INTERACTION OF A GRB-IR SPLICE VARIANT (A HUMAN GRB10 HOMOLOG) WITH THE INSULIN AND INSULIN-LIKE GROWTH-FACTOR-I RECEPTORS - EVIDENCE FOR AROLE IN MITOGENIC SIGNALING

We have utilized the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. We iden tified a human cDNA that is a splice variant of the human GRB10 homolo g GRB-IR, which we term GRB10/IR-SV1 (for GRB10/GRB-IR splice variant 1). The protein encoded by the GRB10/IR-SV1 cDNA contains an SH2 domai n and a pleckstrin homology domain. Cloning of a full-length human cDN A revealed a predicted coding sequence that was similar to the mouse G RB10 protein, although GRB10/IR-SV1 contained an 80-amino acid deletio n. The GRB10/IR-SV1 cDNA is a splice variant of the GRB-IR cDNA such t hat GRB10/IR-SV1 contains an intact pleckstrin homology domain and a d istinct amino terminus. The interaction of GRB10/IR-SV1 with the insul in receptor and the insulin-like growth factor I (IGF-I) receptor is m ediated by the SH2 domain, and we show that glutathione S-transferase- SH2 domain fusion proteins interact specifically in vitro with the ins ulin receptor derived from mammalian cells. The GRB10/IR-SV1 SH2 domai n also interacted with an similar to 135-kDa phosphoprotein from unsti mulated cell lysates, an interaction that decreased after insulin stim ulation. We present evidence that the GRB10/IR-SV1 protein plays a fun ctional role in insulin and IGF-I signaling by showing that microinjec tion of an SH2 domain fusion protein inhibited insulin- and IGF-I-stim ulated mitogenesis in fibroblasts, yet had no effect on mitogenesis in duced by epidermal growth factor. Our findings suggest that GRB10/IR-S V1 may serve to positively link the insulin and IGF-I receptors to an uncharacterized mitogenic signaling pathway.