The insulin receptor serves to focus the hormone on particular target tissues as well as to initiate the responses of these cells to the hormone. Extensive studies of the insulin receptor by many investigators have told us a great deal about this molecule, including its complete amino acid sequence as well as the role of specific residues in receptor function. In addition to binding insulin, the receptor has been shown to have an intrinsic tyrosine specific kinase activity. This activity is increased after the receptor binds insulin and appears to be critical for insulin to stimulate various biological responses. A reversible decrease in the receptor's intrinsic tyrosine kinase activity has been observed in cells from patients with non-insulin dependent diabetes, possibly contributing to the insulin resistance observed in these patients. In addition, a similar phenomena can be induced in cells in culture by stimulating the serine phosphorylation of the receptor with phorbol esters. In vitro, the receptor can be directly phosphorylated by a specific serine/threonine protein kinase, called protein kinase C. This phosphorylation also appears to decrease the intrinsic tyrosine kinase activity of the receptor without affecting the ability of the receptor to bind insulin. To further study the mechanisms of this regulation, we have identified one of the isozymes of protein kinase C for its ability to phosphorylate the receptor in vitro and have isolated stably transfected cell lines which overexpress the insulin receptor and this protein kinase C. We have found that the ability of insulin to activate the receptor kinase in these cells is inhibited ~ 70%. We therefore propose to further study this process by: 1) Selecting stable transfectants of this protein kinase C and various mutated insulin receptors to identify the specific sites regulated by this enzyme; 2) Test the affect of overexpression of kinase C on the ability of insulin and its receptor to be internalized and on insulin to induce various biological responses; 3) Test the specificity of this affect by isolating cell lines overexpressing the receptor and other protein kinase C isozymes and examining the same processes in these cells; and 4) Test whether insulin activates any of the protein kinase C isozymes. After responding to insulin, a cell must terminate its response. One mechanisms for accomplishing this is to degrade the hormone. One enzyme, called insulin-degrading enzyme, has been implicated in this process. We have produced monoclonal antibodies to this enzyme, and isolated a cDNA clone which codes for this molecule. We now propose to: 1) Identify residues in the active site of this protease by site-directed mutagenesis; 2) Increase and/or decrease the levels of this enzyme in cells to further test the role of this enzyme in terminating the response to insulin; and 3) Identify the regions of the protease involved in recognizing insulin by generating anti-peptide antibodies to specific sequences of the molecule. A further understanding of this enzyme may allow the design of specific inhibitors of insulin degradation.
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