Identifizierung und Charakterisierung von Proteinen, die den Transport von von Aquaporin-2 in renalen Hauptzellen kontrollieren

水通道通道蛋白-2 （AQP2）位于肾集管主细胞的小泡内。抗利尿激素抗利尿激素（AVP）与位于细胞基底侧表面的抗利尿激素V2受体结合，诱导AQP2易位到根尖质膜（AQP2穿梭）。这一过程构成了avp调控的抗利尿的分子基础。avp诱导的AQP2穿梭似乎涉及细胞骨架的两种成分微管和f -肌动蛋白。然而，在av诱导的质膜易位和AQP2的内吞回收过程中，携带AQP2的囊泡与细胞骨架的时空关系尚不清楚。介导囊泡沿细胞骨架运输的蛋白质也是未知的。拟建项目的一个重要目标是可视化和机制解释在原代培养的大鼠内髓集管（IMCD）细胞中携带aqp2的囊泡沿着微管和f -肌动蛋白的运动。特别是，我们的目标是鉴定介导转运的蛋白质，包括直接与AQP2相互作用的蛋白质。我们之前的研究表明，抑制小gtp结合蛋白Rho和由此导致的F-actin的减少是avp诱导的IMCD细胞中AQP2穿梭的先决条件。在拟议的项目中，我们的目标是阐明调控f -肌动蛋白的Rho下游信号级联，从而调控AQP2的细胞定位。我们的数据表明，不仅Rho， Rho家族的另外两个成员Cdc42和Rac也参与控制AQP2的细胞定位。因此，拟议项目的进一步目标是澄清Cdc42和Rac在AQP2穿梭中的作用。

The water channel aquaporin-2 (AQP2) resides on intraceflular vesicles of renal collecting duct principal cells. Binding of the antidiuretic hormone vasopressin (AVP) to vasopressin V2 receptors located on the basolaterai surface of the cells induces the translocation of AQP2 into the apical plasma membrane {AQP2 shuttle). This process constitutes the molecular basis of AVP-regulated antidiuresis. The AVP-induced AQP2 shuttle appears to involve microtubules and F-actin, two components of the cytoskeleton. However, the temporal and spatial relationship of AQP2-bearing vesicles and the cytoskeleton during the AVPinduced translocation to the plasma membrane and the endocytic retrieval of AQP2 is unknown. Proteins mediating the transport of the vesicles along the cytoskeleton are also unknown. An important goal of the proposed project is the visualisation and mechanistic explanation of the movement of AQP2-bearing vesicles along microtubules and F-actin in live primary cultured rat inner medullary collecting duct (IMCD) cells. In particular, we aim at identifying the proteins mediating the transport, including proteins which directly interact with AQP2. We have previously shown that inhibition of the small GTP-binding protein Rho and the resulting reduction of F-actin are prerequisites for the AVP-induced AQP2 shuttle in IMCD cells. In the proposed project we aim to elucidate the signalling cascade downstream of Rho that regulates F-actin and, thereby, the cellular localisation of AQP2. Our data suggest that not only Rho but also Cdc42 and Rac, two other members of the Rho family, are involved in the control of the cellular localisation of AQP2. Thus, a further goal of the proposed project is the clarification of the role of Cdc42 and Rac in the AQP2 shuttle.