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由钠介导的 称为 Npt2a 的依赖性磷酸盐转运蛋白位于近曲小管细胞的顶膜中。 Npt2a 通过我们实验室分离和克隆的 PDZ 结构域接头蛋白（称为钠氢交换调节因子 1 (NHERF-1)）与顶膜相连。 NHERF-1缺失小鼠表现出与肾乳头钙沉积相关的磷酸盐、钙和尿酸排泄增加。经小鼠证实，人类 NHERF-1 突变与肾结石有关。我们实验室最近的研究表明，甲状旁腺激素导致 NHERF-1 的第一个 PDZ 结构域磷酸化，随后 Npt2a/NHERF-1 复合物解离，Npt2a 顶膜丰度减少，磷酸盐减少运输。我们推测这种独特的机制可能是调节 NHERF-1 与其其他一些靶蛋白结合的一般过程。为了研究 NHERF-1 在肾磷酸盐转运中的作用并确定 NHERF-1 的磷酸化是否受到其他激素的调节，我们建议研究多巴胺抑制肾磷酸盐转运的机制。使用野生型和 NHERF-1 缺失动物的原代培养物中新鲜制备的肾切片和肾近端小管细胞，我们将首先使用免疫沉淀和共聚焦显微镜确定 NHERF-1 是否与多巴胺 D1 样受体结合。使用 32P 标记的细胞，我们将确定多巴胺 D1 样受体的占据是否会导致 NHERF-1 的磷酸化，并使用腺病毒介导的野生型和突变型 NHERF-1 的基因转移，确定 NHERF-1 中的哪些残基第一个 PDZ 结构域（Npt2a 的结合位点）被磷酸化。特别感兴趣的是位于 NHERF-1 的 Npt2a 结合沟中的丝氨酸 77。然后我们将确定 通过测量野生型和 NHERF-1 缺失细胞中的 cAMP 积累和 PKC 活性，研究多巴胺 D1 样受体使用的信号通路。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 缺失近端小管细胞中钠依赖性磷酸盐转运的影响。还将在感染腺病毒-NHERF-1的NHERF-1无效细胞中进行研究，以确定NHERF-1在介导多巴胺作用中的具体作用，并在NHERF-1的突变形式中进行研究，以确定特定残基的作用，例如丝氨酸77。为了检查抑制机制，我们将使用定量免疫沉淀、共聚焦显微镜和蔗糖密度梯度超速离心来确定多巴胺是否解离 Npt2a/NHERF-1 复合物。因此，我们的直接目标是检验 NHERF-1 与多巴胺受体相互作用以及多巴胺通过磷酸化 Npt2a 结合蛋白来抑制肾磷酸盐转运的假设 NHERF-1 从而解离 NHERF-1/Npt2a 复合物并减少顶端 Npt2a 的膜丰度。我们预计激素转导的比较 这些途径将为 NHERF-1 对磷酸盐转运的生物学作用提供新的见解，并进一步了解 NHERF-1 在其他生理和病理生理过程中的作用，如肾结石、精神分裂症、牛皮癣、侵袭性雌激素受体阳性乳腺癌、和神经性耳聋（亚瑟综合症）。 。