The role of the cytoskeleton in the vasopressin-induced aquaporin-2 shuttle in renal collecting duct principal cells

水通道蛋白2（AQP 2）位于肾集合管主细胞的胞内囊泡上。抗利尿激素血管加压素（AVP）与位于细胞基底外侧表面的血管加压素V2受体的结合诱导AQP 2易位到顶端质膜（AQP 2穿梭）。这一过程构成了AVP调节的抗利尿作用的分子基础。AVP诱导的AQP 2穿梭似乎涉及微管和F-肌动蛋白（细胞骨架的两种成分）。然而，时间和空间的关系AQP 2轴承囊泡和细胞骨架在AVP诱导的易位到质膜和内吞检索AQP 2是未知的。介导囊泡沿着细胞骨架转运的蛋白质也是未知的。该项目的一个重要目标是对原代培养的大鼠内髓集合管（IMCD）细胞中AQP 2-轴承囊泡沿着微管和F-肌动蛋白的运动进行可视化和机械解释。特别是，我们的目标是确定介导的运输，包括蛋白质直接与AQP 2相互作用的蛋白质。我们先前已经证明，抑制小GTP结合蛋白Rho和由此导致的F-actin减少是AVP诱导的AQP 2穿梭IMCD细胞的先决条件。在拟议的项目中，我们的目标是阐明下游的Rho，调节F-肌动蛋白的信号级联，从而，AQP 2的细胞定位。我们的数据表明，不仅Rho，而且Cdc 42和Rac，Rho家族的另外两个成员，参与控制AQP 2的细胞定位。因此，拟议项目的另一个目标是澄清Cdc 42和Rac在AQP 2穿梭中的作用。

The water channel aquaporin-2 (AQP2) resides on intracellular vesicles of renal collecting duct principal cells. Binding of the antidiuretic hormone vasopressin (AVP) to vasopressin V2 receptors located on the basolateral 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 AVP-induced 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 habe 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.