NHERF-1 and dopamine-mediated regulation of renal phosphate transport.

NHERF-1 和多巴胺介导的肾磷酸盐转运调节。

• 批准号: 8394581
• 负责人: EDWARD J WEINMAN
• 金额: --
• 依托单位: BALTIMORE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8394581
• 关键词: Adaptor Signaling Protein; Address; Adenoviruses; Adenylate Cyclase; Affect; Aftercare; Agonist; Amino Acids; Animals; Apical; Aromatic-L-Amino-Acid Decarboxylases; Binding; Binding Proteins; Biochemical; Biological; C-terminal; Calcium; Carbidopa; Cell Fraction; Cell Fractionation; Cell membrane; Cell model; Cells; Co-Immunoprecipitations; Complex; Confocal Microscopy; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Density Gradient Centrifugation; Deposition; Disease; Dissociation; Dopamine; Dopamine D1 Receptor; Dopamine D2 Receptor; Dopamine Receptor; Estrogen receptor positive; Excretory function; Family; Funding; G-Protein-Coupled Receptors; Gene Transfer; Hormonal; Hormones; Human; Hybrids; Hypophosphatemia; Immunoprecipitation; In Vitro; Individual; Inorganic Phosphate Transporter; Ion Channel; Kidney; Kidney Calculi; Kidney Papilla; Knockout Mice; Label; Laboratories; Laboratory Study; Length; Ligand Binding Domain; Link; MAP Kinase Gene; Measures; Mediating; Membrane; Modeling; Modification; Monitor; Mus; Mutate; Mutation; NHERF-1 protein; Nephrolithiasis; Nerve; Neurofibromin 2; Null Lymphocytes; Parathyroid gland; Pathway interactions; Patients; Phosphorylation; Phosphorylation Site; Physiological; Play; Process; Protein Binding; Protein Family; Protein Kinase; Protein Kinase C; Protein Kinase Inhibitors; Proteins; Proximal Kidney Tubules; Psoriasis; Rattus; Regulation; Relative (related person); Research; Resistance; Retrieval; Role; Schizophrenia; Second Messenger Systems; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Site; Slice; Sodium; Staining method; Stains; Stream; Sucrose; Surface Plasmon Resonance; System; Testing; Tissues; Uric Acid; Usher Syndrome; VSNL1 gene; Vesicle; Veterans; Viral; Yeasts; apical membrane; autocrine; basolateral membrane; brush border membrane; calcium phosphate; citrate carrier; deafness; decarboxylase inhibitor; ezrin; hormone regulation; inorganic phosphate; insight; interest; kidney cell; malignant breast neoplasm; member; moesin; mutant; paracrine; polypeptide; protein kinase inhibitor; radixin protein; receptor; receptor binding; renal tubular reabsorption; renal tubular transport; research study; response; second messenger; sodium-hydrogen exchanger regulatory factor; uptake

The renal tubular reabsorption of phosphate is mediated, in large measure, by a sodium- dependent phosphate transporter called Npt2a located in the apical membrane of the cells of the proximal convoluted tubule. Npt2a is tethered to the apical membrane by a PDZ domain adaptor protein isolated and cloned by our laboratory called the Sodium-Hydrogen Exchanger Regulatory Factor-1 (NHERF-1). NHERF-1 null mice demonstrate increased excretion of phosphate, calcium, and uric acid associated with calcium deposition in the renal papilla. In confirmation with the mouse, NHERF-1 mutations in humans have been associated kidney stones. Recent studies from our laboratory have indicated that Parathyroid hormone results in the phosphorylation of the first PDZ domain of NHERF-1 with the subsequent dissociation of Npt2a/NHERF-1 complexes, a decrease in the apical membrane abundance of Npt2a, and a decrease in phosphate transport. We speculate that this unique mechanism may be a general process that regulates NHERF-1 binding to some of its other target proteins. To study the role of NHERF-1 in renal phosphate transport and to determine if the phosphorylation of NHERF-1 is regulated by other hormones, we propose studies to examine the mechanism by which dopamine inhibits renal phosphate transport. Using freshly prepared kidney slices and renal proximal tubule cells in primary culture from wild-type and NHERF-1 null animals, we will first determine if NHERF-1 binds to the dopamine D1-like receptor using immunoprecipitation and confocal microscopy. Using 32P labeled cells, we will then determine if occupancy of the dopamine D1-like receptor results in the phosphorylation of NHERF-1 and, using adenovirus-mediated gene transfer of wild-type and mutated forms of NHERF-1, determine which residues in the first PDZ domain, the binding site of Npt2a, are phosphorylated. Specific interest will focus on serine77 that lies in the Npt2a binding groove of NHERF-1. We will then determine the signaling pathways used by the dopamine D1-like receptor by measuring cAMP accumulation, and PKC activity in wild-type and NHERF-1 null cells. To determine the physiologic role of NHERF-1, we will measure the effect of dopamine on sodium-dependent phosphate transport in wild-type and NHERF-1 null proximal tubule cells. Studies will also be performed in NHERF-1 null cells infected with adenovirus-NHERF-1 to determine the specific role of NHERF-1 in mediating the effect of dopamine and with mutant forms of NHERF-1 to determine the role of specific residues such as serine77. To examine the mechanism of inhibition, we will determine if dopamine dissociates Npt2a/NHERF-1 complexes using quantitative immunoprecipitation, confocal microscopy, and sucrose density gradient ultracentrifugation. Thus, our immediate objectives are to test the hypothesis that NHERF-1 interacts with the dopamine receptor(s) and that dopamine inhibits renal phosphate transport by phosphorylating the Npt2a binding protein NHERF-1 thereby dissociating NHERF-1/Npt2a complexes and decreasing the apical membrane abundance of Npt2a. We anticipate that comparison of hormonal transduction pathways will provide new insights into the biologic role of NHERF-1 on phosphate transport and, further understandings of the role of NHERF-1 in other physiologic and pathophysiologic processes such as nephrolithiasis, schizophrenia, psoriasis, aggressive forms of estrogen receptor positive breast cancer, and nerve deafness (Usher's syndrome). .

磷酸盐的肾小管重吸收在很大程度上是通过钠介导的 依赖性的磷酸转运蛋白称为NPT2A，位于近端综合小管细胞的顶膜中。 NPT2A通过我们的实验室分离并克隆的PDZ结构域衔接蛋白将NPT2A束缚在顶端膜上，称为氢交换器调节因子1（NHERF-1）。 NHERF-1无效小鼠表明磷酸盐，钙和与钙沉积相关的磷酸盐，钙和尿酸的排泄增加。用小鼠确认，人类的NHERF-1突变与肾结石有关。我们实验室的最新研究表明，甲状旁腺激素导致NHERF-1的第一个PDZ结构域的磷酸化，随后NPT2A/NHERF-1复合物的分离，NPT2A的顶膜丰度降低，并降低了磷酸盐转运的降低。我们推测，这种独特的机制可能是调节NHERF-1与其他靶蛋白的结合的一般过程。为了研究NHERF-1在肾磷酸转运中的作用，并确定NHERF-1的磷酸化是否受其他激素的调节，我们建议研究研究多巴胺抑制肾磷酸转运的机制。使用新鲜制备的肾脏切片和肾近端小管细胞在野生型和NHERF-1无动物的原代培养物中，我们将首先确定NHERF-1是否使用免疫沉淀和共聚焦显微镜结合了多巴胺D1样受体。然后，我们将使用32p标记的细胞确定多巴胺D1样受体的占用是否会导致NHERF-1的磷酸化，并使用腺病毒介导的野生型和突变形式的NHERF-1的基因转移，并确定在PDZ域中的哪些残基，在第一个PDZ域中的哪些残基，是NPT2A的结合位点，是磷酸化的。特定的兴趣将集中在NHERF-1的NPT2A结合凹槽中的丝氨酸77上。然后，我们将确定 多巴胺D1样受体使用的信号传导途径通过测量cAMP的积累和野生型和NHERF-1无效细胞中的PKC活性。为了确定NHERF-1的生理作用，我们将测量多巴胺对野生型和NHERF-1无效近端小管细胞中钠依赖性磷酸转运的影响。研究还将在感染腺病毒NHERF-1感染的NHERF-1无效细胞中进行，以确定NHERF-1在介导多巴胺和NHERF-1突变形式的效果中的特定作用，以确定丝氨酸等特定残基的作用，例如丝网膜。为了检查抑制的机制，我们将使用定量免疫沉淀，共聚焦显微镜和蔗糖密度梯度梯度超细胞分解来确定多巴胺是否解散了NPT2A/NHERF-1复合物。因此，我们的直接目的是检验NHERF-1与多巴胺受体相互作用的假设，而多巴胺通过磷酸化NPT2A结合蛋白来抑制肾磷酸转运 NHERF-1因此分离NHERF-1/NPT2A复合物并减少顶端 NPT2A的膜丰度。我们预计荷尔蒙转导的比较 途径将为NHERF-1在磷酸转运中的生物学作用提供新的见解，并进一步理解NHERF-1在其他生理和病理生理过程中的作用，例如肾上腺素病，精神分裂症，牛皮癣，牛皮癣，积极的雌激素受体阳性乳腺癌和Nerve乳腺癌，聋哑人（User Hersy's androme）。 。