This is a grant application, which focuses on studying the role of tyrosine phosphorylation in the mechanism of insulin action. Under this grant over the past 4 years, the PI and colleagues have studied the role of IRS-1 in insulin signaling, used homologous recombinant gene targeting to produce mice and cell lines deficient in this protein, identified IRS-2, defined several of the molecules which interact with IRS-1 and IRS-2 to produce the diverse down-stream signals generated by insulin action, studied the regulation of IRS-1 and IRS-2 in animal models of diabetes and produced a novel polygenic model of NIDDM heterozygous for defects in the insulin receptor and IRS-1. These findings have lead to the hypothesis that insulin signaling is not a simple linear cascade, but a complex network with both alternative and complementary signals generated by families of molecules interacting in the three dimensional space of the cell. The major goal of this grant over the next 5 years will be to further dissect this multi-dimensional network focusing on the potentially complementary, and possibly unique, roles of the various insulin receptor substrates in insulin action. The PI and colleagues will also explore how these components interact with each other and with other signaling molecules to produce both normal patterns of insulin action and alterations in pathophysiologic states. The specific aims of this grant are to: 1) Study the similarities, differences and potentially complementary roles of various insulin receptor substrates in insulin action by creating and characterizing models of altered insulin signaling using homologous recombination to knockout IRS-2, IRS-3, and IRS-4 with both global and conditional/tissue specific strategies. These mice will be crossbred with IRS-1 knockout mice and with each other to determine the nature of the complementary signaling created by multiple insulin receptor substrates. 2) Characterize insulin and IGF-1 action in 3T3 cells derived from knockout animal models lacking IRS-1 and/or other insulin receptor substrates. These cells will be studied before and after reconstitution with different insulin receptor substrates and chimerical molecules produced from the substrates. Metabolic actions will be studied before and after conversion to adipocyte-like cell lines with PPARg. 3) Determine the subcellular compartmentalization, differential partnering and differences in regulation of the major IRS family members in disease states. Together these studies should provide important insights into the complex network of insulin signaling and its alternations in diabetes.