Dennis A. Ausiello, M. D.

Cell biology of vasopressin receptor signaling and trafficking

In the 30 years since my laboratory has been studying the vasopressin type II receptor (V2R), novel characteristics have been elucidated, suggesting a uniqueness for this receptor’s activity among the G-protein coupled receptors (GPCRs). The V2R is mutated in X-linked nephrogenic diabetes insipidus, underscoring the importance of the vasopressin signaling system in water homeostasis in man. We have recently published the paradigm shifting observation that the V2R is not recycled to the membrane after ligand binding and activation, but instead is degraded in lysosomes. We have now discovered that V2R interacts with Alix, a key component of the endosomal sorting cargo related trafficking (ESCRT) complexes of proteins, to enhance ligand-induced V2R degradation. This raises the novel hypothesis that cargo (V2R) can direct its traffic into the degradation pathway by interacting with a specific member of the ESCRT protein complex. The work in my laboratory is aimed at defining the unique molecular and cellular events that allow V2R to function in its unique native environment in the kidney, and that lead to its eventual degradation in lysosomes. Our goals are to explore conformational changes that allow ligand binding, activation and deactivation of wild type and V2R trafficking mutants at low pH and hypertonicity utilizing biochemical approaches, live cell microscopy and novel FRET techniques to dissect intra- (V2R dimerization) and sequential intermolecular (with b-arrestin and Gas) protein-protein interactions. These projects will make use of the Microscopy Core and its FRET capability. We also hope to reveal functionally important interactions of the V2R with accessory proteins during internalization,

including those of the clathrin endocytic complex, and in endosomal compartments after blockade of trafficking in cells expressing wild type and mutant V2R constructs, using spinning disk confocal and TIRF microscopy of live cells, pull down assays, and FRET – again making extensive use of the Microscopy Core facilities. We then plan to explore the novel interaction of V2R and Alix that directs the accelerated entry of ligand-bound V2R into the degradative pathway using microscopy, cell-fractionation and protein interaction techniques, and a novel FLAG-V2R transgenic mouse model. Extensive use of the Microscopy Core will be integral to these studies. Our goal is to continue to provide unique information on the signaling and trafficking of the V2R that make it uniquely able to signal in the harsh native conditions of the kidney, where it undergoes degradation rather than recycling after internalization. The new studies will be relevant to the specific problems of water homeostasis and AQP2 mobilization, and will contribute to dissecting the physiology of  GPCRs in general.