Cell biology of vasopressin-induced water channels

加压素诱导的水通道的细胞生物学

基本信息

批准号：
9176186
负责人：
Dennis Brown
金额：
$ 52.33万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-20 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9176186
关键词：
AVPR2 gene Actins Animals Antineoplastic Agents Apical Area Back Biological Assay Biology Brattleboro Rats Bypass Cell Culture Techniques Cell Line Cell membrane Cell model Cell physiology Cells Cellular biology Chemicals Clinical Complex Cyclic AMP-Dependent Protein Kinases Data Dehydration Disease Duct (organ) structure EGF gene Endocytosis Endosomes Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Epithelial Cells Equilibrium Erlotinib Event Exocytosis FDA approved Fluorescence Funding Goals Human Hypertension Hyponatremia In Situ In Vitro Kidney Kidney Concentrating Ability Knockout Mice Knowledge Lead Liquid substance Lithium Maps Membrane Modeling Mus Mutate Mutation Output Pathway interactions Pattern Pharmaceutical Preparations Phosphorylation Phosphotransferases Preclinical Drug Evaluation Process Production Protein phosphatase Proteins Receptor Inhibition Receptor Signaling Recycling Role Signal Pathway Signal Transduction Site Small Interfering RNA Sodium Temperature Testing Tissues Urine Vasopressin Receptor Vasopressins Water Water consumption Work apical membrane aquaporin-2 base depolymerization design drug discovery drug testing equilibration disorder high throughput screening in vivo inhibitor/antagonist kidney cell live cell imaging mutant novel novel therapeutics polymerization prevent protein expression response salt intake screening sildenafil small molecule libraries trafficking transcytosis translational medicine tyrphostin AG-490 vasopressin resistant diabetes insipidus water channel

项目摘要

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.

项目概要/摘要我们认为，有必要结合知情、有针对性和公正的药物筛选来制定策略使水平衡紊乱正常化，包括肾性尿崩症和低钠血症。总体策略本续签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 生物学的几个重要且之前未被认识的方面：1) AQP2 催化肌动蛋白 AVP 引起的解聚：2) AQP2 运输至顶膜涉及瞬时基底外侧插入和通过转胞吞作用重定向：3) AQP2 在完全不存在任何已知磷酸化的情况下组成型循环事件。目标 1 利用这一新知识并探究 AQP2 磷酸化、肌动蛋白组织，以及肾上皮细胞中 AQP2 膜递送和积累的极性。这种做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品在上一个资助周期的筛选中，我们发现FDA批准了抗癌药物厄洛替尼（Erlotinib），一种EGFR 抑制剂，使锂处理的 NDI 小鼠的尿量减少 50%。目标2将探索EGFR的作用机制在完全没有 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。我们需要确定哪个信号通路负责，以便充分理解如何厄洛替尼在这种情况下发挥作用，并提出替代的靶向方法。最后，我们寻求更多新的调节 AQP2 膜积累的化合物将继续在目标 3 中进行，其中将进行荧光测定用于无偏见地筛选化学文库中的内吞作用抑制剂或刺激剂。内吞作用的同时抑制剂是用于 NDI 的候选者，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。我们还将测试胞吐作用抑制剂化合物在我们之前的筛选中鉴定出它们具有防止 AQP2 膜积聚的能力，这也是药物的一个特征这将防止水超载。我们之前的研究和本次更新申请中提出的工作范围包括通过细胞培养测定到整体动物研究，对蛋白质相互作用进行体外表征。我们开发了用于高通量化学筛选的新细胞系，用于活细胞成像的新 AQP2-EGFP 构建体，我们有我们的设施中新建立了条件性加压素受体基因敲除小鼠群体，用于体内药物测试。我们的工作结合了更好地理解基本机制的需要，以推动转化医学和临床进展，使用新颖的细胞测定法提供更直接的药物发现方法。