CELLULAR BIOLOGY OF RENAL FUNCTION AND DISEASE

肾功能和疾病的细胞生物学

基本信息

批准号：
7907365
负责人：
Dennis Brown
金额：
$ 19.2万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
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：
2009-09-30 至 2010-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7907365
关键词：
AVPR2 gene Acids Actins Address Adenylate Cyclase Adrenergic Receptor Affect Albumins Animals Antibodies Apical Apoptosis Arachidonic Acids Area Award Bicarbonates Binding Biochemical Biogenesis Biological Biological Assay Biological Models Body Fluids Calcitonin Capsid Proteins Carrier Proteins Cell Cycle Cell Fractionation Cell Line Cell Proliferation Cell membrane Cell physiology Cell surface Cells Cellular biology Chloride Channels Clathrin Clear Cell Clinical Trials Collaborations Collagen Complementary DNA Complex Confocal Microscopy Cues Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cyclic GMP Cytoplasmic Tail Cytoskeleton Cytosolic Phospholipase A2 Data Degradation Pathway Development Dinoprostone Disease Down-Regulation Duct (organ) structure Educational process of instructing Endocytosis Endosomes Environment Enzyme-Linked Immunosorbent Assay Enzymes Epididymis Epithelial Epithelial Cells Epithelium Event Faculty Feedback Fertility Fertilization Fibrosis Fluorescence Resonance Energy Transfer Funding Mechanisms Future G Protein-Coupled Receptor Genes Gelsolin Gene Proteins Generations Genitourinary system Glomerular Mesangial Cell Goals Growth Growth Factor Guanine Nucleotide Exchange Factors Guanosine Triphosphate Phosphohydrolases Head Heart Heart Diseases Heart Hypertrophy Homeostasis Hydrogen Peroxide Image In Situ In Vitro Individual Inflammation Inflammation Mediators Inflammatory Inflammatory Response Intercalated Cell Intracellular Signaling Proteins Kidney Kidney Diseases Knockout Mice Laboratories Lasers Lead Length Life Ligands Location Lung Lysosomes Male Genital Organs Mass Spectrum Analysis Mediating Membrane Membrane Proteins Messenger RNA Microdissection Microscope Microscopy Modeling Molecular Monomeric GTP-Binding Proteins Multivesicular Body Mus National Institute of Child Health and Human Development Nature Netherlands Organ Paper Pathway interactions Phase Phospholipase A2 Phosphorylation Physiological Play Predisposition Procedures Process Production Protein Isoforms Proteins Proteomics Protons Publications Publishing Reagent Receptor Signaling Recruitment Activity Recycling Regulation Regulatory Pathway Renal function Research Role Route Sampling Screening procedure Serum Signal Pathway Signal Transduction Signaling Protein Site Sperm Maturation Staging Steroids Stimulus Stress Striated Muscles System Tail Techniques Technology Testing Time Tissues Total Internal Reflection Fluorescent Touch sensation Transgenic Mice Translating United States National Institutes of Health Upper arm Ureteral obstruction Urine Utah Variant Vas deferens structure Vasopressin Receptor Vasopressins Vesicle Viagra Work aquaporin-2 base body system cell type cytokine data sharing enzyme activity extracellular follow-up genetic regulatory protein hormone regulation human HTATIP protein human RIPK1 protein in vivo inhibitor/antagonist insight kidney cell kidney epithelial cell laser capture microdissection lectures lung tumorigenesis mRNA Expression male mesangial cell mouse model movie mutant new therapeutic target news novel overexpression oxidant stress phospholipase A2-II polymerization programs promoter protein degradation protein expression protein protein interaction protein transport receptor reproductive reproductive function response sensor success therapeutic target trafficking trans-Golgi Network uptake vacuolar H+-ATPase water channel yeast two hybrid system

项目摘要

DESCRIPTION, OVERALL (provided by applicant): Paradigm-shifting observations on aquaporin 2 (AQP2) and vasopressin receptor (V2R) trafficking, and vacuolar ATPase (V-ATPase) pH sensing and recycling have lead to new hypotheses to be addressed in this renewal. Project I will define VP-dependent and independent regulation of AQP2, and will identify proteins whose interaction with AQP2 is modified by phosphorylation to regulate trafficking. A novel role for AQP2 in regulating actin polymerization via interaction with RhoGAPS will be explored. Use of PDE5 inhibitors and statins to achieve VP-independent urine concentration will be tested as a potential strategy for future treatment of NDI. Project II will examine ligand induced conformational changes of the V2R at varying pH and tonicity using FRET techniques to dissect intra- and intermolecular protein interactions. Association of V2R with accessory proteins during internalization will be defined in cells expressing wild type and mutant V2R. Our novel observation that V2R interacts with the ESCRT protein Alix to accelerate V2R degradation will be pursued in these studies that address the regulation of body fluid homeostasis. Project III will pursue the breakthrough finding that the V-ATPase is an endosomal pH sensor by defining conformational changes in the V-ATPase tail that result in pH-dependent recruitment of small GTPases to membranes. It will identify pH sensitive residues on the luminal domains of the V-ATPase, and use albumin uptake to show relevance of the pH sensing mechanism to proximal tubule function. Project IV will elucidate downstream effectors (PKA, Epac) of soluble adenylate cyclase in modulating V-ATPase recycling and proton secretion in the epididymis, a "model" epithelium in which luminal acidic pH is critical for sperm maturation and storage. It will examine the role of cGMP-induced proton secretion in this tissue, and will address the exciting hypothesis that the V-ATPase is also an extracellular pH sensor that provides feedback control of luminal pH. These studies will allow a better understanding of male fertility, and uncover mechanisms that regulate V-ATPase in acidifying cells in general, including renal intercalated cells. The Microscopy Core B facility has been a major contributor to the success of this PPG. All projects gain considerable added value from extensive intellectual and technical collaborations that characterize our efforts to understand the relationship between cellular signaling, protein trafficking, and the responses of urogenital epithelial cells to their environment.

描述，总体（由申请人提供）：关于水通道蛋白2（AQP2）和加压素受体（V2R）运输的范式转移观察，以及液泡ATPase（V-ATPase）pH感应和回收利用已导致在此续签中解决新的假设。项目我将定义AQP2的VP依赖性和独立的调节，并将确定其与AQP2相互作用的蛋白质通过磷酸化来改变以调节运输的蛋白质。将探讨AQP2在通过与Rhogaps相互作用来调节肌动蛋白聚合中的新作用。使用PDE5抑制剂和他汀类药物来实现与VP无关的尿液浓度，将被检验，作为NDI将来治疗的潜在策略。项目II将使用FRET技术在不同的pH和张力下检查配体诱导V2R的构象变化，以剖析分子内和分子间蛋白质相互作用。在表达野生型和突变体V2R的细胞中，将定义V2R与辅助蛋白的缔合。我们对V2R与ESCRT蛋白ALIX相互作用以加速V2R降解的新颖观察结果将在这些研究中涉及调节体液稳态的调节。 III项目将通过定义V-ATPase尾巴中的构象变化来追求突破性的发现，即V-ATPase是一种内体pH传感器，从而导致pH依赖性GTPase募集到膜上。它将在V-ATPase的腔内域上识别pH敏感残基，并使用白蛋白摄取来显示pH传感机理与近端小管功能的相关性。 IV项目将在调节子助剂中调节V-ATPase回收和质子分泌方面阐明可溶性腺苷酸环化酶的下游效应子（PKA，EPAC），其中腔室酸性pH值对精子成熟和存储至关重要。它将检查CGMP诱导的质子分泌在该组织中的作用，并将解决令人兴奋的假设：V-ATPase也是一个细胞外pH传感器，可提供对腔内pH的反馈控制。这些研究将使人们对男性生育能力有更好的了解，并发现通常在酸化细胞中调节V-ATPase的机制，包括肾脏插入的细胞。显微镜核心B设施一直是该PPG成功的主要贡献者。所有项目都从广泛的智力和技术合作中获得了可观的附加值，这些合作是我们为了解细胞信号传导，蛋白质运输以及泌尿生殖器上皮细胞对环境的反应之间关系的努力。