CELLULAR BIOLOGY OF RENAL FUNCTION AND DISEASE

总体描述(由申请人提供)： 水通道蛋白2(AQP2)和加压素受体(V2R)转运的范式转换观察，以及 液泡ATPase(V-ATPase)的pH感知和再循环导致了新的假说需要在这次更新中解决。项目I将定义AQP2依赖于VP的和独立的调节，并将确定其与AQP2的相互作用被磷酸化修饰以调节运输的蛋白质。我们将探索AQP2通过与RhoGAPS相互作用来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VP非依赖性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技术来研究配体在不同pH和张力下诱导的V2R的构象变化，以剖析分子内和分子间的蛋白质相互作用。在表达野生型和突变型V2R的细胞中，V2R与内化过程中的辅助蛋白的联系将被定义。我们新的观察到V2R与ESCRT蛋白Alix相互作用以加速V2R的降解，这些研究将致力于体液稳态的调节。项目III将通过定义V-ATPase尾部的构象变化来追求这一突破性发现，即V-ATPase是一种内体pH传感器，这种构象变化导致小的GTP酶依赖于pH重新募集到膜上。它将识别V-ATPase管腔结构域上的pH敏感残基，并使用白蛋白摄取来显示pH敏感机制与近端小管功能的相关性。项目IV将阐明可溶腺苷环化酶下游效应因子(PKA、EPAC)在调节附睾中V-ATPase循环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对精子成熟和储存至关重要。它将研究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，即V-ATPase也是一个细胞外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的生育能力，并揭示调节酸化细胞(包括肾脏嵌入细胞)中V-ATPase的机制。显微镜核心B设施是这一PPG成功的主要贡献。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，这些合作是我们努力了解细胞信号、蛋白质运输和泌尿生殖系统上皮细胞对其环境的反应之间的关系的特征。

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.