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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Biochemical Endocrinology Study Section (BCE)
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Sato, Sheryl M
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University of California Los Angeles
Schools of Medicine
Los Angeles
United States
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Gouill, Christian Le; Darden, Thomas; Madziva, Michael T et al. (2005) A role for K268 in V2R folding. FEBS Lett 579:4985-90
Innamorati, Giulio; Whang, Michael Insuk; Molteni, Raffaella et al. (2002) GIP, a G-protein-coupled receptor interacting protein. Regul Pept 109:173-9
Le Gouill, Christian; Innamorati, Giulio; Birnbaumer, Mariel (2002) An expanded V2 receptor retention signal. FEBS Lett 532:363-6
Innamorati, G; Le Gouill, C; Balamotis, M et al. (2001) The long and the short cycle. Alternative intracellular routes for trafficking of G-protein-coupled receptors. J Biol Chem 276:13096-103
Cilluffo, M C; Esqueda, E; Farahbakhsh, N A (2000) Multiple receptor activation elicits synergistic IP formation in nonpigmented ciliary body epithelial cells. Am J Physiol Cell Physiol 279:C734-43
Sadeghi, H; Birnbaumer, M (1999) O-Glycosylation of the V2 vasopressin receptor. Glycobiology 9:731-7
Boulay, G; Brown, D M; Qin, N et al. (1999) Modulation of Ca(2+) entry by polypeptides of the inositol 1,4, 5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for roles of TRP and IP3R in store depletion-activated Ca(2+) entry. Proc Natl Acad Sci U S A 96:14955-60
Rajagopalan-Gupta, R M; Mukherjee, S; Zhu, X et al. (1999) Roles of Gi and Gq/11 in mediating desensitization of the luteinizing hormone/choriogonadotropin receptor in porcine ovarian follicular membranes. Endocrinology 140:1612-21
Innamorati, G; Sadeghi, H M; Tran, N T et al. (1998) A serine cluster prevents recycling of the V2 vasopressin receptor. Proc Natl Acad Sci U S A 95:2222-6
Innamorati, G; Sadeghi, H; Birnbaumer, M (1998) Transient phosphorylation of the V1a vasopressin receptor. J Biol Chem 273:7155-61

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