The long-term objective of this proposal is to provide a fundamental understanding of the cell biology of the neurohypophyseal hormone, vasopressin, which plays a major role in the regulation of salt and water balance through its action on the kidney. Its dysfunction is a primary cause of congenital nephrogenic diabetes insipidus (CNDI) and a secondary cause of fluid and electrolyte abnormalities in such diseases as congestive heart failure, cirrhosis of the liver, and nephrotic syndrome. The initial aims of this proposal are to provide an analysis of the structural motifs of the vasopressin V2 receptor (V2R) responsible for 1) proper folding and processing in the ER and Golgi and appropriate targeting to basolateral (or apical) membranes of kidney cells; and 2) regulation of desensitization, downregulation or resensitization of the V2R that we propose is mediated by G protein receptor kinase phosphorylation, arrestin binding, and endocytosis and exocytosis initiated by clathrin-coated pits or caveolae. We also propose that one or more of these events is coordinated by the heterotrimeric G protein subunit, G/alpha/i3. These studies will make use of epitope-tagged or green fluorescent protein-tagged V2Rs or its mutations, CNDI mutants, or """"""""split"""""""" V2Rs to monitor these processes by immunocytochemistry, confocal and electron microscopy or cell surface biotinylation in fixed and living cells. Association of the V2R with similarly tagged auxiliary proteins (e.g. G/alpha/i3, clathrin and adaptor proteins, dynamin or caveolin) will be determined by immunocytochemistry and immunoprecipation or by a new method of surface plasmon resonance. Transduction of the vasopressin receptor signaling pathway to the distal elements in its physiologic action also requires G/alpha/i3 regulation of vasopressin-sensitive Na+ channels, referred to as the 5 pS (rENaC) or the 9 pS channel.
The final aim of our proposal is to determine the ability of G/alpha/i3 to regulate the 5 pS rENaC, evaluated by electrophysiological techniques in oocytes or MDCK cells co-expressing rENaC and a constitutively active, pertussis-toxin insensitive G/alpha/i3 (G/alpha/i3*PTneg). Studies will be conducted to determine if G/alpha/i3 action depends on its membrane targeting sequences previously determined in our laboratory. Direct demonstration of the action of G/alpha/i3*PTneg on its mutations on the 5 pS and 9 pS Na+ channels will be determined by electrophysiological techniques after the addition of the purified protein to the cytosolic surface of membranes of either A6 cells or MDCK cells expressing rENaC, or following addition to purified bovine renal 9 pS Na+ channels reconstituted into lipid bilayers. Finally, utilizing G/alpha/i3*PTneg coupled via a hexahistidine to nickel-agarose beads, effector proteins for G/alpha/i3 action on Na+ channels will be expression cloned.
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