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：
7798943
负责人：
Dennis Brown
金额：
$ 180.73万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-04-01 至 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7798943
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 Ureteral obstruction Urine Utah Variant Vas deferens structure Vasopressin Receptor Vasopressins Vesicle Viagra Work aquaporin-2 arm 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.

总体描述（由申请人提供）：对水通道蛋白 2 (AQP2) 和加压素受体 (V2R) 运输的范式转变观察，以及液泡 ATP 酶 (V-ATP 酶) pH 传感和回收导致了本次更新中需要解决的新假设。项目 I 将定义 AQP2 的 VP 依赖性和独立调节，并将鉴定与 AQP2 相互作用通过磷酸化进行修饰以调节运输的蛋白质。我们将探讨 AQP2 通过与 RhoGAPS 相互作用调节肌动蛋白聚合的新作用。使用 PDE5 抑制剂和他汀类药物来实现与 VP 无关的尿液浓度将作为未来 NDI 治疗的潜在策略进行测试。项目 II 将使用 FRET 技术检查配体诱导的 V2R 在不同 pH 和张力下的构象变化，以剖析分子内和分子间蛋白质相互作用。内化过程中 V2R 与辅助蛋白的关联将在表达野生型和突变型 V2R 的细胞中定义。我们的新观察结果是，V2R 与 ESCRT 蛋白 Alix 相互作用，加速 V2R 降解，这些研究将致力于解决体液稳态的调节问题。项目 III 将通过定义 V-ATP 酶尾部的构象变化来追求 V-ATP 酶是内体 pH 传感器的突破性发现，从而导致小 GTP 酶依赖于 pH 值招募到膜上。它将识别 V-ATP 酶腔域上的 pH 敏感残基，并利用白蛋白摄取来显示 pH 传感机制与近端小管功能的相关性。项目 IV 将阐明可溶性腺苷酸环化酶的下游效应器（PKA、Epac）在调节附睾中 V-ATP 酶回收和质子分泌方面的作用，附睾是一种“模型”上皮，其中管腔酸性 pH 值对于精子成熟和储存至关重要。它将检查 cGMP 诱导的质子分泌在该组织中的作用，并将解决一个令人兴奋的假设，即 V-ATP 酶也是一种细胞外 pH 传感器，提供管腔 pH 的反馈控制。这些研究将有助于更好地了解男性生育能力，并揭示一般酸化细胞（包括肾闰细胞）中调节 V-ATP 酶的机制。显微镜核心 B 设施是本次 PPG 成功的主要贡献者。所有项目都从广泛的知识和技术合作中获得了相当大的附加值，这些合作体现了我们努力了解细胞信号传导、蛋白质运输和泌尿生殖上皮细胞对其环境的反应之间的关系。