IN-VITRO BINDING AND PHOSPHORYLATION OF INSULIN-RECEPTOR SUBSTRATE-1 BY THE INSULIN-RECEPTOR - ROLE OF INTERACTIONS MEDIATED BY THE PHOSPHOTYROSINE-BINDING DOMAIN AND THE PLECKSTRIN-HOMOLOGY DOMAIN

Insulin receptor substrate 1 (IRS-1) is a major substrate of the insul in receptor in most cells. The N terminus of IRS-1 contains a phosphot yrosine binding (PTB) domain and a pleckstrin homology (PH) domain, bo th of which have been identified as important for insulin-stimulated p hosphorylation in intact cells. The PTB domain binds to a phosphorylat ed motif, NPEY(P)960, that is present in the juxtamembrane region of t he insulin receptor. A direct interaction between the PH domain of IRS -1 and the receptor has not been demonstrated. In this study, we exami ne the role of the IRS-1 PTB and PH domains during IRS-1-receptor bind ing and IRS-1 phosphorylation in intact cells and in vitro. Abrogation of binding of the PTB domain to NPXY(P) by mutation of Tyr960 of the insulin receptor did not reduce the binding of phosphorylated IRS-1 to insulin receptors in intact cells, and had no effect on binding of in sulin receptors to IRS-1 or on IRS-1 phosphorylation in vitro. We exam ined the phosphorylation and receptor binding of a mutant recombinant IRS-1 that lacks the N-terminal PH domain (Delta PH-IRS-1). Although p hosphorylation of Delta PH-IRS-1 by wild-type or [Ala960]insulin recep tors was similar to that of IRS-1, binding of insulin receptor to Delt a PH-IRS-1 was markedly reduced relative to that to IRS-1. We conclude that stable association of IRS-1 with the insulin receptor is unaffec ted by disruption of PTB-domain-Tyr960 interactions but requires the I RS-1 PH domain, and that efficient phosphorylation of IRS-1 in intact cells correlates with the formation of stable receptor . IRS-1 complex es.