REGULATION OF ION FLUXES BY THE INSULIN RECEPTOR

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Changes in ion fluxes represent one of the earliest events activated by the interaction of insulin with its receptor. This proposal wants to define the molecular mechanism by which the insulin receptor modifies ion fluxes in target cells. It will be tested if the kinase activity of the receptor is required for insulin stimulation of ion fluxes and if the insulin receptor modifies the phosphorylation and/or subcellular distribution of ion transporters. To test these hypotheses, this study will define: 1. The effect of insulin on K fluxes and Na/K homeostasis in cells expressing normal insulin receptors, focusing on insulin effect on the Na,K-ATPase and the Na/K/Cl cotransporter. 2. Insulin stimulation of ion fluxes and Na/K homeostasis in cells with activated or defective insulin receptor kinase to determine whether the kinase activity of the receptor is required for the stimulation of ion fluxes. 3. The molecular mechanism by which insulin activates ion transporters, and specifically, whether insulin recruits preformed ion transporters on the plasma membrane and/or affects their phosphorylation. This will be determined by a combined approach involving measurement of insulin-induced changes in ouabain binding and biotin labeling of ion transporters on the plasma membrane of intact cells, Western blot analysis on subcellular fractions (using antibodies specific for each transporter and anti- phosphotyrosine antibodies), immunoprecipitation of 32P-transporters, phosphoamino acid analysis and tryptic phosphopeptide mapping. 4. Insulin stimulation of the alpha1, alpha2, and beta1 isoforms of Na,K- ATPase after their transfection into mammalian cells. The great abundance of Na,K-pumps in transfected cells should facilitate the study of changes of insulin-induced changes in their phosphorylation and subcellular distribution. Very little is currently known on the molecular mechanism(s) by which insulin regulates ion fluxes. Ion transport across the plasma membrane by the Na,K-ATPase is an energy-requiring process whose reduction has been implicated in the pathogenesis of human obesity. In addition, changes in insulin-stimulated ion fluxes contribute to acute and long-term complications of diabetes. This study will define the molecular mechanism by which insulin modifies ion fluxes and Na,K-ATPase activity and may allow the design of better therapy to prevent obesity and acute or chronic complications of diabetes.