Human growth factor receptor bound 14 binds the activated insulin receptorand alters the insulin-stimulated tyrosine phosphorylation levels of multiple proteins

To identify proteins interacting in the insulin-signaling pathway that migh t define new pathways or regulate existing ones, we have employed the yeast two-hybrid system. In a two-hybrid screen of a human liver cDNA library, w e identified the human growth factor receptor bound 14 (hGrb14) adaptor pro tein as a partner of the activated insulin receptor. Additional analysis of the insulin receptor - hGrb14 interaction in the yeast two-hybrid system r evealed that the SH2 domain of hGrb14 was not the sole region involved in b inding the activated insulin receptor. The insulin-stimulated interaction b etween hGrb14 and the insulin receptor was also observed in different mamma lian cultured cell lines. This association was detected at 1 min of insulin stimulation and was maximal at 10 nM and greater concentrations of insulin . Chinese hamster ovary cells stably expressing the insulin receptor (CHO-I R) and hGrb14 were used to examine the effects of hGrb14 overexpression on insulin-stimulated tyrosine phosphorylation of proteins; in general, increa sing levels of hGrb14 expression resulted in a reduction in tyrosine phosph orylation. This decrease was demonstrated for the specific proteins src hom ology-containing and collagen-related protein (Shc), insulin receptor subst rate-1 (IRS-1), and Downstream of tyrosine Kinase (Dok). The broad effects of hGrb14 overexpression on insulin-stimulated tyrosine phosphorylation sug gest that it acts early in the insulin-signaling pathway.