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

水通道水通道蛋白-2(AQP2)位于肾集合管主细胞的小泡上。抗利尿激素加压素(AVP)与位于细胞基底外侧表面的加压素V2受体结合，诱导AQP2移位到顶端质膜(AQP2穿梭)。这一过程构成了AVP调节的抗利尿的分子基础。AVP诱导的AQP2穿梭似乎涉及微管和F-肌动蛋白，这是细胞骨架的两个组成部分。然而，AQP2转位到质膜和AQP2的内吞回收过程中，AQP2的囊泡和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。该项目的一个重要目标是可视化和机械地解释活的原代培养的大鼠内髓集合管(IMCD)细胞中携带AQP2的囊泡和F-肌动蛋白沿微管的运动。特别是，我们的目标是确定介导转运的蛋白质，包括与AQP2直接相互作用的蛋白质。我们以前已经证明，抑制小的GTP结合蛋白Rho和由此导致的F-肌动蛋白的减少是AVP诱导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Rho下游调节F-肌动蛋白的信号级联，从而调节AQP2的细胞定位。我们的数据表明，不仅Rho，而且Rho家族的另外两个成员--CDC42和Rac也参与了AQP2细胞定位的控制。因此，拟议项目的另一个目标是澄清CDC42和RAC在AQP2航天飞机中的作用。

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.