The overall goal of this research is to help define the initial steps in the biochemical mechanism by which insulin mediates its metabolic effects. A clear understanding of insulin signal transduction is necessary to elucidate the pathogenesis of the cellular insulin resistance of diabetes mellitus and related disorders. The specific focus of this research is the purification and functional analysis of pp60, a putative endogenous cellular substrate of the insulin receptor tyrosine kinase. pp60 is a 60 kDa protein, of unknown identity, which rapidly becomes tyrosine phosphorylated in insulin-stimulated adipocytes. Tyrosine phosphorylation is hypothesized to activate pp60, enabling it to then relay the insulin signal from the cell surface insulin receptor to intracellular metabolic pathways. To evaluate this hypothesis, an experimental approach is proposed which parallels that already proven successful for pp185 (IRS-1), a distinct insulin receptor substrate. pp60 will be preparatively purified from insulin-treated adipocytes by antiphosphotyrosine immunoaffinity chromatography. Direct identification of pp60 by partial amino acid sequencing will allow molecular cloning of the full-length pp60 cDNA, to establish whether pp60 is homologous to proteins of known function. Antibodies to pp60 will be raised for use in immunopurifying pp60 and analyzing biochemical properties of pp60 relevant to insulin action, including: (a) determining whether pp60 is a direct in vitro substrate of the insulin receptor kinase, (b) determining whether pp60 complexes with other cellular signalling proteins, and (c) determining the tissue and subcellular distribution of pp60. Analyzing pp60 is a logical next step towards a complete definition of the insulin signaling mechanism and may ultimately contribute to the development of improved therapeutic approaches to disease states of impaired insulin responsiveness, such as diabetes mellitus.
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