The first event in the response of target cells to insulin is the binding of the hormone to its specific receptor on the surface of cells. This receptor is a glycoprotein whose structure has been well defined. In addition to an insulin-binding domain, the receptor has an intrinsic enzymatic activity; it catalyzes the transfer of the terminal phosphate of ATP to tyrosine residues of specific proteins. This activity is increased after the receptor binds insulin. Recent evidence from several different experimental approaches has implicated this intrinsic enzymatic activity of the receptor in mediating the biological responses to insulin. That is, if the receptor's enzymatic activity is inhibited, the ability of cells to respond to insulin is decreased. Defects in the receptor's intrinsic kinase activity have recently been noted in individuals with non-insulin-dependent diabetes. This decreased activity of the receptor may partly explain the lack of responsiveness to insulin observed in this disorder. The next major step in understanding insulin action now requires us to determine what protein(s) are phosphorylated by the insulin receptor kinase and whether such protein(s) mediate(s) the biological responses to insulin. The goal of the present research proposal is therefore directed at isolating potential substrates of the insulin receptor kinase and producing monoclonal antibodies to them. In preliminary studies, we have identified a number of endogenous substrates of the insulin receptor kinase in cells overexpressing the insulin antibodies. Microinjection of a pool of these purified substrates into frog oocytes was found to duplicate one of the biological responses to insulin, an increase in the phosphorylation of ribosomal protein S6. We therefore propose to generate monoclonal antibodies to the different substrates. These antibodies will then be used to test the role of individual proteins mediating various responses to insulin by testing the antibodies for the ability to remove the active component in the substrate preparations or to block the ability of insulin to stimulate responses in oocytes and mammalian cells. The identification of the physiologically important substrates for the insulin receptor kinase may allow us to design drugs which directly activate these proteins in cells whose receptors are impaired.
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