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，是必要的辅酶 cAMP 依赖性蛋白激酶 (PKA) 抑制肾功能的因素 Na/H交换剂，NHE3。 NHE-RF 与 NHERF、NHE3 和埃兹蛋白结合 表明 NHE-RF 与其细胞靶标具有独特的相互作用 形成信号复合物。 目前的资助重点是 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 NHE3的抑制。 具体目标 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，但其机制 二聚化的生理效应尚未被探索。 生化 （远西方）和生物物理（生物传感器）测定将用于定义 NHE-RF/NHE-RF 结合动力学。 酵母双杂交筛选 突变体迷你文库将用于确定结合氨基酸 序列并选择未能二聚化的突变体。 生理学 二聚化对 cAMP 对 Na/H 交换的影响为 在表达 NHE3 的 PS120 细胞中使用荧光技术进行了研究 不二聚化的 NHE-RF 突变体。 具体目标 2：通过以下方式描述 NHE3 调节的分子基础： NHE-RF。 NHE-RF 与 NHE3 结合，两种蛋白质发生共免疫沉淀。 生物化学和生物物理测定将用于定义动力学 NHE-RF/NHE3 结合的研究，以及突变型 mini-酵母双杂交筛选 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 NHE3的磷酸化状态和NHE-RF的免疫共沉淀 NHE3 将在表达突变 NHE-RF 的 PS120 细胞中进行检查和/或 NHE3 不结合。 具体目标 3. 定义 NHE-RF 在 NHE3 的 PKA 调节。 新数据表明 NHE-RF 与 PKA 结合 锚定蛋白，埃兹蛋白。 将使用生物化学和生物物理测定 研究 NHE-RF/ezrin 结合和酵母双杂交的动力学 研究定义结合序列。 NHE-的生理作用 RF/ezrin 结合将在表达 loss-of- 的 PS120 细胞中进行研究 代表ERM区域的功能NHE-RF突变体或多肽 埃兹林。