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• W2046124930 abstract "We cloned by interaction with the β-subunit of the insulin receptor the rat variant of the human adapter Grb14 (rGrb14). rGrb14 is specifically expressed in rat insulin-sensitive tissues and in the brain. The binding of rGrb14 to insulin receptors is insulin-dependent in vivo in Chinese hamster ovary (CHO) cells overexpressing both proteins and importantly, in rat liver expressing physiological levels of proteins. However, rGrb14 is not a substrate of the tyrosine kinase of the receptor. In the two-hybrid system, two domains of rGrb14 can mediate the interaction with insulin receptors: the Src homology 2 (SH2) domain and a region between the PH and SH2 domains that we named PIR (forphosphorylated insulin receptor-interactingregion). In vitro interaction assays using deletion mutants of rGrb14 show that the PIR, but not the SH2 domain, is able to coprecipitate insulin receptors, suggesting that the PIR is the major binding domain of rGrb14. The interaction between rGrb14 and the insulin receptors is almost abolished by mutating tyrosine residue Tyr1150 or Tyr1151 of the receptor. The overexpression of rGrb14 in CHO-IR cells decreases insulin stimulation of both DNA and glycogen synthesis. These effects are accompanied by a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1, but insulin receptor autophosphorylation is unaltered. These findings suggest that rGrb14 could be a new downstream signaling component of the insulin-mediated pathways. We cloned by interaction with the β-subunit of the insulin receptor the rat variant of the human adapter Grb14 (rGrb14). rGrb14 is specifically expressed in rat insulin-sensitive tissues and in the brain. The binding of rGrb14 to insulin receptors is insulin-dependent in vivo in Chinese hamster ovary (CHO) cells overexpressing both proteins and importantly, in rat liver expressing physiological levels of proteins. However, rGrb14 is not a substrate of the tyrosine kinase of the receptor. In the two-hybrid system, two domains of rGrb14 can mediate the interaction with insulin receptors: the Src homology 2 (SH2) domain and a region between the PH and SH2 domains that we named PIR (forphosphorylated insulin receptor-interactingregion). In vitro interaction assays using deletion mutants of rGrb14 show that the PIR, but not the SH2 domain, is able to coprecipitate insulin receptors, suggesting that the PIR is the major binding domain of rGrb14. The interaction between rGrb14 and the insulin receptors is almost abolished by mutating tyrosine residue Tyr1150 or Tyr1151 of the receptor. The overexpression of rGrb14 in CHO-IR cells decreases insulin stimulation of both DNA and glycogen synthesis. These effects are accompanied by a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1, but insulin receptor autophosphorylation is unaltered. These findings suggest that rGrb14 could be a new downstream signaling component of the insulin-mediated pathways. insulin receptor substrate phosphotyrosine binding Src homology 2 pleckstrin homology growth factor-binding protein rat Grb14 epidermal growth factor Chinese hamster ovary insulin receptor polymerase chain reaction 5′-rapid amplification of cDNA ends kilobases polyacrylamide gel electrophoresis glutathione S-transferase. Insulin is the principal hormone controlling energy metabolism, by modulating metabolic pathways in different target tissues. The liver occupies a central position in the regulation of glucose homeostasis by insulin; insulin inhibits hepatic gluconeogenesis and stimulates glycogen and lipid synthesis. On the other hand, insulin stimulates glucose transport and utilization in skeletal muscle and adipose tissue. These actions of insulin are mediated through a membrane-bound receptor. The insulin receptor is a member of the receptor tyrosine kinase family, members of which contain an intrinsic tyrosine kinase, which is activated after ligand binding. The best characterized substrates of the insulin receptor are insulin receptor substrate-1 and -2 (IRS-1 and IRS-2),1 and Shc (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Waters S.B. Pessin J.E. Trends Cell Biol. 1996; 6: 1-4Abstract Full Text PDF PubMed Scopus (63) Google Scholar). They are all known to bind to the phosphorylated Tyr960 residue of the receptor via their phosphotyrosine binding (PTB) domain (3Gustafson T.A. He W. Craparo A. Schaub C.D. O'Neill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (327) Google Scholar, 4Tartare-Deckert S. Sawka-Verhelle D. Murdaca J. Van Obberghen E. J. Biol. Chem. 1995; 270: 23456-23460Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 5Sawka-Verhelle D. Tartare-Deckert S. White M.F. Van Obberghen E. J. Biol. Chem. 1996; 271: 5980-5983Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 6He W. Craparo A. Zhu Y. O'Neill T.J. Wang L.-M. Pierce J.H. Gustafson T.A. J. Biol. Chem. 1996; 271: 11641-11645Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Unlike most receptor tyrosine kinases, tyrosine-phosphorylated residues of the insulin receptor do not seem to recruit directly a number of SH2-containing proteins. These proteins are recruited by IRSs, which are considered to be docking proteins, and by Shc (2Waters S.B. Pessin J.E. Trends Cell Biol. 1996; 6: 1-4Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 7Myers Jr., M.G. White M.F. Trends Endocrinol. Metab. 1995; 6: 209-215Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 8Bonfini L. Migliaccio E. Pelicci G. Lanfrancone L. Pelicci P. Trends Biochem. Sci. 1996; 21: 257-261Abstract Full Text PDF PubMed Scopus (235) Google Scholar). For example, IRS-1 interacts with the SH2 domains of the tyrosine phosphatase Shp2 and of the regulatory subunit (p85) of phosphatidylinositol 3-kinase to activate this enzyme. Phosphatidylinositol 3-kinase is likely to be implicated in insulin-stimulated translocation of GLUT4, the isoform of glucose transporters expressed in skeletal muscle and adipose tissue (9Bell G.I. Burant C.F. Takeda J. Gould G.W. J. Biol. Chem. 1993; 268: 19161-19164Abstract Full Text PDF PubMed Google Scholar). However, at the present time, few other effectors of insulin signaling that could participate specifically in metabolic effects of insulin have been characterized. Shc and IRSs are ubiquitously expressed, not specifically in insulin-sensitive tissues, and they are also phosphorylated after activation of a number of receptors, including receptor tyrosine kinases, cytokine receptors, and G protein-coupled receptors (8Bonfini L. Migliaccio E. Pelicci G. Lanfrancone L. Pelicci P. Trends Biochem. Sci. 1996; 21: 257-261Abstract Full Text PDF PubMed Scopus (235) Google Scholar,10Souza S.C. Frick G.P. Yip R. Lobo R.B. Tai L.-R. Goodman H.M. J. Biol. Chem. 1994; 269: 30085-30088Abstract Full Text PDF PubMed Google Scholar, 11Argetsinger L.S. Hsu G.W. Myers Jr., M.G. Billestrup N. White M.F. Carter-Su C. J. Biol. Chem. 1995; 270: 14685-14692Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 12Platanias L.C. Uddin S. Yetter A. Sun X.J. White M.F. J. Biol. Chem. 1996; 271: 278-282Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 13Velloso L.A. Folli F. Sun X.J. White M.F. Saad M.J.A. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12490-12495Crossref PubMed Scopus (353) Google Scholar, 14Kowalski-Chauvel A. Pradayrol L. Vaysse N. Seva C. J. Biol. Chem. 1996; 271: 26356-26361Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). It is therefore possible that other proteins, possibly implicated more specifically in insulin signal transduction, might exist. Recently, different groups have reported the cloning of new proteins supposed to be involved in insulin signaling, since they have been identified by two-hybrid screening using the insulin receptor as bait (15Liu F. Roth R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10287-10291Crossref PubMed Scopus (152) Google Scholar, 16O'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, 17Hansen H. Svensson U. Zhu J. Laviola L. Giorgino F. Wolf G. Smith R.J. Riedel H. J. Biol. Chem. 1996; 271: 8882-8886Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 18Laviola L. Giorgino F. Chow J.C. Baquero J.A. Hansen H. Ooi J. Zhu J. Riedel H. Smith R.J. J. Clin. Invest. 1997; 99: 830-837Crossref PubMed Scopus (88) Google Scholar, 19O'Neill T.J. Zhu Y. Gustafson T.A. J. Biol. Chem. 1997; 272: 10035-10040Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 20Chen J. Sadowski H.B. Kohanski R.A. Wang L.-H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2295-2300Crossref PubMed Scopus (113) Google Scholar). All but two (human MAD2 (19O'Neill T.J. Zhu Y. Gustafson T.A. J. Biol. Chem. 1997; 272: 10035-10040Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and Stat5 (20Chen J. Sadowski H.B. Kohanski R.A. Wang L.-H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2295-2300Crossref PubMed Scopus (113) Google Scholar, 21Sawka-Verhelle D. Filloux C. Tartare-Deckert S. Mothe I. Van Obberghen E. Eur. J. Biochem. 1997; 250: 411-417Crossref PubMed Scopus (44) Google Scholar)) are spliced variants of the Grb10 protein. Grb10 was originally cloned as a growth factor receptor-binding protein by interaction with the EGF receptor (22Ooi J. Yajnik V. Immanuel D. Gordon M. Moskow J.J. Buchberg A.M. Margolis B. Oncogene. 1995; 10: 1621-1630PubMed Google Scholar). It is a molecular adapter and a member of the recently emerged Grb7 family of proteins, which comprises Grb7, Grb10, and Grb14 (23Margolis B. Prog. Biophys. Mol. Biol. 1994; 62: 223-244Crossref PubMed Scopus (48) Google Scholar, 24Daly 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 its precise role remains to be clarified, Grb10 is likely to be implicated in insulin- and insulin-like growth factor-1-induced mitogenesis (16O'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, 25Morrione A. Valentinis B. Resnicoff M. Xu S.-Q. Baserga R. J. Biol. Chem. 1997; 272: 26382-26387Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). To identify new proteins implicated in insulin signal transduction, we performed a two-hybrid screen of a rat liver cDNA library, using the activated cytoplasmic domain of the insulin receptor as bait. To favor the identification of metabolic effectors, the rat used for the construction of the liver cDNA library had been starved for 48 h and refed for 10 h with a diet designed to stimulate transcription of genes implicated in insulin-regulated metabolism. We have cloned a protein displaying a high homology with the human Grb14, a member of the Grb7 subfamily of adapters (24Daly 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). This protein was then called rGrb14. The data presented in this study suggest that rGrb14 is potentially a new effector of the insulin receptor. In addition, we have identified in rGrb14 a region different from the SH2 domain, which is the main binding domain with the Insulin receptor. This region, named PIR (for phosphorylated insulin receptor-interacting region), is homologous to the BPS domain recently described in Grb10 (26He W. Rose D.W. Olefsky J.M. Gustafson T.A. J. Biol. Chem. 1998; 273: 6860-6867Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Synthetic defined dropout yeast media lacking the appropriate amino acids were obtained from Bio 101, Inc. (Vista, CA). Oligonucleotides were purchased at the Pasteur Institut (Paris, France) and Life Technologies, Inc. Monoclonal anti-Myc antibody (9E10 clone) was from Boehringer Mannheim. Monoclonal anti-phosphotyrosine antibody (pY20), and polyclonal antibodies against insulin receptor β-subunit were from Transduction Laboratories. Anti-LexA and anti-Gal4 activation domain antibodies were from CLONTECH (Palo Alto, Ca). Polyclonal anti-rGrb14 antibodies were raised against the N-terminal 17 amino acids of rGrb14 (Neosystem) and purified on protein A-Sepharose before use. All chemicals were from Sigma France, and enzymes were from New England Biolabs (Beverly, MA). The intracellular domains of the rat insulin receptor and of the human insulin receptor ATP binding site mutant (IR K1018A) were amplified by polymerase chain reaction (PCR) using Pfu polymerase (Stratagene, La Jolla, CA) and inserted in frame at the BamHI site of the pLex9 plasmid (pLex-IR and pLex-IR K1018). Other insulin receptor mutants in pLex9 vector and pACTII-Shc construct were generated as described previously (4Tartare-Deckert S. Sawka-Verhelle D. Murdaca J. Van Obberghen E. J. Biol. Chem. 1995; 270: 23456-23460Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 5Sawka-Verhelle D. Tartare-Deckert S. White M.F. Van Obberghen E. J. Biol. Chem. 1996; 271: 5980-5983Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). rGrb14 deletion constructs were generated by PCR and inserted at theBamHI site of pACTII and of pGEX3X (Amersham Pharmacia Biotech). The rGrb14 mutation of the arginine 464 into lysine (rGrb14 R464K) was performed by site-directed mutagenesis using the Quick Change site-directed mutagenesis kit (Stratagene, La Jolla, CA). Sequences and constructions were verified by DNA sequence analysis. The yeast two-hybrid screen was performed in the yeast strain L40 using on one hand pLexIR, which encodes a constitutively activated insulin receptor β-subunit (27O'Neill T.J. Craparo A. Gustafson T.A. Mol. Cell. Biol. 1994; 14: 6433-6442Crossref PubMed Scopus (166) Google Scholar), and on the other hand an oligo(dT)-primed cDNA library from rat liver, cloned in fusion with the Gal4 activation domain in the pGAD3S2X plasmid (gift from M. Cognet-Vasseur, INSERM U129, Paris, France). After transformation by the lithium acetate procedure (28Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425-1426Crossref PubMed Scopus (2895) Google Scholar), yeasts were plated on a tryptophan-leucine-histidine-deficient medium. Colonies growing in the absence of histidine (the first reporter gene) were subsequently tested for β-galactosidase activity (second reporter gene). The plasmids of the library producing yeast colonies of a His+/LacZ+ phenotype were isolated, and the specificity of association of their products with insulin receptors was tested using pLex-lamin as negative control. The cDNA inserts of these positive plasmids were sequenced, using an Applied Biosystems sequencer (Perkin-Elmer). To determine and clone the 5′-end of the rGrb14 cDNA, the 5′-RACE technique was used on a rat liver Marathon-Ready premade cDNA library (CLONTECH), with the Advantage cDNA PCR kit (CLONTECH) and a primer 5′-GCGGCACACCTGCACTGCCAGC-3′ corresponding to the 5′ sequence determined on library cDNA insert, according to the manufacturer's recommendations. We obtained a 250-base pair fragment, which was sequenced and corresponds to the 5′-end of the cDNA. Since the largest library plasmid was lacking only 21 nucleotides of coding sequence, a full-length cDNA containing KpnI and BamHI sites at both ends and a Myc epitope at the 3′-end was reconstructed by PCR with the Pfu polymerase using this plasmid as template and the two following oligonucleotides as primers: 5′-CCGCGGTACCGGATCCCTACGATCATGACCACGTCCCTGCAAGATGGGCAGAGCGCCGCGGGCCG-3′ and 5′-CCGCGGTACCGGATCCGAGATCTTCCTCGCTGATTAGCTTCTGCTCAACAGCCATCCTAGCACAGTAATGC-3′). The sequence integrity of the full-length rGrb14 cDNA was verified by DNA sequencing. Yeast strains were transformed by the lithium acetate method of Gietz (28Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425-1426Crossref PubMed Scopus (2895) Google Scholar). Quantitative analyses of β-galactosidase activity were performed using a solution assay as described previously (29Kasus-Jacobi A. Perdereau D. Tartare-Deckert S. Van Obberghen E. Girard J. Burnol A.-F. J. Biol. Chem. 1997; 272: 17166-17170Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Total RNA was purified from rat tissues or the adipocyte cell line 3T3-F442A using the method of Chomczynski and Sacchi (30Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63187) Google Scholar). Northern blot analysis was performed as described previously (31Burnol A.-F. Leturque A. Loizeau M. Postic C. Girard J. Biochem. J. 1990; 270: 277-279Crossref PubMed Scopus (56) Google Scholar) using as a probe a 32P-radiolabeled 500-base pair XbaI fragment corresponding to the 3′-end of the rGrb14 cDNA. Rat tissues and 3T3-F442A cells were homogenized in a sucrose buffer (250 mm sucrose, 5 mmTris-HCl, pH 7.5, 1 mm phenylmethylsulfonyl fluoride, 1 mm pepstatin A, 10 μm aprotinin, 10 μg/ml leupeptin). These cell extracts were subjected to SDS-PAGE and immunoblotted with polyclonal anti-rGrb14 antibodies. rGrb14 cDNA was inserted into the KpnI site of the pECE vector (32Ellis L. Clauser E. Morgan D.O. Edery M. Roth R.A. Rutter W.J. Cell. 1986; 45: 721-732Abstract Full Text PDF PubMed Scopus (696) Google Scholar). Stable expression of rGrb14 was achieved in CHO-IR cell lines after cotransfection of the pE-rGrb14 plasmid with a plasmid conferring hygromycin resistance by the calcium phosphate procedure. After limiting dilution, pure clones were identified by Northern blot analysis and Western blot analysis using an anti-Myc antibody. We have used the clone 8A9 for CHO-IR/rGrb14 cells. Preliminary experiments have established that endogenous rGrb14 mRNA can be detected by Northern blot in CHO cells using the rat radiolabeled cDNA probe. Confluent CHO-IR cells were serum-deprived for 48 h and stimulated or not by insulin (10−7m) for 10 min at 37 °C. Cells were solubilized at 4 °C, in 20 mm Tris-HCl (pH 7.4) buffer containing 150 mm NaCl, 10 mm EDTA, 1% Triton X-100, 0.1% bovine serum albumin, and a standard mixture of protease inhibitors (Complete; Boehringer Mannheim), in addition to phenylmethylsulfonyl fluoride (2 mm), 20 mmNaF, and 20 mm NaVO3. After a 15-min centrifugation at 15,000 × g, the supernatant was incubated overnight at 4 °C with anti-phosphotyrosine, anti-IR, anti-rGrb14, or anti-IRS-1 antibodies in the presence of protein A-Sepharose. The resulting immunoprecipitates were subjected to SDS-PAGE electrophoresis and immunoblotted with the indicated antibodies. The immunoreactive bands were revealed using the ECL detection kit (Amersham Pharmacia Biotech). For in vivo studies in rats, animals were starved for 24 h, anesthetized, and injected with saline or insulin via the saphenous vein. After 10 min, the liver proteins were extracted in lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mmNaCl, 5 mm EDTA, 30 mm sodium pyrophosphate, 50 mm NaF, 0.5% Nonidet P-40, 0.2% Triton X-100, 1 mm NaVO3, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 2 μg/ml leupeptin, 5 μg/ml aprotinin) and centrifuged for 10 min at 6000 rpm at 4 °C. Immunoprecipitation and Western blot analysis were performed as described above. GST fusion proteins were produced as described previously (29Kasus-Jacobi A. Perdereau D. Tartare-Deckert S. Van Obberghen E. Girard J. Burnol A.-F. J. Biol. Chem. 1997; 272: 17166-17170Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). CHO-IR cells were serum-starved for 24 h and stimulated or not stimulated with insulin (10−7m) for 10 min at 37 °C. The cell lysates (4 × 105 cells) were prepared as described above and incubated overnight at 4 °C with 3 μg of immobilized GST fusion proteins. After extensive washing, bound proteins were eluted by heating in SDS sample buffer, separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with the indicated antibody, and immunoreactive bands were revealed using the ECL detection kit (Amersham Pharmacia Biotech). [14C]Glucose incorporation into glycogen and [3H]thymidine incorporation into DNA were measured as described previously (33Leconte I. Auzan C. Debant A. Rossi B. Clauser E. J. Biol. Chem. 1992; 267: 17415-17423Abstract Full Text PDF PubMed Google Scholar). Briefly, confluent cells were stimulated with increasing concentrations of insulin for 1 h prior to incubation with 2 μCi of [14C]glucose (Amersham Pharmacia Biotech) for 3 h. After two phosphate-buffered saline washes, cells were lysed with 30% KOH, and the endogenous [14C]glycogen was precipitated and counted for radioactivity. After 72 h of serum depletion, cells were treated for 16 h with increasing concentrations of insulin and then exposed to 0.5 μCi of [3H]thymidine (Amersham Pharmacia Biotech) for 45 min. After three phosphate-buffered saline washes, the DNA was precipitated with 10% trichloracetic acid, and the radioactive material was dissolved in 1 m NaOH and counted. Seven million independent yeast colonies were tested; 104 clones contained plasmids encoding proteins that exhibited a specific interaction with the insulin receptor β-subunit and not with the kinase-inactive insulin receptor mutated in the ATP binding site (IR K1018A) or with unrelated proteins like lamin. After DNA sequencing, these specific clones were classified into six different groups encoding distinct proteins (Fig. 1). Three of these proteins were already described as interacting with the insulin receptor: p85α, p85β, and Shc p52 (3Gustafson T.A. He W. Craparo A. Schaub C.D. O'Neill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (327) Google Scholar, 4Tartare-Deckert S. Sawka-Verhelle D. Murdaca J. Van Obberghen E. J. Biol. Chem. 1995; 270: 23456-23460Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 34Levy-Toledano R. Taouis M. Blaettler D.H. Gorden P. Taylor S.I. J. Biol. Chem. 1994; 269: 31178-31182Abstract Full Text PDF PubMed Google Scholar, 35Staubs P.A. Reichart D.R. Saltiel A.R. Milarski K.L. Maegawa H. Bernahu P. Olefsky J.M. Seely B.L. J. Biol. Chem. 1994; 269: 27186-27192Abstract Full Text PDF PubMed Google Scholar, 36Lamothe B. Bucchini D. Jami J. Joshi R.L. FEBS Lett. 1995; 373: 51-55Crossref PubMed Scopus (30) Google Scholar, 37Tartare-Deckert S. Murdaca J. Sawka-Verhelle D. Holt K.H. Pessin J.E. Van Obberghen E. Endocrinology. 1996; 137: 1019-1024Crossref PubMed Scopus (1) Google Scholar, 38Kavanaugh W.M. Williams L.T. Science. 1994; 266: 1862-1865Crossref PubMed Scopus (451) Google Scholar, 39Blaikie P. Immanuel D. Wu J. Li N. Yajnik V. Margolis B. J. Biol. Chem. 1994; 269: 32031-32034Abstract Full Text PDF PubMed Google Scholar). Other clones encode the C terminus domain of a splice variant of SH2B, an Src homology-2 domain-containing adapter (40Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar, 41Riedel H. Wang J. Hansen H. Yousaf N. J. Biochem. (Tokyo). 1997; 122: 1105-1113Crossref PubMed Scopus (66) Google Scholar). Clones encoding the full-length Grb7 were also found. Grb7 is a molecular adapter, first isolated by interaction with the EGF receptor (42Margolis 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 (150) Google Scholar). Another group corresponds to 14 plasmids containing inserts of the same cDNA, varying in length from 0.9 to 2.0 kb. This cDNA is the subject of the present study. The longest insert encodes a 531-amino acid protein, which lacks its N terminus. The missing sequence was cloned and identified using the 5′-RACE technique. The full-length protein is 538 amino acids long, as shown in Fig. 2 A. The first methionine codon was unambiguously identified by its fairly good context for initiating translation (43Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2810) Google Scholar) and by the presence of an in frame stop codon 21 nucleotides upstream. This new protein is an adapter, characterized by the succession of various interacting domains: a central PH domain, a C terminus SH2 domain, and a proline-rich region in the N terminus of the protein. GenBankTM data base searches revealed significant homology of this protein with the Grb7 family of proteins (including Grb14, Grb7, Grb10, Grb-IR, and Grb10-IR/SV1; the last three are spliced variants of the same gene (16O'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, 44Frantz J.D. Giorgetti-Peraldi S. Ottinger E.A. Shoelson S.E. J. Biol. Chem. 1997; 272: 2659-2667Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar)). The percentage of amino acid identity of the different domains of this protein with members of the Grb7 family is schematized on Fig. 2 B. Given the high identity with the amino acid sequence of the human Grb14 (see Fig. 2 B) and the homology in nucleotide sequence with the human Grb14 (83%), it is likely that this protein is the rat variant of human Grb14. It was therefore named rGrb14. The tissue distribution of rGrb14 was studied by Northern and Western blot analysis. The main transcript is approximately 2.5 kb long, and a second smaller transcript (1.9 kb) is also present in some tissues, as shown in Fig. 3 A. rGrb14 mRNAs are expressed in liver, heart, skeletal muscle, pancreas, brain, and white adipose tissue. On Western blot, the rGrb14 protein is a 60-kDa band, which is specifically displaced by preincubation of the antibodies with the antigenic peptide. The protein is present in liver, heart, and brain (Fig. 3 B) and can also be detected in skeletal muscle (data not shown). Thus, the expression of rGrb14 seems to be restricted to insulin target tissues and brain. This does not fully correlate with the human Grb14, which is also expressed in kidney and placenta (24Daly 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). In the adipose cell line 3T3-F442A, rGrb14 mRNA are absent in undifferentiated fibroblasts (Fig. 3 C). After confluence, when fibroblasts begin to differentiate in adipose cells, there is a slight increase in rGrb14 mRNA expression. Maximum accumulation of rGrb14 mRNA is observed in fully differentiated adipose cells, 8 days after confluence. A similar pattern of expression was observed at the protein level (Fig. 3 D), indicating that rGrb14 may be considered as a marker of adipose cell differentiation. In the same cells, Grb7 mRNA expression did not vary with the differentiation state (data not shown). Thus, the parallelism between the level of expression and adipose cell differentiation is specific for rGrb14 among members of the Grb7 family of proteins. The rGrb14-insulin receptor interaction was first investigated in CHO-IR cells (expressing high levels of human insulin receptors) stably overexpressing a Myc-tagged rGrb14 recombinant protein. CHO-IR/rGrb14 cell lysate was either immunoprecipitated using anti-insulin receptor antibodies and immunodetected with anti-Myc antibodies (Fig. 4 A) or immunoprecipitated using anti-rGrb14 antibodies and immunodetected with anti-phosphotyrosine antibodies (Fig. 4 B). Under basal conditions, rGrb14 is not coprecipitated with the insulin receptors. After stimulation by insulin, the association of the activated receptors with rGrb14 is induced. The association between the insulin receptor and rGrb14 was also investigated in rat liver. Anesthetized rats were injected intravenously with insulin or saline, and after 10 min, liver proteins were extracted and immunodetected with anti-phosphotyrosine antibodies. In liver crude extracts, insulin stimulates tyrosine phosphorylation of three major proteins of 180, 95, and 50 kDa (Fig. 4 C, left part). Immunoblotting with corresponding antibodies confirmed that the 180- and 95-kDa bands are, respectively, IRS-1 and the β-subunit of the insulin receptor (data not shown). These liver extracts were immunoprecipitated with anti-rGrb14 antibodies prior to immunodetection with anti-phosphotyrosine antibodies. As shown in the right part of Fig. 4 C, insulin stimulates the association between rGrb14 and the activated insulin receptors in rat liver. These experiments clearly show that rGrb14 binds to the activated insulin receptors, not only in two overexpressing systems (the two-hybrid system and the CHO-IR/rGrb14 cell line) but also in cells expressing physiological levels of both insulin receptors and rGrb14. In insulin-stimulated liver, tyrosine-phosphorylated IRS-1 is also detected in the anti-rGrb14 immunoprecipitate. rGrb14 is not revealed by anti-phosphotyrosine antibodies in CHO-IR/rGrb14 cells or rat liver lysates immunoprecipitated with anti-rGrb14 antibodies (Fig. 4,B and C). Thus, rGrb14 is not a substrate of the insulin receptor tyrosine kinase. In the two-hybrid system, a kinase-inactive insulin receptor mutant (K1018A, mutated in the ATP binding site) was unable to interact with either rGrb14 or Shc, an insulin receptor substrate taken as control (Fig. 5) (27O'Neill T.J. Craparo A. Gustafson T.A. Mol. Cell. Biol. 1994; 14: 6433-6442Crossref PubMed Scopus (166) Google Scholar). This underlines the importance of the receptor activation for the interaction between the insulin receptors and rGrb14. To identify the tyrosyl residues of the insulin receptor that are necessary for this association, we measured the interaction between rGrb14 and insulin receptors mutated" @default.
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