Human GRB-IRβ/GRB10
1997; Elsevier BV; Volume: 272; Issue: 5 Linguagem: Inglês
10.1074/jbc.272.5.2659
ISSN1083-351X
AutoresJ. Daniel Frantz, Sophie Giorgetti‐Peraldi, Elizabeth A. Ottinger, Steven E. Shoelson,
Tópico(s)Diabetes and associated disorders
ResumocDNA clones encoding human (h) Grb7 and a previously unknown protein with high homology to hGrb-IR and mGrb10 (where m indicates mouse) were found by screening expressed sequence tag data bases. hGrb7 mRNA expression is greatest in pancreas and restricted to a few other tissues. The second protein termed hGrb-IRβ/Grb10 contains an intact PH domain and lacks the 80-residue mGrb10 insertion. Expression is greatest in pancreas and muscle but occurs in nearly all tissues. hGrb-IRβ/Grb10 and hGrb-IR likely arise as alternative mRNA splicing products of a common gene. Reverse transcriptase-coupled polymerase chain reaction shows both mRNAs in muscle. In cells, Grb-IRβ/Grb10 protein translocates from cytosol to membrane upon insulin stimulation, most likely due to direct interactions with the insulin receptor. These interactions are mediated by the SH2 domain and additional regions of the protein. Studies with mutated receptors and synthetic phosphopeptides show that the hGrb-IRβ/Grb10 SH2 domain binds at least two sites in the insulin receptor: the kinase activation loop > the juxtamembrane site. hGrb-IRβ/Grb10 also binds a 135-kDa phosphoprotein in unstimulated 3T3-L1 adipocytes; binding is reduced upon insulin stimulation. In addition, the c-Abl SH3 domain binds Grb-IR/Grb10, whereas Fyn, phosphatidylinositol 3-kinase p85, and Grb2 SH3 domains do not. The site of c-Abl SH3 domain interaction is highly conserved within the Grb-IR/Grb10/Grb7/Grb14 family. hGrb-IRβ/Grb10 also binds platelet-derived growth factor and epidermal growth factor receptors, suggesting a broader role in the signaling pathways of numerous receptors. We conclude that hGrb-IRβ/Grb10 is a widely expressed, PH and SH2 domain-containing, SH3 domain-binding protein that functions downstream from activated insulin and growth factor receptors. cDNA clones encoding human (h) Grb7 and a previously unknown protein with high homology to hGrb-IR and mGrb10 (where m indicates mouse) were found by screening expressed sequence tag data bases. hGrb7 mRNA expression is greatest in pancreas and restricted to a few other tissues. The second protein termed hGrb-IRβ/Grb10 contains an intact PH domain and lacks the 80-residue mGrb10 insertion. Expression is greatest in pancreas and muscle but occurs in nearly all tissues. hGrb-IRβ/Grb10 and hGrb-IR likely arise as alternative mRNA splicing products of a common gene. Reverse transcriptase-coupled polymerase chain reaction shows both mRNAs in muscle. In cells, Grb-IRβ/Grb10 protein translocates from cytosol to membrane upon insulin stimulation, most likely due to direct interactions with the insulin receptor. These interactions are mediated by the SH2 domain and additional regions of the protein. Studies with mutated receptors and synthetic phosphopeptides show that the hGrb-IRβ/Grb10 SH2 domain binds at least two sites in the insulin receptor: the kinase activation loop > the juxtamembrane site. hGrb-IRβ/Grb10 also binds a 135-kDa phosphoprotein in unstimulated 3T3-L1 adipocytes; binding is reduced upon insulin stimulation. In addition, the c-Abl SH3 domain binds Grb-IR/Grb10, whereas Fyn, phosphatidylinositol 3-kinase p85, and Grb2 SH3 domains do not. The site of c-Abl SH3 domain interaction is highly conserved within the Grb-IR/Grb10/Grb7/Grb14 family. hGrb-IRβ/Grb10 also binds platelet-derived growth factor and epidermal growth factor receptors, suggesting a broader role in the signaling pathways of numerous receptors. We conclude that hGrb-IRβ/Grb10 is a widely expressed, PH and SH2 domain-containing, SH3 domain-binding protein that functions downstream from activated insulin and growth factor receptors. INTRODUCTIONMany of the effects of activated tyrosine kinase-linked receptors are mediated by cascades of intracellular tyrosine phosphorylation reactions. Receptors with intrinsic kinase activity typically phosphorylate themselves, and in many cases the phosphorylated receptor tyrosines serve as docking sites for SH2 domain proteins (1Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2217) Google Scholar, 2Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (924) Google Scholar). Since many SH2 domain proteins either are enzymes or associate with enzymes, these interactions provide a mechanism for recruiting catalytic effectors to the activated receptor. In the case of insulin signaling, autophosphorylation activates the receptor kinase (3White M.F. Shoelson S.E. Keutmann H. Kahn C.R. J. Biol. Chem. 1988; 263: 2969-2980Abstract Full Text PDF PubMed Google Scholar, 4Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (954) Google Scholar) and creates a docking site for substrate protein PTB domains (5Wolf G. Trub T. Ottinger E. Groninga L. Lynch A. White M.F. Miyazaki M. Lee J. Shoelson S.E. J. Biol. Chem. 1995; 270: 27407-27410Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 6Eck M.J. Dhe-Paganon S. Trub T. Nolte R. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Both effects are necessary to trigger intracellular pathways via the substrates IRS-1 and Shc. However, the SH2 domain effectors of insulin action bind primarily to the phosphorylated substrate proteins rather than the insulin receptor itself.The recent discovery of an SH2 domain protein called Grb-IR was met with considerable interest because it binds the insulin receptor and not its substrates (7Liu F. Roth R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10287-10291Crossref PubMed Scopus (150) Google Scholar). However, this Grb7-like protein reportedly inhibits insulin signaling and contains an unusual 46-residue deletion within its apparent PH domain. Therefore, we have considered the possibility that additional related proteins (potentially with intact domains) might exist to provide positive signals downstream from the insulin receptor. Three members of the Grb7 family (Grb7, Grb10, and Grb14) have been identified by screening cDNA expression libraries with phosphorylated fragments of the EGF 1The abbreviations used are: EGFepidermal growth factorRT-PCRreverse transcriptase-coupled PCR5′-RACE5′-rapid amplification of cDNA endsDMEMDulbecco's modified Eagle's mediumGSTglutathione S-transferasekbkilobase pair(s)bpbase pair(s)PDGFplatelet-derived growth factorPI 3-kinasephosphatidylinositol 3-kinasePAGEpolyacrylamide gel electrophoresis. receptor (8Margolis B. Silvennoinen O. Comoglio F. Roonprapunt C. Skolnik E. Ullrich A. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8894-8898Crossref PubMed Scopus (149) Google Scholar, 9Ooi J. Yajnik V. Immanuel D. Gordon M. Moskow J.J. Buchberg A.M. Margolis B. Oncogene. 1995; 10: 1621-1630PubMed Google Scholar, 10Daly R.J. Sanderson G.M. Janes P.W. Sutherland R.L. J. Biol. Chem. 1996; 271: 12502-12510Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Although each is derived from a distinct genetic locus, they share a common domain architecture: a C-terminal SH2 domain and >300 residues of extended homology that encompasses a PH domain. mGrb10 also contains an 80-residue insertion, relative to mGrb7 and hGrb14. Its function is unknown. Although the presence of SH2 and PH domains strongly implies a role for these proteins in cellular signaling, their physiologic functions remain vague. Nevertheless, all have been implicated in neoplastic conditions. mGrb7 binds and is coamplified with HER2/neu in certain types of breast cancer (11Stein D. Wu J. Fuqua S.A.W. Roonprapunt C. Yajnik V. D'Eustachio P. Moskow J.J. Buchberg A.M. Osborne C.K. Margolis B. EMBO J. 1994; 13: 1331-1340Crossref PubMed Scopus (219) Google Scholar). mGrb10 binds the Ret receptor (12Pandey A. Duan H. Di Fiore P.P. Dixit V.M. J. Biol. Chem. 1995; 270: 21461-21463Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), whose gene (the ret protooncogene) is rearranged and activated in certain thyroid carcinomas and contains germ line mutations associated with syndromes of multiple endocrine neoplasias (13Grieco M. Santoro M. Berlingieri M.T. Melillo R.M. Donghi R. Bongarzone I. Pierotti M.A. Della Porta G. Fusco A. Vecchio G. Cell. 1990; 60: 557-563Abstract Full Text PDF PubMed Scopus (832) Google Scholar, 14Santoro M. Carlomagno F. Romano A. Bottaro D.P. Dathan N.A. Grieco M. Fusco A. Vecchio G. Matoskova B. Kraus M.H. Di Fiore P.P. Science. 1995; 267: 381-383Crossref PubMed Scopus (794) Google Scholar). And expression of Grb14 may be elevated in estrogen receptor-positive breast cancer cell lines and certain prostate cancer cells (10Daly R.J. Sanderson G.M. Janes P.W. Sutherland R.L. J. Biol. Chem. 1996; 271: 12502-12510Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). hGrb-IR and mGrb10 proteins share regions of high homology, although the two are not simple species variants. hGrb-IR has not been implicated in oncogenesis.Expressed sequence tag data bases (dbEST) were screened as a strategy to identify related proteins. Two clones were found. 2Damien Dunnington (SmithKline Beecham Pharmaceuticals) first identified these clones in the Human Genome Sciences data base. One encodes human Grb7. Further sequencing of the second clone revealed a previously unknown protein with high homology to hGrb-IR and mGrb10, which we refer to as hGrb-IRβ/Grb10. It has an intact PH domain and lacks the mGrb10 insertion. hGrb-IRβ/Grb10 and hGrb-IR probably represent alternative mRNA splicing products of a common gene. Both mRNAs are expressed in muscle, a major site of insulin action. hGrb-IRβ/Grb10 protein is present in the cytosol of unstimulated Rat1 fibroblasts and translocates to the membrane following insulin stimulation. We have characterized hGrb-IRβ/Grb10 interactions with activated insulin, EGF, and PDGF receptors and SH3 domain proteins. We conclude that Grb-IRβ/Grb10 is a previously unknown signaling protein that may function downstream from activated insulin and growth factor receptors.DISCUSSIONWe have identified a variant transcript of the human Grb-IR/Grb10 gene. The encoded protein has high sequence homology with hGrb-IR and mGrb10, although its domain architecture is more similar to that of Grb7 and Grb14. This is because hGrb-IR contains a 46-residue deletion within its PH domain and 58-residue extension at its N terminus, relative to our protein, Grb7 and Grb14. mGrb10 contains an 80-residue insertion, relative to the other proteins. The functions of these insertions, deletions, and extensions are unknown. We have analyzed functions of Grb-IR/Grb10 proteins in vitro and in cells. The protein is present in the cytosol of quiescent cells but translocates to the membrane upon insulin stimulation. This redistribution is likely through interactions with the insulin receptor. Receptor interactions with hGrb-IRβ/Grb10 are mediated in part through the SH2 domain, which binds to the phosphorylated kinase activation loop and the phosphorylated juxtamembrane region. This may explain why overexpression of Grb-IR inhibits insulin signaling (hGrb-IR and hGrb-IRβ/Grb10 SH2 domains are identical) (7Liu F. Roth R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10287-10291Crossref PubMed Scopus (150) Google Scholar). Binding at juxtamembrane Tyr-960 must block PTB domain interactions and prevent phosphorylation of substrates such as IRS-1 and Shc. Binding at the kinase loop would prevent substrate phosphorylation, as well.It is also clear from our results that regions outside of the SH2 domain participate in receptor binding. The PH domain may have a role, perhaps analogous to the way tandem PH and PTB domains of IRS proteins participate in receptor recognition (5Wolf G. Trub T. Ottinger E. Groninga L. Lynch A. White M.F. Miyazaki M. Lee J. Shoelson S.E. J. Biol. Chem. 1995; 270: 27407-27410Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 6Eck M.J. Dhe-Paganon S. Trub T. Nolte R. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 29Gustafson T.A. He W. Craparo A. Schaub C.D. O'Neill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (321) Google Scholar, 41Voliovitch H. Schindler D.G. Hadari Y.R. Taylor S.I. Accili D. Zick Y. J. Biol. Chem. 1995; 270: 18083-18087Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 42Myers M.G. Grammer T.C. Brooks J. Glasheen E.M. Wang L.-M. Sun X.J. Blenis J. Pierce J.H. White M.F. J. Biol. Chem. 1995; 270: 11715-11718Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). 3S. Dhe-Paganon and S. E. Shoelson, unpublished observations. However, it is difficult to predict the function of PH domains (26Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). This one is located within a longer (≈320-residue) region of extended homology with the Grb7/Grb10/Grb14 family and the product of the Caenorhabditis elegans gene mig10 (9Ooi J. Yajnik V. Immanuel D. Gordon M. Moskow J.J. Buchberg A.M. Margolis B. Oncogene. 1995; 10: 1621-1630PubMed Google Scholar). This may suggest that the PH domain is embedded in a larger structure or flanked by one or two other functional domains. It has not been recognized previously that regions outside of the SH2 domains of Grb7, Grb10, Grb14, or Grb-IR participate in their interactions with phosphoproteins.Grb-IR lacks 40 residues of its apparent PH domain yet interacts avidly with the insulin receptor. This may imply that the PH is not involved. However, the N-terminal Grb-IR extension may compensate by completing the PH domain. Phospholipase C-γ1 contains such a "split" PH domain with its entire SH2-SH2-SH3 domain region inserted between strands β3 and β4 of the PH domain β-sandwich. Strands β1-β3 are separated from the remainder of the domain by over 300 residues. Even though Grb-IR is a stable, folded protein, its PH domain appears to be missing strands β1-β3. Since one cannot generally remove large pieces of a stable protein fold without denaturing the protein and altering its physicochemical characteristics, something seems to have taken the place of strands β1-β3. The N-terminal extension would be one obvious candidate, although its sequence does not match the PH domain consensus. 4T. Gibson, personal communication.Our findings also show selective in vitro interactions between SH3 domains and Grb-IR/Grb10. We identified a high affinity site for c-Abl SH3 domain binding that is common to the known members of the Grb7/Grb10/Grb14/Grb-IR family. While further studies are needed to determine if these proteins bind c-Abl or the Abelson oncoprotein in cells, and which additional SH3 domain proteins bind Grb7/Grb10/Grb14/Grb-IR family members, it is tempting to speculate that these interactions might have potential roles in normal signaling and oncogenesis. Interestingly, the 3BS sequence (SLPAIPNPFPEL) does not conform to the known class I specificity of the Abl SH3 domain: (N)PXΘXΨPXΨP(C) (Θ and Ψ represent residues with aromatic and hydrophobic side chains, respectively) (43Sparks A.B. Rider J.E. Hoffman N.G. Fowlkes D.M. Quillam L.A. Kay B.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1540-1544Crossref PubMed Scopus (331) Google Scholar, 44Rickles R.J. Botfield M.C. Weng Z. Taylor J.A. Green O.M. Brugge J.S. Zoller M.J. EMBO J. 1994; 13: 5598-5604Crossref PubMed Scopus (223) Google Scholar). If the orientation of 3BS is flipped, however, then it fits a class II consensus remarkably well: (C)PΘXXPΨXPΨ(N). Several SH3 domains (e.g. Src and Grb2) bind polyproline peptides in two orientations (43Sparks A.B. Rider J.E. Hoffman N.G. Fowlkes D.M. Quillam L.A. Kay B.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1540-1544Crossref PubMed Scopus (331) Google Scholar, 44Rickles R.J. Botfield M.C. Weng Z. Taylor J.A. Green O.M. Brugge J.S. Zoller M.J. EMBO J. 1994; 13: 5598-5604Crossref PubMed Scopus (223) Google Scholar, 45Feng S. Chen J.K. Yu H. Simon J.A. Schreiber S.J. Science. 1994; 266: 1241-1247Crossref PubMed Scopus (739) Google Scholar). While Abl has not been shown to do so previously, our data suggest that it can. The high resolution structure of the liganded Abl SH3 domain supports this possibility (46Musacchio A. Saraste M. Wilmanns M. Nat. Struct. Biol. 1994; 1: 546-551Crossref PubMed Scopus (280) Google Scholar). 5A. Masacchio, personal communication.While this manuscript was being reviewed a related study was published (47O'Neill T.J. Rose D.W. Pillay T.S. Hotta K. Olefsky J.M. Gustafson T.A. J. Biol. Chem. 1996; 271: 22506-22513Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The two studies agree on most points. The sequences and domain architectures of the two predicted proteins are identical, and both studies show prominent expression of hGrb-IRβ/Grb10 mRNA in skeletal muscle and pancreas, an activation-dependent interaction of Grb-IRβ/Grb10 with the insulin receptor, and a decrease in binding to pp135 with insulin stimulation. Both studies also conclude that the activation loop of the insulin receptor kinase is a primary site of binding to the hGrb-IRβ/Grb10 SH2 domain. We report an additional interaction with the juxtamembrane region of the receptor, but our results are not in conflict. 6T. Gustafson, personal communication. O'Neill et al. (47O'Neill T.J. Rose D.W. Pillay T.S. Hotta K. Olefsky J.M. Gustafson T.A. J. Biol. Chem. 1996; 271: 22506-22513Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) demonstrated interactions of Grb-IRβ/Grb10 with insulin-like growth factor-1 receptors, whereas we showed binding to activated PDGF and EGF receptors. They showed that the mitogenic effects of insulin and insulin-like growth factor-1 were blocked by microinjecting the Grb-IRβ/Grb10 SH2 domain into fibroblasts. We did not study this effect. Additional findings that we report include the Northern analyses of hGrb7, the presence of distinct transcripts encoding Grb-IR and hGrb-IRβ/Grb10, the insulin-induced translocation of Grb-IRβ/Grb10 from cytosol to membrane, the interaction of hGrb-IRβ/Grb10 with the insulin receptor at a site outside of its SH2 domain, the interaction of Grb-IR/Grb10 with selected SH3 domains, and the demonstration that the conserved 3BS sequence binds SH3 domains.Many SH2 domain proteins are enzymes, such as the Src, Abl, and ZAP70/Syk kinases, the SHP1/SHP2 phosphatases, the phospholipase C-γ1, and RasGAP. Others associate with enzymes such as the p85 subunit of PI 3-kinase and Grb2 and Crk binding to guanyl nucleotide exchange factors Sos and C3G, respectively. Still other SH2 domain proteins like Shc and Shb act as substrates of receptor tyrosine kinases. The identification of intrinsic or associated activity provides valuable clues about function, but these are lacking for the entire Grb7 family of proteins. Potential clues to Grb-IR/Grb10 functions should be provided by cellular studies. Stable expression of Grb-IR inhibited insulin signaling in Chinese hamster ovary cells, as assessed by substrate phosphorylation and associated PI 3-kinase activity (7Liu F. Roth R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10287-10291Crossref PubMed Scopus (150) Google Scholar). In contrast, microinjection of its SH2 domain (a potential dominant-negative) inhibits DNA synthesis in fibroblasts, suggesting that Grb-IR/Grb10 is a positive mediator of mitogenesis (47O'Neill T.J. Rose D.W. Pillay T.S. Hotta K. Olefsky J.M. Gustafson T.A. J. Biol. Chem. 1996; 271: 22506-22513Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Although Grb-IR and Grb-IRβ/Grb10 differ in domain structure and may have distinct or even opposing biological functions, it is not easy to reconcile these findings. There also appears to be a third related transcript in skeletal muscle, suggesting that yet another form of the protein may be involved in signaling downstream from insulin and possibly growth factor receptors. Additional studies are needed to elucidate functions of this intriguing family of proteins. INTRODUCTIONMany of the effects of activated tyrosine kinase-linked receptors are mediated by cascades of intracellular tyrosine phosphorylation reactions. Receptors with intrinsic kinase activity typically phosphorylate themselves, and in many cases the phosphorylated receptor tyrosines serve as docking sites for SH2 domain proteins (1Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2217) Google Scholar, 2Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (924) Google Scholar). Since many SH2 domain proteins either are enzymes or associate with enzymes, these interactions provide a mechanism for recruiting catalytic effectors to the activated receptor. In the case of insulin signaling, autophosphorylation activates the receptor kinase (3White M.F. Shoelson S.E. Keutmann H. Kahn C.R. J. Biol. Chem. 1988; 263: 2969-2980Abstract Full Text PDF PubMed Google Scholar, 4Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (954) Google Scholar) and creates a docking site for substrate protein PTB domains (5Wolf G. Trub T. Ottinger E. Groninga L. Lynch A. White M.F. Miyazaki M. Lee J. Shoelson S.E. J. Biol. Chem. 1995; 270: 27407-27410Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 6Eck M.J. Dhe-Paganon S. Trub T. Nolte R. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Both effects are necessary to trigger intracellular pathways via the substrates IRS-1 and Shc. However, the SH2 domain effectors of insulin action bind primarily to the phosphorylated substrate proteins rather than the insulin receptor itself.The recent discovery of an SH2 domain protein called Grb-IR was met with considerable interest because it binds the insulin receptor and not its substrates (7Liu F. Roth R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10287-10291Crossref PubMed Scopus (150) Google Scholar). However, this Grb7-like protein reportedly inhibits insulin signaling and contains an unusual 46-residue deletion within its apparent PH domain. Therefore, we have considered the possibility that additional related proteins (potentially with intact domains) might exist to provide positive signals downstream from the insulin receptor. Three members of the Grb7 family (Grb7, Grb10, and Grb14) have been identified by screening cDNA expression libraries with phosphorylated fragments of the EGF 1The abbreviations used are: EGFepidermal growth factorRT-PCRreverse transcriptase-coupled PCR5′-RACE5′-rapid amplification of cDNA endsDMEMDulbecco's modified Eagle's mediumGSTglutathione S-transferasekbkilobase pair(s)bpbase pair(s)PDGFplatelet-derived growth factorPI 3-kinasephosphatidylinositol 3-kinasePAGEpolyacrylamide gel electrophoresis. receptor (8Margolis B. Silvennoinen O. Comoglio F. Roonprapunt C. Skolnik E. Ullrich A. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8894-8898Crossref PubMed Scopus (149) Google Scholar, 9Ooi J. Yajnik V. Immanuel D. Gordon M. Moskow J.J. Buchberg A.M. Margolis B. Oncogene. 1995; 10: 1621-1630PubMed Google Scholar, 10Daly R.J. Sanderson G.M. Janes P.W. Sutherland R.L. J. Biol. Chem. 1996; 271: 12502-12510Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Although each is derived from a distinct genetic locus, they share a common domain architecture: a C-terminal SH2 domain and >300 residues of extended homology that encompasses a PH domain. mGrb10 also contains an 80-residue insertion, relative to mGrb7 and hGrb14. Its function is unknown. Although the presence of SH2 and PH domains strongly implies a role for these proteins in cellular signaling, their physiologic functions remain vague. Nevertheless, all have been implicated in neoplastic conditions. mGrb7 binds and is coamplified with HER2/neu in certain types of breast cancer (11Stein D. Wu J. Fuqua S.A.W. Roonprapunt C. Yajnik V. D'Eustachio P. Moskow J.J. Buchberg A.M. Osborne C.K. Margolis B. EMBO J. 1994; 13: 1331-1340Crossref PubMed Scopus (219) Google Scholar). mGrb10 binds the Ret receptor (12Pandey A. Duan H. Di Fiore P.P. Dixit V.M. J. Biol. Chem. 1995; 270: 21461-21463Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), whose gene (the ret protooncogene) is rearranged and activated in certain thyroid carcinomas and contains germ line mutations associated with syndromes of multiple endocrine neoplasias (13Grieco M. Santoro M. Berlingieri M.T. Melillo R.M. Donghi R. Bongarzone I. Pierotti M.A. Della Porta G. Fusco A. Vecchio G. Cell. 1990; 60: 557-563Abstract Full Text PDF PubMed Scopus (832) Google Scholar, 14Santoro M. Carlomagno F. Romano A. Bottaro D.P. Dathan N.A. Grieco M. Fusco A. Vecchio G. Matoskova B. Kraus M.H. Di Fiore P.P. Science. 1995; 267: 381-383Crossref PubMed Scopus (794) Google Scholar). And expression of Grb14 may be elevated in estrogen receptor-positive breast cancer cell lines and certain prostate cancer cells (10Daly R.J. Sanderson G.M. Janes P.W. Sutherland R.L. J. Biol. Chem. 1996; 271: 12502-12510Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). hGrb-IR and mGrb10 proteins share regions of high homology, although the two are not simple species variants. hGrb-IR has not been implicated in oncogenesis.Expressed sequence tag data bases (dbEST) were screened as a strategy to identify related proteins. Two clones were found. 2Damien Dunnington (SmithKline Beecham Pharmaceuticals) first identified these clones in the Human Genome Sciences data base. One encodes human Grb7. Further sequencing of the second clone revealed a previously unknown protein with high homology to hGrb-IR and mGrb10, which we refer to as hGrb-IRβ/Grb10. It has an intact PH domain and lacks the mGrb10 insertion. hGrb-IRβ/Grb10 and hGrb-IR probably represent alternative mRNA splicing products of a common gene. Both mRNAs are expressed in muscle, a major site of insulin action. hGrb-IRβ/Grb10 protein is present in the cytosol of unstimulated Rat1 fibroblasts and translocates to the membrane following insulin stimulation. We have characterized hGrb-IRβ/Grb10 interactions with activated insulin, EGF, and PDGF receptors and SH3 domain proteins. We conclude that Grb-IRβ/Grb10 is a previously unknown signaling protein that may function downstream from activated insulin and growth factor receptors.
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