SODIUM-H+ TRANSPORT IN RENAL APICAL MEMBRANES

钠-H 在肾尖膜中的转运

• 批准号: 6517619
• 负责人: EDWARD J WEINMAN
• 金额: $ 25.53万
• 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/6517619
• 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 hydrogen exchanger; 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，它是一种必需的协同蛋白， 肾脏cAMP依赖性蛋白激酶（PKA）抑制因子 Na/H交换器，NHE 3。 NHE-RF与NHERF、NHE 3和埃兹蛋白结合 这表明NHE-RF与其细胞靶点独特地相互作用 形成信号复合物。 目前的补助金集中在 的生化要求和生理意义 参与PKA介导的蛋白质部分之间的物理相互作用 抑制NHE 3。 具体目的1：确定NHE-RF二聚化的功能作用。 NHE-RF与NHE-RF结合形成头对头二聚体，但是NHE-RF结合的机制是不确定的。 和二聚化的生理学效应尚未探索。 生化 (Far Western）和生物物理学（生物传感器）测定将用于定义 NHE-RF/NHE-RF结合的动力学。 酵母双杂交筛选 突变体微型文库将用于确定结合氨基酸 序列并选择不能二聚化的突变体。 生理 二聚化对cAMP对Na/H交换的影响将是 在表达NHE 3的PS120细胞中使用荧光技术研究， 不二聚化的NHE-RF突变体。 具体目标2：描述NHE 3调节的分子基础， NHE-RF。 NHE-RF与NHE 3结合，两种蛋白质发生免疫共沉淀。 将使用生物化学和生物物理测定来确定动力学 的NHE-RF/NHE 3结合，和酵母双杂交筛选突变的小- 文库以确定结合氨基酸序列并选择 功能丧失突变体 cAMP对Na/H转运的影响， NHE 3的磷酸化状态和NHE-RF的免疫共沉淀 将在表达突变NHE-RF和/或NHE 3的PS120细胞中检查NHE-RF和/或NHE 3。 NHE 3不结合。 具体目标3。 为了确定NHE-RF在治疗中的生理重要性， NHE 3的PKA调节。 新数据表明NHE-RF与PKA结合 锚蛋白，埃兹蛋白。 将使用生化和生物物理分析 研究NHE-RF/ezrin结合动力学和酵母双杂交 研究以确定结合序列。NHE的生理效应- RF/ezrin结合将在表达缺失- 功能NHE-RF突变体或多肽，其代表 ezrin。