Cell Biology of Vasopressin-induced Water Channels-Research Supplement

由于缺乏安全、有效和特异性的水平衡失调治疗方法， 我们的工作是确定新的细胞内途径，通过这些途径， 肾脏主细胞中的水通道（AQP 2）可以在缺乏正常功能的情况下正常化。 加压素受体（V2 R）信号通路。这项提案的总体目标是重新剖析- 确定水通道蛋白2（AQP 2）的贩运和调控途径，以提供可操作的，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）是由于肾脏对VP不敏感而导致的尿过多 生产，而水潴留，往往是由于不适当的VP分泌，发生在条件， 充血性心力衰竭目的1解决了AQP 2本身指导其细胞内运输的假设 行程作为“主动”货物蛋白，而不是被动的旁观者。我们认为AQP 2“催化” 隔室特异性肌动蛋白重塑通过直接和间接的相互作用，与不同的队列肌动蛋白- 调节蛋白这些包括肌动蛋白本身，胞吐过程中的Arp 2/3肌动蛋白重塑复合物，以及 肌动蛋白结合蛋白ezrin在胞吞作用。目标2将确定蜂窝串扰机制， 加压素受体（VP/V2 R-正作用）和表皮生长因子受体之间的拉锯战 (EGF/EGFR -负作用）调节AQP 2运输和水平衡。首先，我们来看看EGF是如何发挥作用的。 其它EGFR配体通过受体活化和下游信号传导抑制VP的抗利尿作用。然后 我们将探讨EGFR通路的抑制是如何导致VP非依赖性AQP 2磷酸化和膜 通过激活非典型激酶P90核糖体S6激酶（RSK）来抑制细胞的积累。技术中心 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，酶活性测定，培养的上皮细胞中修饰蛋白的表达 细胞和整个动物生理学。了解AQP 2调节的新细胞机制并定义 细胞内信号传导中更具体的参与者将为研究调节打开未探索的途径 体液和电解质的平衡我们希望我们的数据将允许开发更具选择性和 细胞特异性药理学策略调节水平衡紊乱中的AQP 2运输。

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.