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 insulin receptor in most cells. The N terminus of IRS-1 contains a phosphotyrosine binding (PTB) domain and a pleckstrin homology (PH) domain, both of which have been identified as important for insulin-stimulated phosphorylation in intact cells. The PTB domain binds to a phosphorylated motif, NPEY(P)960, that is present in the juxtamembrane region of the insulin receptor. A direct interaction between the PH domain of IRS-1 and the receptor has not been demonstrated. In this study, we examine the role of the IRS-1 PTB and PH domains during IRS-1 receptor binding 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 insulin receptors to IRS-1 or on IRS-1 phosphorylation in vitro. We examined the phosphorylation and receptor binding of a mutant recombinant IRS-1 that lacks the N-terminal PH domain (delta PH-IRS-1). Although phosphorylation of delta PH-IRS-1 by wild-type or [Ala960]insulin receptors was similar to that of IRS-1, binding of insulin receptor to delta 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 unaffected by disruption of PTB-domain-Tyr960 interactions but requires the IRS-1 PH domain, and that efficient phosphorylation of IRS-1 in intact cells correlates with the formation of stable receptor IRS complexes.
Backer
J M
JM
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Wjasow
C
C
Zhang
Y
Y
D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D055504:Insulin Receptor Substrate Proteins; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014443:Tyrosine
D001798:Blood Proteins; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; C039077:platelet protein P47; D019000:Phosphotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin