Identification of the rat adapter Grb14 as an inhibitor of insulin actions

We cloned by interaction with the beta-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, rGrbl4 is not a substrate of the tyrosine kinase of the receptor. In the two-hybrid system, two domains of rGrbl4 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 (for (p) under bar hosphorylated insulin receptor-(i) under bar nteracting (r) under bar egion). In vitro interaction assays using deletion mutants of rGrbl4 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 Tyr(1150) Or Tyr(1151) 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 rGrbl4 could be a new downstream signaling component of the insulin-mediated pathways.