Cell Biology of Vasopressin-induced Water Channels

由于缺乏安全、有效和特效的水平衡失调治疗方法， 我们的工作是确定细胞膜上水通道蛋白2积累的新的细胞内途径 肾主细胞的水通道(AQP2)在缺乏正常功能的情况下可以恢复正常 加压素受体(V2R)信号通路。这项建议的总体目标是对新的- 确定水通道蛋白2(AQP2)的运输和调节途径，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。 肾源性尿崩症(NDI)是由肾脏对VP不敏感引起的，并导致尿量过多 产生，而水分保留，通常是不适当的VP分泌的结果，在以下情况下发生 充血性心力衰竭目标1解决了AQP2本身指导其细胞内转运的假设 行程是一个“主动”的货物蛋白质，而不是一个被动的旁观者。我们认为AQP2“催化” 通过与不同的肌动蛋白队列的直接和间接相互作用，肌动蛋白特异性的重塑。 调节蛋白。这些包括肌动蛋白本身，胞吐过程中的Arp2/3肌动蛋白重塑复合体，以及 肌动蛋白结合蛋白Ezrin在内吞作用中。目标2将确定蜂窝串扰机制(S)，通过该机制 加压素受体(VP/V2R阳性作用)与表皮生长因子受体之间的拉锯战 (EGF/EGFR负效应)调节AQP2的运输和水平衡。我们将首先探讨EGF和 其他EGFR配体通过激活受体和下游信号通路抑制VP的抗利尿作用。然后 我们将询问抑制EGFR通路如何导致非VP依赖的AQP2磷酸化和膜 通过激活非规范的P90核糖体S6激酶(RSK)而积聚。技术的核心 建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、酶活性分析、培养上皮中修饰蛋白的表达 细胞和整个动物生理学。理解AQP2调节的新细胞机制和定义 细胞内信号转导的更多特定参与者将开辟研究该调控的未知途径 体液和电解质的动态平衡。我们希望我们的数据将允许开发更有选择性和 调节水平衡失调中AQP2转运的细胞特异性药理学策略。

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.