H+-ATPase And AQP2: Regulation Of Targeting And Recycling In IMCD Cells

描述（由申请人提供）：体内和培养的IMCD细胞分泌H+，由H+- atp酶介导，并吸收水通道蛋白-2 （AQP2）介导的水。在培养的大鼠IMCD细胞和完整的IMCD片段中，运输是通过SNARE介导的胞外插入和携带H+- atp酶或AQP2的囊泡的内吞回收来调节的。虽然H+- atp酶和AQP2的胞外分泌是独立调节的，但它们利用类似的SNARE系统进行膜靶向融合。在本提案中，我们将描述这些蛋白的靶向过程，并确定SNARE调节剂对H+- atp酶和AQP2膜循环的不同影响。我们将验证质膜H+- atp酶的B1亚基包含将组装的H+- atp酶靶向顶膜的分子信息的假设。B1是B亚基的同工异构体，存在于专门用于质子分泌的细胞的H+- atp酶中。另一种异构体B2存在于所有细胞的囊泡膜H+- atp酶中。我们预测，质膜H+- atp酶的靶向信息编码在N端或c端氨基酸中，这是ATP6V1B1与B2最不同的两个区域。我们的研究还将确定Munc 18-2和snapin作为H+- atp酶和AQP2的SNARE调节因子的作用。Munc 18-2通过PKC信号传导，通过调节H+- atp酶与syntaxin的相互作用，控制IMCD细胞中H+- atp酶的顶端靶向和插入。我们假设Munc 18-2通过调节AQP2胞吐和内吞作用来控制质膜AQP2非常不同。我们提出以下循环：Munc通过与囊泡AQP2-VAMP结合来阻止融合复合物的形成。当PKA磷酸化AQP2时，Munc与AQP2和VAMP分离，发生囊泡融合，将AQP2、VAMP和Munc传递到质膜。在质膜AQP2去磷酸化后，Munc与AQP2重新结合，将AQP2和VAMP浓缩在网格蛋白包被的凹坑中，在那里Munc-AQP2-VAMP复合物被内吞进入细胞内部。最后，我们将对最近发现的SNAP与腺苷酸环化酶结合蛋白snapin的作用进行评估。我们认为，在IMCD细胞中，snapin调节H+- atp酶和AQP2的胞吐。当snapin与SNAP-23结合时，形成了数量增加的SNAP-syntaxin复合物，这些复合物是水泡状VAMP的t-SNARE受体，将H+- atp酶传递到顶膜。Snapin也可能通过放大抗利尿激素第二信息（cAMP）来调节AQP2胞吐，因为已有研究表明，在PKA磷酸化后，Snapin结合并激活腺苷酸环化酶。我们相信这些研究将为这些顶端膜转运蛋白的调控提供新的范例，并为液体和酸碱疾病的治疗提供新的方法。

DESCRIPTION (provided by applicant): IMCD cells in vivo and in culture secrete H+, mediated by an H+-ATPase, and absorb H2O mediated by aquaporin-2 (AQP2). In cultured rat IMCD cells and in intact IMCD segments, transport is regulated by SNARE mediated exocytic insertion and endocytic retrieval of vesicles carrying either the H+-ATPase or AQP2. Although exocytosis of H+-ATPase and AQP2 are independently regulated, they utilize a similar SNARE system for membrane targeting-fusion. In this proposal we will characterize the targeting process for these proteins and identify the disparate effects of SNARE regulators on H+-ATPase versus AQP2 membrane cycling. We will test the hypothesis that the B1 subunit of the plasma membrane H+-ATPase contains molecular information for targeting of the assembled H+-ATPase to the apical membrane. B1 is the isoform of the B subunit that is present in the H+-ATPase of cells specialized for proton secretion. The other isoform, B2, is present in the vesicular membrane H+-ATPase of all cells. We predict that the targeting information for the plasma membrane H+-ATPase is encoded in either the N or the C-terminal amino acids, the two regions of ATP6V1B1 that are most dissimilar from B2. Our studies will also determine the role of Munc 18-2 and snapin as SNARE regulators for H+- ATPase and AQP2. Munc 18-2, through PKC signaling, controls the apical targeting and insertion of H+-ATPase in IMCD cells by regulating its interaction with syntaxin. We hypothesize that Munc 18-2 controls plasma membrane AQP2 very differently by regulating both AQP2 exocytosis and endocytosis. We propose the following cycle: Munc prevents formation of a fusion complex by association with vesicular AQP2-VAMP. Upon PKA phosphorylation of AQP2, Munc dissociates from AQP2 and VAMP and vesicle fusion occurs delivering AQP2, VAMP and Munc to the plasma membrane. Upon dephosphorylation of plasma membrane AQP2, Munc reassociates with AQP2, concentrates AQP2 and VAMP in clathrin-coated pits where this complex of Munc-AQP2-VAMP is endocytosed into the cell interior. Lastly, the role of snapin, a recently identified SNAP and adenylate cyclase binding protein, will be evaluated. We propose that snapin regulates exocytosis of both H+-ATPase and AQP2 in IMCD cells. Upon snapin binding to SNAP-23, an increased number of SNAP-syntaxin complexes are formed which are the t-SNARE receptor for the vesicular VAMP, delivering H+-ATPase to the apical membrane. Snapin may also regulate AQP2 exocytosis by amplifying the vasopressin second message (cAMP) since it has been shown that upon PKA phosphorylation, snapin binds and activates adenylate cyclase. We believe these studies will provide novel paradigms for the regulation of these apical membrane transporters and provide newer modalities for the treatment of fluid and acid-base disorders. PUBLIC HEALTH RELEVANCE These studies will provide important new information concerning the mechanisms by which the kidney regulates water and acid-base balance and how these mechanisms are disrupted by genetic and acquired disease states. An enhanced understanding of these abnormalities could lead to the development of improved therapeutic interventions.