Cell biology of vasopressin-induced water channels

项目摘要/摘要 我们认为，有必要结合知情、有针对性和不偏不倚的药物筛查来制定战略，以 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 在此续期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，我们已经 揭示了水通道蛋白2生物学中几个以前未被认识的重要方面：1)水通道蛋白2催化肌动蛋白 对AVP反应的解聚：2)AQP2转运到根尖膜涉及一过性基底侧插入 和跨细胞重定向：3)AQP2在完全没有任何已知的磷酸化的情况下进行结构性回收 事件。目的1利用这一新知识并询问AQP2磷酸化之间的关系， 肌动蛋白的组织，以及AQP2膜在肾上皮细胞中传递和积聚的极性。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，我们使用高通量的化学品 在前一个资助周期的筛查中，我们发现FDA批准了抗癌药物Erlotinib，一种EGFR 抑制剂，使锂治疗NDI小鼠的尿量减少50%。目标2将探索EGFR 在完全没有VP的情况下，单独抑制会导致AQP2磷酸化和膜积聚。 缺乏PKA刺激。我们需要确定哪条信号通路负责，以便充分了解 厄洛替尼在这种情况下起作用，并提出替代的有针对性的方法。最后，我们对更多新事物的追求 在目标3中，将继续使用调节AQP2膜积累的化合物，其中将进行荧光分析 用于对内吞作用的抑制剂或刺激剂的化学库进行无偏筛选。而内吞作用 抑制剂是NDI的候选药物，AQP2内吞作用的特异性刺激剂在水条件下可能有用 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物化合物 在我们之前的筛查中发现了它们阻止AQP2膜堆积的能力，这也是药物的一个特征 这将防止水超载。我们先前的研究和在这次续期申请中建议的工作范围从 蛋白质相互作用的体外表征，通过细胞培养试验，到整个动物研究。我们已经开发出 用于高通量化学筛选的新细胞系，用于活细胞成像的新的AQP2-EGFP构建，以及我们有 本中心新建立的条件性加压素受体基因敲除小鼠群体，用于体内药物试验。我们的 工作结合了对基本机制的更好理解的需要，以推动转化医学和 临床进展，使用新的细胞分析的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.