The long term goal of this research is to understand the molecular basis of the activation of G proteins by G protein coupled receptors. The V2 vasopressin receptor that serves as a model for these studies mediates the antidiuretic response of the kidney to ADH. The development of efficient V2R antagonists to control the syndrome of inappropriate ADH secretion are ongoing, and information about receptor structure and function will contribute towards the understanding of the mechanism of action of agonists and antagonists. Two areas of receptor biology will be addressed: ligand mediated regulation of receptor activity, and the mechanism of receptor activation of G proteins. Preliminary studies identified a segment of 15 amino acids at the C-terminus of the V2 vasopressin receptor that is the substrate of ligand-promoted phosphorylation and, when present, blocks the recycling of the receptor to the cell surface.
Specific Aim 1 deals with the identification of the protein(s) that mediate the retention of the phosphorylated V2 vasopressin receptor inside the cells, that probably fulfills a similar role for other proteins sequestered within the cells. A """"""""bait"""""""" modeled on this segment shall be used to search for the retaining protein by screening lambda gt11 expression libraries and applying the yeast two hybrid system.
Specific Aim 2 applies a variety of methods to define segments of the receptor involved in G protein activation: 1) proteolytic """"""""footprinting"""""""" to identify amino acids of the receptor that make direct contact with heterotrimeric G proteins under conditions known to favor receptor/G protein interaction; 2) determination of the vicinity of amino acids in the cytoplasmic portion of the receptor by creating disulfide bonds between cysteines, singles or in pairs, engineered at locations expected to be proximal to each other; 3) introduction of histidines, singles or in pairs, at chosen locations of the cytoplasmic face of the receptor to create metal ion binding sites that complex with nickel or zinc, successful formation of bonds in 2) and complexes in 3) is expected to destroy G protein activation by the receptor; 4) creating disulfide bonds between single cysteines introduced in the receptor in the regions protected from proteolysis identified in 1) and single cysteines introduced into Gsalpha to better define the contact points between both proteins.
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