Characterization of AQP2 interacting proteins and novel functions of AQP2

描述(由申请人提供)： 这位申请者提出了一个研究计划，为她在肾脏生理学/病理生理学领域的学术肾病学和基础科学研究领域的职业生涯做准备，特别是AQP2贩运。本申请者将提议鉴定AQP2相互作用的蛋白质，并研究这些相互作用的生理意义，以更好地了解AQP2运输的分子机制，并探索AQP2在细胞生物学和病理生理过程中可能的新作用。这项研究将在马萨诸塞州综合医院膜生物学项目(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)是哺乳动物肾脏调节水分重吸收的主要抗利尿激素。它的功能是将AQP2水通道从细胞质小泡募集到集合管主细胞的质膜上。VP-AQP2信号通路的损伤导致充血性心力衰竭、肝硬变和尿崩症的液体滞留。AQP2通过复杂的贩运途径进行调控，但这些途径尚未得到很好的描述。我们的假设是，AQP2的受控运输需要在细胞内转运、胞吐和内吞过程中直接和间接的蛋白质-蛋白质相互作用。具体地说，我们将1)通过进一步向下追踪AQP2转运途径中的亚细胞室/步骤(S)来扩展我们目前对AQP2与热休克蛋白70(Hsc70)相互作用的研究，并继续鉴定从我们的酵母双杂交筛选中发现的其他AQP2结合候选基因；2)研究磷酸化和去磷酸化在AQP2转运中作用的分子机制；3)探索AQP2在细胞黏附、迁移和肾小管形成/畸形中的潜在新角色。我们将调查AQP2在一般生物过程中的分布和贩运，以及AQP2贩运的改变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、3D培养系统、斑马鱼模型系统和AQP2突变小鼠模型。将使用多学科方法和强大的技术来开展这些研究，以阐明AQP2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.