cytogenet Oncol Haematol. http://www.inforbiogen.fr/services/chromcancer/ Genes/ABL.html Angeloni, D., Danilkovitch-Miagkova, A., Ivanov, S. V., Breathnach, R., Johnson, B. E., Leonard, E. J., and Lerman, M. I. (2000). Gene structure of the human receptor tyrosine kinase RON and mutation analysis in lung cancer samples. Genes Chromosomes Cancer 29, 147±156. Banu, N., Price, D. J., London, R., Deng, B., Mark, M., Godowski, P. J., and Avraham, H. (1996). Modulation of megakaryocytopoiesis by human macrophage-stimulating protein, the ligand for the RON receptor. J. Immunol. 156, 2933±2940. Barr, P. J. (1991). Mammalian subtilisins: the long-sought dibasic processing endoproteases. Cell 66, 1±3. Best, S., Le Tissier, P., Towers, G., and Stoye, J. P. (1996). Positional cloning of the mouse retrovirus restriction gene Fv1. Nature 382, 826±829. Bezerra, J. A., Carrick, T. L., Degen, J. L., Witte, D., and Degen, S. J. F. (1998). Biological effects of targeted inactivation of hepatocyte growth factor-like protein in mice. J. Clin. Invest. 101, 1175±1183. Bork, P., Doerks, T., Springer, T. A., and Snel, B. (1999). Domains in plexins: links to integrins and transcription factors. Trends. Biochem. Sci. 24, 261±263. Broxmeyer, H. E., Cooper, S., Li, Z. H., Lu, L., Sarris, A., Wang, M. H., Chang, M. S., Donner, D. B., and Leonard, E. J. (1996). Macrophage-stimulating protein, a ligand for the RON receptor protein tyrosine kinase, suppresses myeloid progenitor cell proliferation and synergizes with vascular endothelial cell growth factor and members of the chemokine family. Ann. Hematol. 73, 1±9. Chen, Q., Seol, D. W., Carr, B., and Zarnegar, R. (1997). Coexpression and regulation of Met and Ron proto-oncogenes in human hepatocellular carcinoma tissues and cell lines. Hepatology 26, 59±66. Chen, W. S., Kung, H. J., Yang, W. K., and Lin, W. (1999). Comparative tyrosine-kinase profiles in colorectal cancers: enhanced arg expression in carcinoma as compared with adenoma and normal mucosa. Int. J. Cancer 83, 579±584. Chen, Y. Q., Fisher, J. H., and Wang, M. H. (1998). Activation of the RON receptor tyrosine kinase inhibits inducible nitric oxide synthase (iNOS) expression by murine peritoneal exudate macrophages: phosphatidylinositol±3 kinase is required for RON-mediated inhibition of iNOS expression. J. Immunol. 161, 4950±4959.

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Chen, Y. Q., Zhou, Y. Q., Angeloni, D., Kurtz, A. L., Qiang, X. Z., and Wang, M. H. (2000). Overexpression and activation of the RON receptor tyrosine kinase in a panel of human colorectal carcinoma cell lines. Exp. Cell Res. 261, 229±238. Collesi, C., Santoro, M. M., Gaudino, G., and Comoglio, P. M. (1996). A splicing variant of the RON transcript induces constitutive tyrosine kinase activity and an invasive phenotype [published erratum appears in Mol. Cell. Biol. 1997 Jan;17(1):528]. Mol. Cell. Biol. 16, 5518±5526. Comoglio, P. M., Tamagnone, L., and Boccaccio, C. (1999). Plasminogen-related growth factor and semaphorin receptors: a gene superfamily controlling invasive growth. Exp. Cell Res. 253, 88±99. Correll, P. H., Iwama, A., Tondat, S., Mayrhofer, G., Suda, T., and Bernstein, A. (1997). Deregulated inflammatory response in mice lacking the STK/RON receptor tyrosine kinase. Genes Funct. 1, 69±83. Danilkovitch, A., and Leonard, E. J. (1999). Kinases involved in MSP/RON signaling. J. Leukoc. Biol. 65, 345±348. Danilkovitch, A., Skeel, A. and Leonard, E. J. (1999a). Macrophage stimulating protein-induced epithelial cell adhesion is mediated by a PI3-K-dependent, but FAK-independent mechanism. Exp. Cell Res. 248, 575±582. Danilkovitch, A., Miller, M., and Leonard, E. J. (1999b). Interaction of macrophage-stimulating protein with its receptor. Residues critical for beta chain binding and evidence for independent alpha chain binding. J. Biol. Chem. 274, 29937±29943. Danilkovitch, A., Donley, S., Skeel, A., and Leonard, E. J. (2000). Two independent signaling pathways mediate the antiapoptotic action of macrophage-stimulating protein on epithelial cells. Mol. Cell. Biol. 20, 2218±2227. Danilkovitch-Miagkova, A., Angeloni, D., Skeel, A., Donley, S., Lerman, M., and Leonard, E. J. (2000). Integrin-mediated RON growth factor receptor phosphorylation requires tyrosine kinase activity of both the receptor and c-Src. J. Biol. Chem. 275, 14783±14786. Danilkovitch-Miagkova, A., and Leonard, E. J. (2001). Cross-talk between RON receptor tyrosine kinase and other transmembrane receptors. Histol. Histopathol. 16, 623±631. Del Gatto, F., Gilbert, E., Ronsin, C., and Breathnach, R. (1995). Structure of the promoter for the human macrophage stimulating protein receptor gene. Biochim. Biophys. Acta 1263, 93±95. Follenzi, A., Bakovic, S., Gual, P., Stella, M. C., Longati, P., and Comoglio, P. M. (2000). Cross-talk between the protooncogenes Met and Ron. Oncogene 19, 3041±3049. Gaudino, G., Follenzi, A., Naldini, L., Collesi, C., Santoro, M., Gallo, K. A., Godowski, P. J., and Comoglio, P. M. (1994). RON is a heterodimeric tyrosine kinase receptor activated by the HGF homologue MSP. EMBO J. 13, 3524±3532. Gaudino, G., Avantaggiato, V., Follenzi, A., Acampora, D., Simeone, A., and Comoglio, P. M. (1995). The proto-oncogene RON is involved in development of epithelial, bone and neuroendocrine tissues. Oncogene 11, 2627±2637. Grigoriadis, A. E., Wang, Z. Q., Cecchini, M. G., Hofstetter, W., Felix, R., Fleisch, H. A., and Wagner, E. F. (1994). c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 266, 443±448. Han, S., Stuart, L. A., and Degen, S. J. (1991). Characterization of the DNF15S2 locus on human chromosome 3: identification of a gene coding for four kringle domains with homology to hepatocyte growth factor. Biochemistry 30, 9768±9780. Herley, M.T., D'Andrea, A.D., and Ney, P.A. (1999). Physical and functional interactions between the erythropoetin receptor and a truncated form of the STK/RON receptor tyrosine kinase. Blood 94, 652a.

18 Alla Danilkovitch-Miagkova and Edward J. Leonard Herley, M. T., D'Andrea, A. D., and Ney, P. A. (2000). Physical and functional interactions between the erythropoietin receptor and a truncated form of the STK/RON receptor tyrosine kinase. Blood 94, 652a/652a. Huff, J. L., Jelinek, M. A., Borgman, C. A., Lansing, T. J., and Parsons, J. T. (1993). The proto-oncogene c-sea encodes a transmembrane protein-tyrosine kinase related to the Met/hepatocyte growth factor/scatter factor receptor. Proc. Natl Acad. Sci. USA 90, 6140±6144. Iwama, A., Okano, K., Sudo, T., Matsuda, Y., and Suda, T. (1994). Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells. Blood 83, 3160±3169. Iwama, A., Wang, M. H., Yamaguchi, N., Ohno, N., Okano, K., Sudo, T., Takeya, M., Gervais, F., Morissette, C., and Leonard, E. J. (1995). Terminal differentiation of murine resident peritoneal macrophages is characterized by expression of the STK protein tyrosine kinase, a receptor for macrophage-stimulating protein. Blood 86, 3394±3403. Iwama, A., Yamaguchi, N., and Suda, T. (1996). STK/RON receptor tyrosine kinase mediates both apoptotic and growth signals via the multifunctional docking site conserved among the HGF receptor family. EMBO J. 15, 5866±5875. Kolodkin, A. L., Matthes, D. J., and Goodman, C. S. (1993). The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell 75, 1389± 1399. Kumar, V., Resnick, P., Eastcott, J. W., and Bennett, M. (1978). Mechanism of genetic resistance to Friend virus leukemia in mice. V. Relevance of Fv-3 gene in the regulation of in vivo immunosuppression. J. Natl Cancer Inst. 61, 1117±1123. Kurihara, N., Iwama, A., Tatsumi, J., Ikeda, K., and Suda, T. (1996). Macrophage-stimulating protein activates STK receptor tyrosine kinase on osteoclasts and facilitates bone resorption by osteoclast-like cells. Blood 87, 3704±3710. Leonard, E. J., and Danilkovitch, A. (2000). Macrophage stimulating protein. Adv. Cancer Res. 77, 139±167. Leonard, E. J., and Skeel, A. (1980). Functional differences between resident and exudates peritoneal mouse macrophages: specific serum protein requirements for responsiveness to chemotaxins J.Reticuloendothel. Soc. 28, 437±447. Leonard, E. J., and Skeel, A. (1976). A serum protein that stimulates macrophage movement, chemotaxis and spreading. Exp. Cell Res. 102, 434±438. Li, B. Q., Wang, M. H., Kung, H. F., Ronsin, C., Breathnach, R., Leonard, E. J., and Kamata, T. (1995). Macrophage-stimulating protein activates Ras by both activation and translocation of SOS nucleotide exchange factor. Biochem. Biophys. Res. Commun. 216, 110±118. Liu, Q. P., Fruit, K., Ward, J., and Correll, P. H. (1999). Negative regulation of macrophage activation in response to IFN-gamma and lipopolysaccharide by the STK/RON receptor tyrosine kinase. J. Immunol. 163, 6606±6613. Longati, P., Bardelli, A., Ponzetto, C., Naldini, L., and Comoglio, P. M. (1994). Tyrosines1234±1235 are critical for activation of the tyrosine kinase encoded by the MET proto-oncogene (HGF receptor). Oncogene 9, 49±57. Ludwig, H., Meran, J., and Zojer, N. (1999). Multiple myeloma: an update on biology and treatment. Ann. Oncol. 10 (Suppl. 6), 31±43. Maestrini, E., Tamagnone, L., Longati, P., Cremona, O., Gulisano, M., Bione, S., Tamanini, F., Neel, B. G., Toniolo, D., and Comoglio, P. M. (1996). A family of transmembrane proteins with homology to the MET-hepatocyte growth factor receptor. Proc. Natl Acad. Sci. USA 93, 674±678.

Maggiora, P., Marchio, S., Stella, M. C., Giai, M., Belfiore, A., De Bortoli, M., Di Renzo, M. F., Costantino, A., Sismondi, P., and Comoglio, P. M. (1998). Overexpression of the RON gene in human breast carcinoma. Oncogene 16, 2927±2933. Mark, M. R., Lokker, N. A., Zioncheck, T. F., Luis, E. A., and Godowski, P. J. (1992). Expression and characterization of hepatocyte growth factor receptor-IgG fusion proteins. Effects of mutations in the potential proteolytic cleavage site on processing and ligand binding. J. Biol. Chem. 267, 26166± 26171. Mera, A., Suga, M., Ando, M., Suda, T., and Yamaguchi, N. (1999). Induction of cell shape changes through activation of the interleukin-3 common beta chain receptor by the RON receptor-type tyrosine kinase. J. Biol. Chem. 274, 15766±15774. Montero-Julian, F. A., Dauny, I., Flavetta, S., Ronsin, C., Andre, F., Xerri, L., Wang, M. H., Marvaldi, J., Breathnach, R., and Brailly, H. (1998). Characterization of two monoclonal antibodies against the RON tyrosine kinase receptor. Hybridoma 17, 541±551. Muraoka, R. S., Sun, W. Y., Colbert, M. C., Waltz, S. E., Witte, D. P., Degen, J. L., and Friezner, D. S. (1999). The Ron/STK receptor tyrosine kinase is essential for peri-implantation development in the mouse. J. Clin. Invest. 103, 1277±1285. Nagata, H., Worobec, A. S., Oh, C. K., Chowdhury, B. A., Tannenbaum, S., Suzuki, Y., and Metcalfe, D. D. (1995). Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder. Proc. Natl Acad. Sci. USA 92, 10560±10564. Nakamura, T., Aoki, S., Takahashi, T., Matsumoto, K., and Kiyohara, T. (1996). Cloning and expression of Xenopus HGF-like protein (HLP) and Ron/HLP receptor implicate their involvement in early neural development. Biochem. Biophys. Res. Commun. 224, 564±573. Nanney, L. B., Skeel, A., Luan, J., Polis, S., Richmond, A., Wang, M. H., and Leonard, E. J. (1998). Proteolytic cleavage and activation of pro-macrophage-stimulating protein and upregulation of its receptor in tissue injury. J. Invest. Dermatol. 111, 573±581. Ohshiro, K., Iwama, A., Matsuno, K., Ezaki, T., Sakamoto, O., Hamaguchi, I., Takasu, N., and Suda, T. (1996). Molecular cloning of rat macrophage-stimulating protein and its involvement in the male reproductive system. Biochem. Biophys. Res. Commun. 227, 273±280. Okino, T., Egami, H., Ohmachi, H., Takai, E., Tamori, Y., Nakagawa, K., Nakano, S., Akagi, J., Sakamoto, O., Suda, T., and Ogawa, M. (1999). Presence of RON receptor tyrosine kinase and its splicing variant in malignant and non-malignant human colonic mucosa. Int. J. Oncol. 15, 709±714. Persons, D. A., Paulson, R. F., Loyd, M. R., Herley, M. T., Bodner, S. M., Bernstein, A., Correll, P. H., and Ney, P. A. (1999). Fv2 encodes a truncated form of the Stk receptor tyrosine kinase. Nature Genet. 23, 159±165. Ponzetto, C., Bardelli, A., Zhen, Z., Maina, F., Dalla, Z. P., Giordano, S., Graziani, A., Panayotou, G., and Comoglio, P. M. (1994). A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell 77, 261±271. Quantin, B., Schuhbaur, B., Gesnel, M. C., Doll'e, P., and Breathnach, R. (1995). Restricted expression of the ron gene encoding the macrophage stimulating protein receptor during mouse development. Dev. Dyn. 204, 383±390.

RON Receptor Ronsin, C., Muscatelli, F., Mattei, M. G., and Breathnach, R. (1993). A novel putative receptor protein tyrosine kinase of the met family. Oncogene 8, 1195±1202. Sakamoto, O., Iwama, A., Amitani, R., Takehara, T., Yamaguchi, N., Yamamoto, T., Masuyama, K., Yamanaka, T., Ando, M., and Suda, T. (1997). Role of macrophage-stimulating protein and its receptor, RON tyrosine kinase, in ciliary motility. J. Clin. Invest. 99, 701±709. Santoro, M., Carlomagno, F., Romano, A., Bottaro, D. P., Dathan, N. A., Grieco, M., Fusco, A., Vecchio, G., Matoskova, B., and Kraus, M. H. (1995). Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B. Science 267, 381±383. Santoro, M., Collesi, C., Grisendi, S., Gaudino, G., and Comoglio, P.M. (1996). Constitutive activation of the RON gene promotes invasive growth but not transformation. [Erratum, 17, 1758, 1997.] Mol. Cell. Biol. 16, 7072±7083. Santoro, M. M., Penengo, L., Minetto, M., Orecchia, S., Cilli, M., and Gaudino, G. (1998). Point mutations in the tyrosine kinase domain release the oncogenic and metastatic potential of the Ron receptor. Oncogene 17, 741±749. Schmidt, L., Duh, F. M., Chen, F., Kishida, T., Glenn, G., Choyke, P., Scherer, S. W., Zhuang, Z., Lubensky, I., Dean, M., Allikmets, R., Chidambaram, A., Bergerheim, U. R., Feltis, J. T., Casadevall, C., Zamarron, A., Bernues, M., Richard, S., Lips, C. J., Walther, M. M., Tsui, L. C., Geil, L., Orcutt, M. L., Stackhouse, T., and Zbar, B. (1997). Germline and somatic mutations in the tyrosine kinase domain of the MET protooncogene in papillary renal carcinomas. Nature Genet. 16, 68±73. Shibuya, T., and Mak, T. W. (1982). A host gene controlling early anaemia or polycythaemia induced by Friend erythroleukaemia virus. Nature 296, 577±579. Skeel, A., and Leonard, E. J. (1994). Action and target cell specificity of human macrophage-stimulating protein (MSP). J. Immunol. 152, 4618±4623. Skeel, A., Yoshimura, T., Showalter, S. D., Tanaka, S., Appella, E., and Leonard, E. J. (1991). Macrophage stimulating protein: purification, partial amino acid sequence, and cellular activity. J. Exp. Med. 173, 1227±1234. Tamagnone, L., Artigiani, S., Chen, H., He, Z., Ming, G. I., Song, H., Chedotal, A., Winberg, M. L., Goodman, C. S., Poo, M., Tessier-Lavigne, M., and Comoglio, P. M. (1999). Plexins are a large family of receptors for transmembrane, secreted, and GPIanchored semaphorins in vertebrates. Cell 99, 71±80. Tsujimura, T. (1996). Role of c-kit receptor tyrosine kinase in the development, survival and neoplastic transformation of mast cells. Pathol. Int. 46, 933±938. Wahl, R. C., Hsu, R. Y., Huff, J. L., Jelinek, M. A., Chen, K., Courchesne, P., Patterson, S. D., Parsons, J. T., and Welcher, A. A. (1999). Chicken macrophage stimulating protein is a ligand of the receptor protein-tyrosine kinase Sea. J. Biol. Chem. 274, 26361±26368. Waltz, S. E., McDowell, S. A., Muraoka, R. S., Air, E. L., Flick, L. M., Chen, Y. Q., Wang, M. H., and Degen, S. J. (1997). Functional characterization of domains contained in hepatocyte growth factor-like protein. J. Biol. Chem. 272, 30526±30537. Waltz, S. E., Toms, C. L., McDowell, S. A., Clay, L. A., Muraoka, R. S., Air, E. L., Sun, W. Y., Thomas, M. B., and Degen, S. J. (1998). Characterization of the mouse Ron/Stk receptor tyrosine kinase gene. Oncogene 16, 27±42. Wang, M. H., Cox, G. W., Yoshimura, T., Sheffler, L. A., Skeel, A., and Leonard, E. J. (1994a). Macrophage-stimulating protein

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inhibits induction of nitric oxide production by endotoxin- or cytokine-stimulated mouse macrophages. J. Biol. Chem. 269, 14027±14031. Wang, M. H., Ronsin, C., Gesnel, M. C., Coupey, L., Skeel, A., Leonard, E. J., and Breathnach, R. (1994b). Identification of the ron gene product as the receptor for the human macrophage stimulating protein. Science 266, 117±119. Wang, M. H., Iwama, A., Skeel, A., Suda, T., and Leonard, E. J. (1995). The murine stk gene product, a transmembrane protein tyrosine kinase, is a receptor for macrophage-stimulating protein. Proc. Natl Acad. Sci. USA 92, 3933±3937. Wang, M. H., Dlugosz, A. A., Sun, Y., Suda, T., Skeel, A., and Leonard, E. J. (1996a). Macrophage-stimulating protein induces proliferation and migration of murine keratinocytes. Exp. Cell Res. 226, 39±46. Wang, M. H., Montero-Julian, F. A., Dauny, I., and Leonard, E. J. (1996b). Requirement of phosphatidylinositol-3 kinase for epithelial cell migration activated by human macrophage stimulating protein. Oncogene 13, 2167±2175. Wang, M. H., Julian, F. M., Breathnach, R., Godowski, P. J., Takehara, T., Yoshikawa, W., Hagiya, M., and Leonard, E. J. (1997). Macrophage stimulating protein (MSP) binds to its receptor via the MSP beta chain. J. Biol. Chem. 272, 16999± 17004. Wang, M. H., Fung, H. L., and Chen, Y. Q. (2000a). Regulation of the RON receptor tyrosine kinase expression in macrophages: blocking the RON gene transcription by endotoxininduced nitric oxide. J. Immunol. 164, 3815±3821. Wang, M. H., Kurtz, A. L., and Chen, Y. (2000b). Identification of a novel splicing product of the RON receptor tyrosine kinase in human colorectal carcinoma cells. Carcinogenesis 21, 1507± 1512. Willett, C. G., Smith, D. I., Shridhar, V., Wang, M. H., Emanuel, R. L., Patidar, K., Graham, S. A., Zhang, F., Hatch, V., Sugarbaker, D. J., and Sunday, M. E. (1997). Differential screening of a human chromosome 3 library identifies hepatocyte growth factor-like/macrophage-stimulating protein and its receptor in injured lung. Possible implications for neuroendocrine cell survival. J. Clin. Invest. 99, 2979±2991. Willett, C. G., Wang, M. H., Emanuel, R. L., Graham, S. A., Smith, D. I., Shridhar, V., Sugarbaker, D. J., and Sunday, M. E. (1998). Macrophage-stimulating protein and its receptor in non-small-cell lung tumors: induction of receptor tyrosine phosphorylation and cell migration. Am. J. Respir. Cell Mol. Biol. 18, 489±496. Williams, T. A., Longati, P., Pugliese, L., Gual, P., Bardelli, A., and Michieli, P. (1999). MET(PRC) mutations in the Ron receptor result in upregulation of tyrosine kinase activity and acquisition of oncogenic potential. J. Cell Physiol. 181, 507±514. Xiao, Z. Q., Chen, Y. Q., and Wang, M. H. (2000). Requirement of both tyrosine residues 1330 and 1337 in the C-terminal tail of the RON receptor tyrosine kinase for epithelial cell scattering and migration. Biochem. Biophys. Res. Commun. 267, 669±675. Yoshimura, T., Yuhki, N., Wang, M. H., Skeel, A., and Leonard, E. J. (1993). Cloning, sequencing, and expression of human macrophage stimulating protein (MSP, MST1) confirms MSP as a member of the family of kringle proteins and locates the MSP gene on chromosome 3. J. Biol. Chem. 268, 15461± 15468. Yu, H. H., and Kolodkin, A. L. (1999). Semaphorin signaling: a little less per-plexin. Neuron 22, 11±14.

20 Alla Danilkovitch-Miagkova and Edward J. Leonard

LICENSED PRODUCTS Anti-human RON rabbit polyclonal antibodies (C-20, Cat# sc-322, Santa Cruz, Santa Clarita, CA, USA)

Anti-mouse RON goat polyclonal antibodies (Cat# AF431, R&D Systems, Minneapolis, MN, USA)

Anti-human RON mouse monoclonal antibodies (ID1 and ID2, Immunotech, France) (Montero-Julian et al., 1998)

Anti-phospho RON rabbit polyclonal antibodies (Cat#44-442, Biosource International, Camarillo, CA, USA)

Anti-RON mouse monoclonal antibodies (Cat#R61120 , Transduction Lab., Lexington, KY, USA) Recombinant mouse RON receptor/Fc Chimera (Cat#431-MS, R&D Systems, Minneapolis, MN, USA)

These antibodies were developed against a synthetic peptide corresponding to the C-terminal part of human RON (blocking peptide is available). Antibodies recognize human RON, do not crossreact with MET and mouse RON. Antibodies can be used for IP, WB, IH. These antibodies, developed against mouse recombinant RON, were produced by the mouse myeloma NSO cell line. Antibodies recognize the extracellular domain of mouse RON, and do not crossreact with MET and human RON. Antibodies can be used for ELISA, IP, WB, MSP neutralizing assays. These antibodies were developed by immunizing mice with MDCK cells overexpressing human RON (RE7 clone). Antibodies recognize the extracellular domain of human RON, and do not crossreact with MET and mouse RON. Antibodies can be used for IP, FC, IH. They block MSP binding to RON and MSP biological activities. These antibodies were developed against a synthetic peptide of mouse RON containing phosphorylated tyrosines Y1215 and 1216. Antibodies recognize tyrosine phosphorylated mouse and human RON. Antibodies can be used for WB. These antibodies were developed against the  chain of human RON. Antibodies recognize the 40 kDa  chain of mouse, rat, frog, dog and human RON. Antibodies can be used for WB and IF. A DNA sequence encoding the extracellular domain of mouse RON (corresponding to 1±960 amino acids) was fused to the C-terminal 6X histidine-tagged Fc region of human IgG1 via a polypeptide linker. The chimeric protein was expressed in the NSO mouse myeloma cell line. The product can be used for ELISA, MSP binding assays.

WB, western blotting; IP, immunoprecipitation; IH, immunohistochemistry; FC, flow cytometry; IF, immunofluorescence.