Cell Biology of Vasopressin-induced Water Channels

加压素诱导的水通道的细胞生物学

• 批准号: 10652774
• 负责人: Dennis Brown
• 金额: $ 52.7万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-20 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10652774
• 关键词: Actin-Binding Protein; Actins; Address; Affect; Animals; Biological Assay; Biological Products; Cell membrane; Cell physiology; Cell surface; Cells; Cellular biology; Clinical; Clinical Treatment; Clinical Trials; Complex; Congestive Heart Failure; Cytoskeleton; Data; Defect; Dehydration; Development; Disease; Down-Regulation; Duct (organ) structure; EGF gene; Electrolytes; Endocytosis; Epidermal Growth Factor Receptor; Epithelial Cells; Equilibrium; Erlotinib; Event; Exocytosis; F-Actin; Failure; Follow-Up Studies; Funding; Goals; Growth Factor Receptors; Health; Homeostasis; Hypertension; Hyponatremia; Inherited; Ion Channel; Kidney; Kidney Diseases; Knowledge; Ligands; Link; Liquid substance; Membrane; Modification; Molecular; Participant; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphorylation; Physiology; Play; Process; Production; Proteins; Public Health; RPS6KA gene; Receptor Activation; Receptor Cross-Talk; Receptor Signaling; Recycling; Regulation; Regulatory Pathway; Renal function; Research; Retrieval; Ribosomal Protein S6 Kinase; Role; Signal Pathway; Signal Transduction; Specificity; Techniques; Translating; Urine; Vasopressin Receptor; Vasopressins; War; Water; Work; absorption; antidiuretic; aquaporin-2; arm; cohort; depolymerization; design; enzyme activity; equilibration disorder; ezrin; genetic regulatory protein; inhibitor; kidney cell; live cell imaging; novel; novel therapeutic intervention; novel therapeutics; pharmacologic; protein transport; receptor; superresolution microscopy; therapeutic target; trafficking; vasopressin resistant diabetes insipidus; water channel; water treatment

Because of a lack of safe, effective and specific treatments for disorders of water balance, the ultimate goal of our work is to identify novel intracellular pathways by which plasma membrane accumulation of the aquaporin 2 water channel (AQP2) in kidney principal cells can be normalized in the absence of a properly functioning vasopressin receptor (V2R) signaling pathway. The overall objective of this proposal is to dissect newly- identified aquaporin 2 (AQP2) trafficking and regulatory pathways in order to provide actionable, basic information that can be translated into cell-specific clinical advances for the treatment of these conditions. Nephrogenic diabetes insipidus (NDI) is caused by renal insensitivity to VP, and results in excessive urine production, whereas water retention, often a result of inappropriate VP secretion, occurs in conditions such as congestive heart failure. Aim 1 addresses the hypothesis that AQP2 itself directs its intracellular trafficking itinerary as an “active” cargo protein rather than a passive bystander. We propose that AQP2 “catalyzes” compartment-specific actin remodeling via direct and indirect interactions with different cohorts of actin- regulatory proteins. These include actin itself, the Arp2/3 actin remodeling complex during exocytosis, and the actin binding protein ezrin during endocytosis. Aim 2 will identify the cellular crosstalk mechanism(s) by which a tug-of-war between the vasopressin receptor (VP/V2R - positive action) and epidermal growth factor receptor (EGF/EGFR - negative effect) regulate AQP2 trafficking and water balance. We will first explore how EGF and other EGFR ligands inhibit the antidiuretic effect of VP by receptor activation and downstream signaling. Then we will ask how inhibition of the EGFR pathway results in VP-independent AQP2 phosphorylation and membrane accumulation by activating a non-canonical kinase, P90 ribosomal S6 kinase (RSK). Techniques central to the proposed work include the molecular characterization of protein interactions, advanced super resolution microscopy and live cell imaging, enzyme activity assays, expression of modified proteins in cultured epithelial cells, and whole animal physiology. Understanding novel cellular mechanisms of AQP2 regulation and defining more specific participants in intracellular signaling will open unexplored avenues of research into the regulation of fluid and electrolyte homeostasis. We expect that our data will allow the development of more selective and cell specific pharmacological strategies to regulate AQP2 trafficking in water balance disorders.

由于缺乏安全、有效和特异的水平衡紊乱治疗方法，最终目标 我们的工作是确定水通道蛋白 2 质膜积累的新细胞内途径 肾主细胞中的水通道（AQP2）可以在缺乏正常功能的情况下正常化 加压素受体 (V2R) 信号通路。该提案的总体目标是剖析新的—— 确定了水通道蛋白 2 (AQP2) 贩运和监管途径，以便提供可操作的、基本的 这些信息可以转化为治疗这些疾病的细胞特异性临床进展。 肾性尿崩症 (NDI) 是由肾脏对 VP 不敏感引起的，并导致尿液过多 生产，而水潴留通常是由于 VP 分泌不当造成的，发生在以下条件下： 充血性心力衰竭。目标 1 提出了 AQP2 本身指导其细胞内运输的假设 作为“主动”货物蛋白而不是被动旁观者的行程。我们建议 AQP2“催化” 通过与不同肌动蛋白群体的直接和间接相互作用来进行室特异性肌动蛋白重塑 调节蛋白。这些包括肌动蛋白本身、胞吐作用期间的 Arp2/3 肌动蛋白重塑复合物以及 胞吞过程中肌动蛋白结合蛋白埃兹蛋白。目标 2 将确定细胞串扰机制，通过该机制 加压素受体（VP/V2R - 积极作用）和表皮生长因子受体之间的拉锯战 （EGF/EGFR - 负面影响）调节 AQP2 运输和水平衡。我们将首先探讨 EGF 和 其他 EGFR 配体通过受体激活和下游信号传导抑制 VP 的抗利尿作用。然后 我们会问抑制 EGFR 通路如何导致不依赖于 VP 的 AQP2 磷酸化和膜 通过激活非经典激酶 P90 核糖体 S6 激酶 (RSK) 来积累。技术的核心 拟议的工作包括蛋白质相互作用的分子表征、先进的超分辨率 显微镜和活细胞成像、酶活性测定、培养上皮细胞中修饰蛋白的表达 细胞和整个动物生理学。了解 AQP2 调节的新细胞机制并定义 细胞内信号传导的更具体参与者将为调节研究开辟未探索的途径 液体和电解质稳态。我们期望我们的数据能够开发出更具选择性和 调节水平衡紊乱中 AQP2 运输的细胞特异性药理学策略。