Insulin is the major anabolic and anti-catabolic hormone of man. In spite of substantial progress in the last several years, the cellular mechanisms of insulin action remain poorly understood. The insulin receptor is an insulin-activated, transmembrane tyrosine-specific protein kinase. Substantial evidence now indicates that the first intracellular step of regulatory significance in insulin action is the tyrosine autophosphorylation of this receptor, and the concomitant activation of its kinase function. Considerable uncertainty, however, prevails as to the nature of the next step. A number of intracellular proteins other than the IR have been shown to undergo insulin-stimulated tyrosine- specific phosphorylation. We proposed that among these are molecules that are crucial to the further transmission of the signal initiated by the insulin receptor. The goal of this work is to identify these physiologically relevant molecular targets of the insulin receptor kinase, and determine how they function to transmit this signal in biochemical terms. The bulk of the work proposed in the next phase of our studies will focus on one candidate target, a Mr 1800,000 polypeptide, which is phosphorylated on tyrosine exclusively in response to insulin and IGF-1 in many cells, which we call p180; p180 may actually represent a family of proteins of similar Mr. Substantial purification of p180 has been accomplished; one monoclonal antibody, and a panel of candidate monoclonal antibodies, has been produced, and the generation of more such antibodies is planned. These monoclonal antibodies will be employed to complete the purification of p180 as well as to isolate by molecular cloning, a cDNA (or cDNA's) encoding this polypeptide (or polypeptides). In collaborative studies, the cell biology of p180 will be characterized by immunoaffinity electron microscopic methods. The amino acid sequence surrounding the sites of insulin-stimulated tyrosine phosphorylation on p180 will be defined, and the phosphorylation of purified p180 by the purified insulin receptor will be characterized. Employing the information and reagents gathered through these efforts, we will determine whether p180 functions as a signalling intermediate in insulin action. This will be accomplished by inhibiting the expression of endogenous p180 with antisense RNA and/or by inhibiting its TYR phosphorylation through the introduction of appropriate inhibitory monoclonal antibodies into intact cells. If p180 proves not to be a signalling intermediate, we will identify other candidate substrates using improved anti-phosphotyrosine antibodies, and insulin receptor affinity chromatography. These studies will have important implications for the design of new pharmacologic interventions in diabetes mellitus, and in the understanding of the disordered growth control which characterized cancer.
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