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Tyr⁶¹² and Tyr⁶³² in Human Insulin Receptor Substrate-1 Are Important for Full Activation of Insulin-Stimulated Phosphatidylinositol 3-Kinase Activity and Translocation of GLUT4 in Adipose Cells¹

Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892; and Department of Oncology and Neurosciences, University G. D’Annunzio (D.L.E., A.C.), Chieti 66013, Italy

Address all correspondence and requests for reprints to: Michael J. Quon, M.D., Ph.D., Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 8C-218, 10 Center Drive, MSC 1755, Bethesda, Maryland 20892-1755. E-mail: quonm{at}nih.gov

To examine contributions of specific YXXM motifs in human insulin receptor substrate-1 (IRS-1) to mediating the metabolic actions of insulin, we studied IRS-1 mutants containing various substitutions of Phe for Tyr. In transfected NIH-3T3^IR cells, insulin stimulation caused a 5-fold increase in phosphatidylinositol 3-kinase (PI3K) activity coimmunoprecipitated with wild-type IRS-1. No PI3K activity was associated with IRS1-F6 (Phe substituted for Tyr at positions 465, 612, 632, 662, 941, and 989). Adding back both Tyr⁶¹² and Tyr⁶³² fully restored IRS-1-associated PI3K activity, whereas adding back either Tyr⁶¹² or Tyr⁶³² alone was associated with intermediate PI3K activity. In rat adipose cells transfected with epitope-tagged GLUT4, insulin stimulation caused a 2-fold increase in cell surface GLUT4-HA. Cotransfection of cells with GLUT4-HA and either wild-type IRS-1 or IRS1-Y612/Y632 increased basal cell surface GLUT4-HA (in the absence of insulin) to approximately 80% of the levels seen in insulin-stimulated control cells, whereas overexpression of IRS1-F6 had no effect on the insulin dose-response curve. Overexpression of IRS1-Y612 or IRS1-Y632 caused intermediate effects. Thus, both Tyr⁶¹² and Tyr⁶³² are important for IRS-1 to fully activate PI3K and mediate translocation of GLUT4 in response to insulin.