INTERACTION BETWEEN THE GRB10 SH2 DOMAIN AND THE INSULIN-RECEPTOR CARBOXYL-TERMINUS

Grb10 is a member of a recently identified family of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated s ignal transduction. We identified and isolated the Grb10 SH2 domain ba sed on its interaction with the intracellular domain of the insulin re ceptor beta-subunit using the yeast two-hybrid system, The interaction was specific for the insulin receptor and the insulin-like growth fac tor-1 receptor, and it required a catalytically active receptor kinase domain and an intact Grb10 SH2 domain. Glutathione S-transferase fusi on proteins containing the Grb10 SH2 domain associated in an insulin-d ependent manner with insulin receptors from cell lysates and with puri fied insulin receptors. Go-precipitation experiments revealed the asso ciation of cellular Grb10 with hormone-stimulated insulin receptors in cell extracts. The Grb10 SH2 domain did not bind to an insulin recept or lacking 43 amino acids at the carboxyl terminus, and it exhibited h ighest affinity for a phosphopeptide containing Tyr(P)-1322. Unlike p8 5 and Syp, which also bind to Tyr(P)-1322, Grb10 was not found to asso ciate with insulin receptor substrate-1, These results suggest that Gr b10 is a novel insulin receptor interactive protein and provide direct evidence for an insulin receptor substrate-1-independent function of the insulin receptor carboxyl terminus in protein binding.