Insulin is the major anabolic and anticatabolic hormone of mammals. In addition, it is representative of a large family of peptide/protein growth factors, some of which are related structurally (e.g., IGF-1 and IGF-2), and others in a broader functional sense (e.g., EGF). The molecular basis for the cellular actions of insulin and these hormones is not understood. Much evidence indicates that as a consequence of insulin's interaction with its cell-surface receptor, a series of regulatory reactions are initiated which, within minutes, modify the activity of a variety of intracellular enzymes and transmembrane transport systems. It appears that a critical intermediate step in this series of regulatory reactions involves an alteration in protein phosphorylation, which is expressed in several ways: many enzymes regulating substrate metabolism undergo a dephosphorylation, which modifies their activity. In addition, insulin promotes the (serine-specific) phosphorylation of a subset of cellular proteins. Finally, the insulin receptor molecule itself is known to function as a tyrosine-specific protein kinase. The goals of the proposed studies are to define the role of the insulin receptor tyrosine kinase in the biologic actions of the hormone and elucidate the specific biochemical steps by which this role is effected. It is hypothesized that augmented serine-specific protein phosphorylation is (one of) the mechanism(s) by which insulin regulatory signals are transmitted and amplified, and the detection and purification of insulin-stimulated (serine-specific) protein kinases will be undertaken. Further studies will entail the purification of the insulin receptor, an analysis of its primary structure and attempts to selectively modify its kinase function. We will develop techniques to reinsert the receptor into cultured cells. Specific peptide substrates and inhibitors for serine and tyrosine kinase function will be devised. Techniques to examine the effects of these peptides or antibodies to these peptides will be established in hormone-responsive cells. We will search for the intracellular targets of the receptor tyrosine kinase, employing antibodies that react with phosphotyrosine residues, and attempt to detect proteins that interact specifically with the insulin receptor. These studies will clarify the cellular mechanism of insulin action and provide new insights into the hormonal regulation of cell metabolism and growth. These results will have implications for the design of pharmacologic intervention in diabetes mellitus, and in understanding the disordered growth control which characterizes cancer.
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