This is the second revision of the competitive renewal of DK38712 entitled """"""""Interaction between insulin receptors and cell proteins"""""""". This proposal focuses on the function of the insulin receptor tyrosyl kinase (IR) and the mechanisms of insulin signal transmission. This work is scientifically and clinically important because diabetes is a contemporary health problem that affects about 2% of the world population. Whereas 10% of these individuals suffer from an absolute lack of insulin, most are diabetic because their cells do not respond fully to normal or elevated amounts of circulating insulin. The biochemical problems causing insulin resistance in these Type II diabetics are not well understood. In a few cases, mutations of the IR appear to be the culprit; however, rational treatments for the majority of diabetics requires a knowledge of IR function and regulation, and the molecular details of IR signal transmission. The experiments outlined in this proposal will define structural features of the juxtamembrane region of the IR that are essential for receptor function and signal transmission. The juxtamembrane region of the IR contains about 23 amino acids and is located immediately after the transmembrane spanning region at the inner face of the plasma membrane. Although considerable amino acid sequence identity exists among tyrosyl kinases, the juxtamembrane regions are frequently unique and may be partially responsible for the specificity of signal transmission. Mutations in the juxtamembrane region have a minimal effect on autophosphorylation and kinase activity of the IR, but significantly impair its biological activity and endocytosis, and markedly reduce insulin-stimulated tyrosine phosphorylation of endogenous substrates. Thus, the juxtamembrane region appears to be essential for specific interactions between the IR and cellular proteins that are involved in these responses. Site-directed mutagenesis of the cDNA of the IR will be carried out to alter specific amino acids in the juxtamembrane region to define some essential features for IR function and signaling: (1) determine the role of Tyr960 and Tyr953 in the juxtamembrane region for the interaction between the IR and cellular proteins; (2) determine the requirement for a predicted beta-turn near these tyrosine residues by altering the amino acid sequence in the NPXY960 and GPXY953 motifs to sequences that predict or do not predict beta-turns; (3) assess the specificity of the juxtamembrane region for biological activity, endocytosis and substrate phosphorylation by replacing the IR juxtamembrane region with the corresponding sequence from the receptors for insulin-like growth factor-1, epidermal growth factor, platelet derived growth factor, c-ros and LDL; and (4) determine whether overexpression of the IR juxtamembrane region in a truncated IR molecule competitively inhibits normal IR function and signaling. These four classes of mutant IR molecules will be studied for biological activity, """"""""affinity"""""""" for the cellular endocytosis system, their ability to interact with and phosphorylate the major IR substrate IRS-1(pp185), and their ability to activate the phosphatidylinositol 3'-kinase. These experiments will provide new information regarding the molecular basis for interactions between the IR and cellular protein, and provide a model to understand the mechanism of insulin receptor signal transmission.
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