The objective of the proposed research is to study the intracellular signaling mechanisms for the insulin receptor (IR). The investigators have recently made the observation that after insulin stimulation of rat HTC hepatoma and other cells one or more signaling complexes form that consists of: the IR; the p85 subunit of phosphatidylinositol-3-kinase; Ras GTPase activating protein (GAP); and p62 GAP-associated protein. In the proposal, it is proposed to test the hypothesis that p85, via its two SH2 and one SH3 domains, serves as an adaptor molecule to directly link the activated IR to GTP binding proteins. First, the investigators will study the Ras pathway. Using cell lysates following insulin treatment, it is proposed to study the cellular proteins (IR and p62) interacting with the SH2 domains of p85. For this study, the investigators will employ fusion proteins containing the SH2 and SH3 domains of p85 incorporated into maltose binding protein. Cellular proteins interacting with the fusion proteins will be analyzed by western blotting with appropriate antibodies. To study direct protein-protein interactions, the investigators will employ purified IR and p62; and fusion proteins containing SH2/3 domains of p85 or GAP. To study the role of GAP and p62 in Ras activation, the investigators will employ purified p62 and antibodies to p62 in permeabilized cells and determine the effects on GAP and Ras activities. Second, PI will study the heterotrimeric G protein. The investigators will identify the 41 kD G protein using antibodies to various G proteins. The investigators will also determine whether the 41 kD G protein that they find associated with p85 is also associated with the IR; and is related to the G protein (G1R41) that is associated with the IR in human placenta. To determine whether the 41 kD G protein is associated with regulatory proteins of the Ras pathway, the investigators will immunoprecipitate the 41 kD G protein with specific antibodies and analyze by western blotting with appropriate antibodies. In cells overexpressing normal Ras or dominant inhibitory Ras, the investigators will also study association of this G protein with both p85 and the IR. These proposed studies therefore should provide new insights into IR signaling mechanisms in target cells.
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