INSULIN RECEPTOR/KINASE REGULATION IN THE INTACT CELLS

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In cultured mouse 3T3-L1 cells, insulin controls autophosphorylation of the insulin receptor/protein kinase. The kinetics and concentration dependence of this reaction suggest it is part of the regulatory pathways that control metabolic responses to insulin. Many of these pathways involve other protein kinases and some form of signal amplification. Placing the insulin receptor/protein kinase in an enzyme cascade would make it subject to multiple levels of regulation. The problem is to understand cellular regulation of insulin receptor-protein kinase activity. The long-term objective is to alter the in situ activity of the insulin receptor/protein kinase by using selective substrates and inhibitors that have been defined in vitro. In vitro kinetic experiments with purified receptor/kinase should establish: 1. The effect of site-specific autophosphorylation on activation and substrate selection; this requires purification of a 180 kDa acidic substrate from 3T3-L1 cells. 2. The mechanisms of inhibition and activation by substrate and nonsubstrate polypeptides; chemical and enzymic modifications of these polypeptides will be used to further alter their effects. 3. An assay for use in identifying regulators of cellular origin. Comparisons with receptor/kinase autophosphorylated in situ and subsequently purified, will be done by 1. peptide mapping of the phosphorylation sites, and 2. determination of substrate selection. For application to the receptor/kinase in situ, methods will be developed to incorporate selected substrates and/or inhibitors into the cells. Measurements will be made of 1. phosphorylation of the "exogenous substrate", 2. autophosphorylation of the receptor/kinase, and 3. altered biological responses. Results of the proposed studies may be directly applicable to Type 2 diabetes (a "post-receptor defect"), to other growth hormone receptor/protein kinases, and to the nontransforming or transforming abilities of cellular versus viral oncogenes.