Characterization of AQP2 interacting proteins and novel functions of AQP2

描述（由申请人提供）： 该申请人提出了一项研究计划，为她在肾脏生理学/病理生理学领域的学术肾脏学和基础科学研究（特别是AQP 2贩运）做好准备。本申请人将提议表征AQP 2相互作用蛋白并研究这些相互作用的生理意义，以更好地理解AQP 2运输的分子机制，并探索AQP 2在细胞生物学和病理生理学过程中可能的新作用。这项研究将在马萨诸塞州总医院膜生物学项目（PMB）和肾脏科的丹尼斯·布朗博士的实验室进行。血管加压素（VP）是哺乳动物肾脏水重吸收调节的主要抗利尿激素。它通过将AQP 2水通道从细胞质囊泡募集到集合管主细胞的质膜来发挥作用。VP-AQP 2信号通路的损伤导致充血性心力衰竭、肝硬化中可见的液体潴留，以及尿崩症中可见的浓缩缺陷。AQP 2通过复杂的运输途径进行调节，这些途径尚未得到很好的表征。我们的假设是，AQP 2的调节运输需要细胞内移位、胞吐和胞吞过程中直接和间接的蛋白质-蛋白质相互作用。具体而言，我们将1）通过进一步追踪AQP 2运输途径中的亚细胞区室/步骤来扩展我们目前关于AQP 2和热休克蛋白70（hsc 70）相互作用的研究，并继续表征我们在酵母双杂交筛选中鉴定的其他AQP 2结合候选物; 2）研究磷酸化和去磷酸化对AQP 2运输作用的分子机制; 3）探索AQP 2在细胞粘附、迁移和肾小管形成/畸形中的新的潜在作用。我们将调查的分布和运输的AQP 2在一般的生物过程中，以及改变AQP 2贩运对这些生物过程的影响。我们将使用稳定或原代细胞培养、组织切片培养、三维培养系统、斑马鱼模型系统和AQP 2突变小鼠模型。多学科的方法和强大的技术将被用来进行这些研究，这将揭示AQP 2的贩运和功能的机制。

DESCRIPTION (provided by applicant): This applicant has proposed a program of research to prepare her for a career in academic nephrology and basic science research in the field of renal physiology/pathophysiology, specifically, AQP2 trafficking. This applicant will propose to characterize AQP2 interacting proteins and investigate the physiological significance of these interactions, to better understand the molecular mechanism underlying AQP2 trafficking, and explore the possible novel role of AQP2 in cellular biological and pathophysiological processes. The research will be conducted in the laboratory of Dr. Dennis Brown at the Program in Membrane Biology (PMB) and Division of Nephrology, Massachusetts General Hospital. Vasopressin (VP) is the major antidiuretic hormone involved in the regulation of water reabsorption by mammalian kidney. It functions by recruiting the AQP2 water channel from cytoplasmic vesicles to the plasma membrane of collecting duct principal cells. The impairment of VP-AQP2 signaling pathways results in fluid retention seen in congestive heart failure, cirrhosis, as well as concentrating defect seen in diabetes insipidus. AQP2 is regulated through complex trafficking pathways which have not been well characterized. Our hypothesis is that regulated trafficking of AQP2 requires direct and indirect protein-protein interactions during intracellular translocation, exocytosis as well as endocytosis. Specifically, we will 1) extend our current study on the interaction of AQP2 and heat shock protein 70 (hsc70) by tracking further down to subcellular compartments/step(s) in AQP2 trafficking pathways, and continue to characterize other AQP2 binding candidates identified from our yeast-two hybrid screen; 2) study the molecular mechanism underlying the role of phosphorylation and dephosphorylation on AQP2 trafficking; 3) explore novel potential roles of AQP2 in cell adhesion, migration, and renal tubular formation/malformation. We will investigate the distribution and trafficking of AQP2 during the general biological processes as well as the effect of alteration of AQP2 trafficking on these biological processes. We will using stable or primary cell culture, tissue slice culture, 3D culture system, a zebrafish model system, and AQP2 mutant mouse model. Multidisciplinary approaches and powerful technologies will be used to carry out these studies that will shed light on the mechanism of AQP2 trafficking and function.