REGULATION OF ION FLUXES BY THE INSULIN RECEPTOR

离子通量的变化代表了由电离层激活的最早事件之一。 胰岛素与其受体的相互作用该提案旨在 定义胰岛素受体修饰离子的分子机制 靶细胞中的通量。将测试所述蛋白质的激酶活性是否与所述蛋白质的激酶活性相同。 受体是胰岛素刺激离子流所必需的， 胰岛素受体改变磷酸化和/或亚细胞 离子转运蛋白的分布。为了验证这些假设，本研究 将定义： 1.胰岛素对细胞钾通量和Na/K稳态的影响 表达正常胰岛素受体，重点研究胰岛素对 Na，K-ATPase和Na/K/Cl协同转运蛋白。 2.胰岛素刺激细胞内离子流和Na/K稳态 激活或缺陷的胰岛素受体激酶，以确定是否 受体的激酶活性是刺激离子 剂. 3.胰岛素激活离子转运蛋白的分子机制， 特别是，胰岛素是否招募了预先形成的离子转运蛋白， 质膜和/或影响其磷酸化。这将是 通过包括测量胰岛素诱导的 哇巴因结合和生物素标记的离子转运蛋白的变化 完整细胞的质膜，亚细胞的Western印迹分析， 组分（使用对每种转运蛋白和抗- 磷酸酪氨酸抗体），32 P-转运蛋白的免疫沉淀， 磷酸氨基酸分析和胰蛋白酶磷酸肽作图。 4.胰岛素刺激Na，K-α 1，α 2和β 1亚型 ATP酶的活性。伟大的丰饶 转染细胞中Na，K-泵的变化应有助于研究 胰岛素诱导的磷酸化和亚细胞 分布 目前对这种现象的分子机制知之甚少， 胰岛素调节离子流。离子通过质膜的转运 Na，K-ATP酶是一个需要能量的过程，其还原已经被 与人类肥胖症的发病机制有关。此外， 胰岛素刺激的离子流有助于急性和长期 糖尿病并发症这项研究将确定分子机制 胰岛素通过该途径改变离子流和Na，K-ATP酶活性， 允许设计更好的治疗方法来预防肥胖和急性或慢性 糖尿病并发症

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.