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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。

基本信息

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
项目类别：
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

项目摘要

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). .

肾小管对磷酸盐的重吸收在很大程度上是由钠离子介导的