NHERF-1 and PTH regulation of the renal transport of phosphate.

NHERF-1 和 PTH 调节磷酸盐的肾脏转运。

基本信息

批准号：
8144168
负责人：
EDWARD J WEINMAN
金额：
$ 31.24万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-05-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8144168
关键词：
8-Bromo Cyclic Adenosine Monophosphate Accounting Adaptor Signaling Protein Adenoviruses Affinity Animals Binding Binding Proteins Biochemical Biological Assay C-terminal Cell Line Cell membrane Cells Co-Immunoprecipitations Complex Confocal Microscopy Cyclic AMP-Dependent Protein Kinases Cytoskeleton Density Gradient Centrifugation Diet Disease Dissociation Elements Estrogen receptor positive Excretory function Family Functional disorder Gel Genes Goals Hormones In Vitro Ingestion Inorganic Phosphate Transporter Ion Channel Kidney Maps Mediating Membrane Mitogen-Activated Protein Kinases Modeling Mus Mutation NHERF-1 protein Nephrolithiasis Neurofibromin 2 Parathyroid Hormone Receptor Parathyroid gland Pathway interactions Phosphoproteins Phosphorylation Phosphorylation Site Physiological Process Protein Binding Protein Dephosphorylation Protein Family Protein Hormone Receptor Protein Kinase Protein Kinase C Proteins Proximal Kidney Tubules Psoriasis Regulation Reporting Resistance Role Scaffolding Protein Schizophrenia Second Messenger Systems Serine Signal Transduction Signaling Protein Site Site-Directed Mutagenesis Slice Sodium Stimulus Sucrose Surface Plasmon Resonance Threonine Tubular formation apical membrane brush border membrane ezrin hormone regulation in vivo inorganic phosphate insight interest malignant breast neoplasm member moesin public health relevance radixin protein response second messenger sodium-hydrogen exchanger regulatory factor trafficking urinary

项目摘要

DESCRIPTION (provided by applicant): The Sodium-Hydrogen Exchanger Regulatory Factor-1 (NHERF-1) is an adaptor protein containing two protein-interactive PDZ domains and a C-terminal ERM binding domain that localizes to the brush border membrane of renal proximal convoluted tubule cells and binds to Npt2a, the major sodium-dependent phosphate transporter. Our recent studies have indicated that sodium-dependent phosphate transport in proximal tubule cells from NHERF-1-/- kidneys are resistant to the inhibitory effect of Parathyroid Hormone (PTH). In this current application, we explore the hypothesis that PTH mediates the phosphorylation of specific residues in PDZ I of NHERF-1 thereby regulating Npt2a/NHERF-1 complexes, the abundance of Npt2a in the apical membrane of renal proximal tubule cells, and as a consequence, the tubular reabsorption of phosphate. Elucidation of the factors that regulate the binding of target proteins to PDZ I of NHERF-1 may also provide broader insights into how regulation of PDZ domains of adaptor proteins impact on biologic responses to hormones and on the pathophysiology of NHERF-1 related diseases. In intact animals and cultured proximal tubule cells, we will use physiologic, biochemical, and cell biologic assays to determine how PTH-mediated phosphorylation of PDZ I of NHERF-1 regulateS the binding affinity of target proteins such as Npt2a and the proximal tubule reabsorption of phosphate. We propose three specific aims. First, we will map the serine and/or threonine residues in PDZ I of NHERF-1 that are phosphorylated in response to PTH and downstream protein kinases. Second, we propose to study the association and dissociation of Npt2a/NHERF-1 complexes in response to PTH-mediated phosphorylation of NHERF-1 using in-vitro and in-vivo assays. Third, we will determine the physiologic role of PTH-mediated NHERF-1 phosphorylation on the regulation of phosphate transport in the proximal tubule of the kidney. PUBLIC HEALTH RELEVANCE NHERF-1 is an adaptor protein that binds multiple transporters in the kidney including Npt2a, the major renal proximal tubule phosphate transporter. We will study the hypothesis that Parathyroid Hormone-mediated regulation of renal phosphate transport involves regulation of the binding of Npt2a to NHERF-1 by site-specific phosphorylation of the PDZ I domain of NHERF-1. These observations may provide mechanistic insights into the processes that regulate the binding of target proteins to adaptors such as NHERF-1 and provide broader insights into the pathophysiology of NHERF-1 related diseases.

描述（由申请人提供）：钠氢交换调节因子-1 (NHERF-1) 是一种衔接蛋白，含有两个蛋白质相互作用的 PDZ 结构域和一个 C 端 ERM 结合结构域，定位于肾近端刷状缘膜曲管细胞并与 Npt2a（主要的钠依赖性磷酸盐转运蛋白）结合。我们最近的研究表明，NHERF-1-/- 肾脏近端小管细胞中钠依赖性磷酸盐转运对甲状旁腺激素 (PTH) 的抑制作用具有抵抗力。在当前的应用中，我们探讨了以下假设：PTH 介导 NHERF-1 的 PDZ I 中特定残基的磷酸化，从而调节 Npt2a/NHERF-1 复合物、肾近曲小管细胞顶膜中 Npt2a 的丰度，并作为结果，磷酸盐的肾小管重吸收。阐明调节靶蛋白与 NHERF-1 的 PDZ I 结合的因素也可能为接头蛋白 PDZ 结构域的调节如何影响激素的生物反应以及 NHERF-1 相关疾病的病理生理学提供更广泛的见解。在完整的动物和培养的近曲小管细胞中，我们将使用生理、生化和细胞生物学测定来确定 PTH 介导的 NHERF-1 的 PDZ I 磷酸化如何调节靶蛋白（如 Npt2a）的结合亲和力以及近曲小管重吸收磷酸盐。我们提出三个具体目标。首先，我们将绘制 NHERF-1 的 PDZ I 中的丝氨酸和/或苏氨酸残基图谱，这些残基响应 PTH 和下游蛋白激酶而被磷酸化。其次，我们建议使用体外和体内测定法研究 Npt2a/NHERF-1 复合物响应 PTH 介导的 NHERF-1 磷酸化的结合和解离。第三，我们将确定 PTH 介导的 NHERF-1 磷酸化对肾近曲小管磷酸盐转运调节的生理作用。公共卫生相关性 NHERF-1 是一种接头蛋白，可结合肾脏中的多种转运蛋白，包括 Npt2a（主要的肾近端小管磷酸转运蛋白）。我们将研究这样的假设：甲状旁腺激素介导的肾磷酸盐转运调节涉及通过 NHERF-1 的 PDZ I 结构域的位点特异性磷酸化来调节 Npt2a 与 NHERF-1 的结合。这些观察结果可能为调节靶蛋白与 NHERF-1 等接头的结合过程提供机制见解，并为 NHERF-1 相关疾病的病理生理学提供更广泛的见解。