Cell biology of vasopressin-induced water channels

项目总结/摘要 我们认为，有必要结合知情、有针对性和无偏见的药物筛选来制定战略， 使水平衡紊乱正常化，包括肾源性尿崩症和低钠血症。总体战略 本更新申请中提出的建议旨在促进这一目标。在过去三年的资助中，我们 揭示了水通道蛋白2生物学的几个重要和以前未被认识的方面：1）AQP 2催化肌动蛋白 AVP引起的解聚：2）AQP 2向顶膜的运输涉及短暂的基底外侧插入 3）AQP 2在完全不存在任何已知磷酸化的情况下组成性地聚集 事件目的1利用这一新的知识，并询问AQP 2磷酸化， 肌动蛋白组织以及肾上皮细胞中AQP 2膜递送和积聚的极性。这种方法 使我们能够设想更明智的方法来解决水平衡失调问题。接下来，我们使用高通量化学 在上一个资助周期的筛选中，我们发现FDA批准的抗癌药物厄洛替尼（EGFR） 在锂处理的NDI小鼠中，抑制剂使尿量减少50%。目的2：探讨EGFR在肿瘤细胞中的作用机制。 在完全不存在VP的情况下，单独的抑制导致AQP 2磷酸化和细胞膜积聚。 没有PKA刺激。我们需要确定哪个信号通路负责，以充分了解如何 厄洛替尼在这种情况下工作，并提出替代的靶向方法。最后，我们寻求更多新的 调节AQP 2膜积累的化合物将在目标3中继续，其中荧光测定将在 用于无偏筛选内吞作用的抑制剂或刺激剂的化学文库。虽然内吞作用 抑制剂是用于NDI的候选物，AQP 2内吞作用的特异性刺激剂可以在水的条件下使用 超负荷可能导致低钠血症甚至高血压。我们还将测试胞吐抑制剂化合物， 在我们之前的筛选中鉴定了它们阻止AQP 2膜积聚的能力，这也是药物的一个特征， 可以防止水超载。我们先前的研究和在这次更新申请中提出的工作范围从 从体外蛋白质相互作用的表征，通过细胞培养试验，到整个动物研究。我们已经开发 用于高通量化学筛选的新细胞系，用于活细胞成像的新AQP 2-EGFP构建体，我们有一个用于高通量化学筛选的新细胞系。 新建立的条件性血管加压素受体敲除小鼠群体在我们的设施，在体内药物测试。我们 工作结合了更好地理解基本机制的需要，以推动转化医学， 临床进展，使用新的细胞测定更直接的药物发现方法。

PROJECT SUMMARY/ABSTRACT We believe that a combination of informed, targeted and unbiased drug screening is necessary to devise strategies to normalize water balance disorders, including nephrogenic diabetes insipidus and hyponatremia. The overall strategy proposed in this renewal application is designed to facilitate this goal. Over the past three years of funding, we have uncovered several important and previously unrecognized aspects of aquaporin 2 biology: 1) AQP2 catalyzes actin depolymerization in response to AVP: 2) AQP2 trafficking to the apical membrane involves transient basolateral insertion and redirection via transcytosis: 3) AQP2 recycles constitutively in the complete absence of any known phosphorylation events. Aim 1 takes advantage of this new knowledge and interrogates the relationship between AQP2 phosphorylation, actin organization, and the polarity of AQP2 membrane delivery and accumulation in renal epithelial cells. This approach allows us to envisage more informed approaches to water balance disorders. Next, our use of high throughput chemical screening in the previous funding cycle led us to discover that the FDA approved cancer drug Erlotinib, an EGFR inhibitor, reduces urine output by 50% in lithium treated NDI mice. Aim 2 will explore the mechanism by which EGFR inhibition alone, in the complete absence of VP, causes AQP2 phosphorylation and membrane accumulation in the absence of PKA stimulation. We need to identify which signaling pathway is responsible in order to fully understand how Erlotinib works in this setting, and to suggest alternative targeted approaches. Finally, our quest for additional new compounds that modulate AQP2 membrane accumulation will continue in Aim 3, in which a fluorescence assay will be used for unbiased screening of chemical libraries for inhibitors or stimulators of endocytosis. While endocytosis inhibitors are candidates for use in NDI, specific stimulators of AQP2 endocytosis could be useful in conditions of water overload that could lead to hyponatremia and even hypertension. We will also test exocytosis-inhibitor compounds that were identified in our previous screen for their ability to prevent AQP2 membrane accumulation, also a feature of drugs that would prevent water overloading. Our prior studies and the work proposed in this renewal application range from the in vitro characterization of protein interactions, through cell culture assays, to whole animal studies. We have developed new cell lines for high throughput chemical screens, a new AQP2-EGFP construct for live cell imaging, and we have a newly-established colony of conditional vasopressin receptor knockout mice in our facility for in vivo drug testing. Our work combines the need for a better understanding of basic mechanisms in order to drive translational medicine and clinical advances, with a more direct drug discovery approach using novel cell assays.