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.

项目摘要/摘要我们认为，必须将知情，有针对性和公正的药物筛查组合起来，以制定策略来制定策略将水平平衡疾病归一化，包括肾脏基糖尿病和低钠血症。总体策略在此更新应用中提出的旨在促进这一目标。在过去三年的资金中，我们有发现了水通道蛋白2生物学的几个重要和以前未被认可的方面：1）AQP2催化肌动蛋白响应AVP的解聚化：2）AQP2运输到根尖膜涉及瞬时基底外侧插入和通过转介毒作用的重定向：3）AQP2在完全没有任何已知的磷酸化的情况下组成型回收事件。 AIM 1利用这一新知识，询问AQP2磷酸化之间的关系，肌动蛋白组织，以及肾上皮细胞中AQP2膜递送和积累的极性。这种方法允许我们设想更明智的水平疾病方法。接下来，我们使用高通量化学物质在先前的资金周期中进行筛查使我们发现FDA批准了EGFR的癌症药物Erlotinib 抑制剂，在锂处理的NDI小鼠中，尿液输出量减少了50％。 AIM 2将探索EGFR的机制仅在完全没有VP的情况下，仅抑制就会导致AQP2磷酸化和膜积累缺乏PKA刺激。我们需要确定哪种信号通路负责，以便完全了解 Erlotinib在这种情况下起作用，并建议采用替代目标方法。最后，我们寻求其他新的调节AQP2膜积累的化合物将在AIM 3中继续进行，其中荧光测定将是用于对内吞作用的抑制剂或刺激剂的化学文库无偏筛选。而内吞作用抑制剂是用于NDI的候选者，AQP2内吞作用的特定刺激剂在水的条件下可能很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剂化合物在我们以前的屏幕上确定了它们预防AQP2膜积累的能力，这也是药物的特征这将防止水超负荷。我们先前的研究以及此续签应用中提出的工作范围从通过细胞培养分析和整个动物研究，蛋白质相互作用的体外表征。我们已经发展了高通量化学屏幕的新细胞系，这是一种新的AQP2-EGFP构造，用于活细胞成像，我们有一个我们设施中有条件加压素受体敲除小鼠的新建立的菌落用于体内药物测试。我们的工作结合了对基本机制的更好理解，以推动转化医学和临床进展，采用新型细胞测定方法采用更直接的药物发现方法。