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

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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.