REGULATION OF ION FLUXES BY THE INSULIN RECEPTOR

胰岛素受体对离子通量的调节

• 批准号: 2149174
• 负责人: NICOLA LONGO
• 金额: $ 10.04万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2149174
• 关键词: CHO cells; adipocytes; biological signal transduction; cell type; immunoprecipitation; insulin; insulin receptor; ion transport; membrane potentials; membrane transport proteins; mutant; northern blottings; phosphorylation; potassium; protein isoforms; protein tyrosine kinase; receptor binding; receptor expression; rubidium; site directed mutagenesis; sodium; sodium potassium exchanging ATPase; transfection; western blottings

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.

离子通量的变化代表了最早激活的事件之一 胰岛素与其受体的相互作用。该提议想 定义胰岛素受体修饰离子的分子机制 靶细胞中的通量。如果它的激酶活性 受体是胰岛素刺激离子通量所必需的，如果 胰岛素受体可修饰磷酸化和/或亚细胞 离子转运蛋白的分布。为了检验这些假设，这项研究 将定义： 1。胰岛素对细胞中K通量和Na/k稳态的影响 表达正常的胰岛素受体，重点是胰岛素对 Na，K-ATPase和Na/K/Cl共转运蛋白。 2。胰岛素刺激离子通量和Na/k的细胞中的Na/k稳态 激活或有缺陷的胰岛素受体激酶，以确定是否是 受体的激酶活性是刺激离子需要的 通量。 3。胰岛素激活离子转运蛋白的分子机制， 具体而言，胰岛素是否在 质膜和/或影响其磷酸化。这将是 由涉及胰岛素诱导的测量的组合方法确定 离子转运蛋白的ouabain结合和生物素标记的变化 完整细胞的质膜，亚细胞的蛋白质印迹分析 分数（使用针对每个转运蛋白和抗体特异的抗体 磷酸酪氨酸抗体），32p转运蛋白的免疫沉淀， 磷酸氨基酸分析和胰蛋白酶磷酸肽图。 4。胰岛素刺激Na，k-的α1，α2和β1同工型 ATPase转染到哺乳动物细胞后。丰富的 在转染细胞中的Na，K-Pumps应促进变化 胰岛素诱导的磷酸化和亚细胞的变化 分配。 目前在分子机制上已经知道的很少 胰岛素调节离子通量。离子跨质膜的离子运输 Na，K-ATPase是一个降低能量的过程 与人肥胖的发病机理有关。另外，变化 胰岛素刺激的离子通量有助于急性和长期 糖尿病并发症。这项研究将定义分子机制 胰岛素修饰离子通量和Na，K-ATPase活性，并且可能 允许设计更好的治疗以防止肥胖和急性或慢性 糖尿病并发症。