SODIUM-H+ TRANSPORT IN RENAL APICAL MEMBRANES

钠-H 在肾尖膜中的转运

• 批准号: 6190944
• 负责人: EDWARD J WEINMAN
• 金额: $ 21.87万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6190944
• 关键词: apical membrane; cell line; cyclic AMP; cytoskeletal proteins; dimer; fluorescent dye /probe; genetic library; hydrogen transport; immunoprecipitation; ion transport; laboratory rabbit; membrane transport proteins; protein binding; protein kinase A; protein protein interaction; renal tubular transport; site directed mutagenesis; sodium ion; yeast two hybrid system

NHE-RF is a new discovered PDZ motif protein that is a necessary co- factor in cAMP-dependent protein kinase (PKA) inhibition of the renal Na/H exchanger, NHE3. NHE-RF binds to NHERF, to NHE3, and to ezrin suggesting that NHE-RF interacts uniquely with its cellular targets forming signaling-complexes. The present grant focuses on the biochemical requirements for and physiologic significance of the physical interaction between proteins moieties involved in PKA mediated inhibition of NHE3. Specific Aim 1: To determine the functional role of NHE-RF dimerization. NHE-RF binds to NHE-RF forming head-to-head dimers but the mechanism of and the physiologic effect of dimerization are unexplored. Biochemical (Far Westerns) and biophysical (biosensor) assays will be used to define the kinetics of NHE-RF/NHE-RF binding. Yeast two-hybrid screening of mutant mini-libraries will be used to determine the binding amino acid sequences and to select mutants that fail to dimerize. The physiologic effect of dimerization on the effect of cAMP on Na/H exchange will be studied using fluorescence techniques in PS120 cells expressing NHE3 and NHE-RF mutants that do not dimerize. Specific Aim 2: To delineate the molecular basis for NHE3 regulation by NHE-RF. NHE-RF binds to NHE3 and the two proteins co-immunoprecipitate. Biochemical and biophysical assays will be used to define the kinetics of NHE-RF/NHE3 binding, and yeast two-hybrid screening of mutant mini- libraries to determine the binding amino acids sequences and to select loss-of-function mutants. The effect of cAMP on Na/H transport, the phosphorylation state of NHE3, and the co-immunoprecipitation of NHE-RF and NHE3 will be examined in PS120 cells expressing mutant NHE-RF and/or NHE3 which do not bind. Specific Aim 3. To define the physiologic importance of NHE-RF in the PKA regulation of NHE3. New data suggests NHE-RF binds to the PKA anchor protein, ezrin. Biochemical and biophysical assays will be used to study the kinetics of NHE-RF/ezrin binding and yeast two-hybrid studies to define the binding sequences. The physiologic effect of NHE- RF/ezrin binding will be studied in PS120 cells expressing loss-of- function NHE-RF mutants or polypeptides representing the ERM region of ezrin.

NHE-RF是一种新发现的PDZ基序蛋白，是必要的共同 肾脏抑制cAMP依赖性蛋白激酶（PKA）的因子 Na/H交换器，NHE3。 NHE-RF与NHERF，NHE3结合，并与Ezrin结合 表明NHE-RF与其细胞靶标有独特的相互作用 形成信号复合物。 目前的赠款专注于 对生物化学要求和生理意义的要求 PKA介导的蛋白质部分之间的物理相互作用 抑制NHE3。 特定目标1：确定NHE-RF二聚体的功能作用。 NHE-RF与NHE-RF结合了头对头二聚体，但机理 二聚化的生理效果未开发。 生化 （远西方）和生物物理（生物传感器）测定法将用于定义 NHE-RF/NHE-RF结合的动力学。 酵母双杂交筛查 突变的迷你纤维将用于确定结合氨基酸 序列并选择无法二聚的突变体。 生理学 二聚化对CAMP对Na/H交换的影响的影响将是 在表达NHE3和的PS120细胞中使用荧光技术进行了研究 不二聚体的NHE-RF突变体。 特定目的2：描述NHE3调控的分子基础 nhe-rf。 NHE-RF与NHE3结合，两种蛋白质共免疫沉淀。 生化和生物物理测定将用于定义动力学 NHE-RF/NHE3结合，以及酵母对突变体微型筛选 库确定结合氨基酸序列并选择 功能丧失突变体。 营地对Na/H运输的影响， NHE3的磷酸化状态，以及NHE-RF的共免疫沉淀 将在表达突变体NHE-RF和/或的PS120细胞中检查NHE3 不绑定的NHE3。 特定目的3。定义NHE-RF的生理重要性 NHE3的PKA调节。 新数据表明NHE-RF与PKA结合 锚蛋白Ezrin。 将使用生化和生物物理测定 研究NHE-RF/Ezrin结合的动力学和酵母双杂交的动力学 研究定义结合序列。 NHE的生理效应 RF/Ezrin结合将在表达损失的PS120细胞中研究 功能NHE-RF突变体或代表ERM区域的多肽 埃兹林。