INTERACTION BETWEEN THE PHOSPHOTYROSINE BINDING DOMAIN OF SHC AND THEINSULIN-RECEPTOR IS REQUIRED FOR SHC PHOSPHORYLATION BY INSULIN IN-VIVO

Stimulation of the insulin receptor (IR) results in tyrosine phosphory lation of the intermediate molecules insulin receptor substrate-1 (IRS -1), IRS-2, and Shc, which then couple the IR to downstream signaling pathways by serving as binding sites for signaling molecules with SH2 domains. It has been proposed that direct binding of IRS-1, IRS-2, and Shc to an NPX-Tyr(P) motif in the juxtamembrane region of the IR is r equired for tyrosine phosphorylation of these molecules by the LR. In this regard, Shc and IRS-1 contain domains that are distinct from SH2 domains, referred to as the phosphotyrosine binding (PTB) or phosphoty rosine interaction (PI) domains, which bind phosphotyrosine in the con text of an NPX-Tyr(P) motif. To further clarify the role of the Shc PT B/PI domain, we identified a mutation in this domain that abrogated bi nding of Shc to the IR in vitro. Interestingly, this mutation complete ly abolished Shc phosphorylation by the IR in vivo whereas mutation of the arginine in the FLVRES motif of the Shc SH2 domain did not affect Shc phosphorylation by insulin. In addition, we identified specific a mino acids on the IR that are required for the IR to stimulate Shc but not IRS-1 phosphorylation in vivo. As with the PTB/PI domain Shc muta nt, the ability of these mutant receptors to phosphorylate Shc correla tes with the binding of the PTB/PI domain of Shc to similar sequences in vitro. These findings support a model in which binding of the PTB/P I domain of Shc directly to the NPX-Tyr(P) motif on the LR mediates Sh c phosphorylation by insulin.