HUMAN GRB-IR-BETA GRB10 - SPLICE VARIANTS OF AN INSULIN AND GROWTH-FACTOR RECEPTOR-BINDING PROTEIN WITH PH AND SH2 DOMAINS/

cDNA clones encoding human (h) Grb7 and a previously unknown protein w ith high homology to hGrb-IR and mGrb10 (where m indicates mouse) were found by screening expressed sequence tag data bases. hGrb7 mRNA expr ession is greatest in pancreas and restricted to a few other tissues. The second protein termed hGrb-IR beta/Grb10 contains an intact PH dom ain and lacks the 80-residue mGrb10 insertion. Expression is greatest in pancreas and muscle but occurs in nearly all tissues. hGrb-IR beta/ Grb10 and hGrb-IR likely arise as alternative mRNA splicing products o f a common gene. Reverse transcriptase-coupled polymerase chain reacti on shows both mRNAs in muscle. In cells, Grb-IR beta/Grb10 protein tra nslocates from cytosol to membrane upon insulin stimulation, most like ly due to direct interactions with the insulin receptor. These interac tions are mediated by the SH2 domain and additional regions of the pro tein. Studies with mutated receptors and synthetic phosphopeptides sho w that the hGrb-IR beta/Grb10 SH2 domain binds at least two sites in t he insulin receptor: the kinase activation loop > the juxtamembrane si te. hGrb-IR beta/Grb10 also binds a 135-kDa phosphoprotein in unstimul ated 3T3-L1 adipocytes; binding is reduced upon insulin stimulation. I n addition, the c-Abl SH3 domain binds Grb-IR/Grb10, whereas Fyn, phos phatidylinositol 3-kinase p85, and Grb2 SH3 domains do not. The site o f c-Abl SH3 domain interaction is highly conserved within the Grb-IR/G rb10/Grb7/Grb14 family. hGrb-IR beta/Grb10 also binds platelet-derived growth factor and epidermal growth factor receptors, suggesting a bro ader role in the signaling pathways of numerous receptors. We conclude that hGrb-IR beta/Grb10 is a widely expressed, PH and SH2 domain-cont aining, SH3 domain-binding protein that functions downstream from acti vated insulin and growth factor receptors.