Characterization of Aquaporin 2 Interacting Proteins

说明(申请人提供)：血管加压素(VP)是主要的抗利尿激素，参与调节哺乳动物肾脏对水的重吸收。大量研究证实，VP通过将AQP2水通道从细胞质小泡募集到集合管主细胞的质膜上发挥作用。VP-AQP2信号通路的损伤导致尿崩症和一些心力衰竭患者的液体滞留。AQP2通过复杂的转运途径进行调控。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。这些研究分为三个特定的目标：鉴定和鉴定新的AQP2相互作用蛋白，以及研究这些新的蛋白相互作用在体内和体外AQP2转运中的功能意义。这项研究将利用多学科方法(分子生物学、细胞生物学、生物化学和免疫细胞化学)和强大的技术(酵母双杂交筛选、结构功能分析、基因转移)来鉴定和表征新的蛋白质-蛋白质相互作用及其生物学功能。这些研究将为AQP2转运背后鲜为人知的分子机制提供重要信息，并最终可能导致对尿液浓度缺陷患者的更好治疗。

DESCRIPTION (provided by applicant): Vasopressin (VP) is the major antidiuretic hormone involved in the regulation of water reabsorption by mammalian kidney. Intensive studies have established that VP 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 diabetes insipidus as well as fluid retention seen in some heart failure patients. AQP2 is regulated through complex trafficking pathways. Our hypothesis is that regulated trafficking of AQP2 requires direct and indirect protein-protein interactions during intracellular translocation, exocytosis as well as endocytosis. The proposed studies are divided into three specific aims addressing identification and characterization of novel AQP2 interacting proteins as well as investigation of the functional significance of these novel protein interactions on AQP2 trafficking in vitro and in vivo. The proposed study will utilize multidisciplinary approaches (molecular biology, cell biology, biochemistry and immunocytochemistry) and powerful technology (yeast two hybrid screen, structural functional analysis, gene transfer) to identify and characterize novel protein-protein interactions and their biologic functions. These studies will provide important information of the poorly understood molecular mechanisms underlying AQP2 trafficking, and may ultimately lead to better therapies for patients with defective urinary concentration.