I propose continued studies on the molecular structure and the mechanism of regulatory activity of the beta-adrenergic receptor. During the last funding cycle, we studied the beta- adrenergic regulation of the stimulatory GTP-binding protein of the adenylate cyclase system, Gs, using purified Gs and beta- adrenergic receptors that were co-reconstituted into unilamellar phospholipid vesicles. (1) We have cloned the cDNA for the receptor and have began to study the organization of the protein. The cloned DNA will be expressed in yeast to provide larger amounts of receptor for biochemical studies and to allow expression of in vitro mutagenized receptor. Expression in a receptor-deficient mammalian cell line will also be developed to allow in situ physiological study of receptor function. (2) The sites of beta-adrenergic agonist binding and of the recently described regulatory cysteine disulfide will be mapped chemically. Functional effects of altering these structures by site-directed mutagenesis will be analyzed after co- reconstituting recombinant receptor with Gs as previously described. (3) The binding of the receptor to Gs will be studied in the vesicles both by covalent crosslinking and by analyzing an isolated agonist-receptor-Gs complex whose formation precedes rapid guanine nucleotide binding. The binding of receptor to Gs will also be assayed directly in the vesicles by measuring fluorescence energy transfer between covalently attached fluorophores on each protein. (4) We will continue enzymologic studies of the mechanism of receptor-mediated regulation of Gs function. The interdependence of receptor and the beta gamma subunits of Gs will be probed in vesicles containing various alpha:beta gamma stoichiometric ratios. Receptor-stimulated binding and release of GTP, GDP, and GTP gamma S will serve as functional assays for receptor function. (5) We will probe the ability of different phospholipids to maintain the activity of the receptor and to support receptor-Gs coupling. We will also test the hypothesis that the rate of receptor-mediated activation of Gs depends on the diffusion-limited collision of receptor and Gs molecules in the membrane.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030355-09
Application #
3278075
Study Section
Biochemistry Study Section (BIO)
Project Start
1981-08-01
Project End
1992-07-31
Budget Start
1989-08-01
Budget End
1990-07-31
Support Year
9
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Navaratnarajah, Punya; Gershenson, Anne; Ross, Elliott M (2017) The binding of activated G?q to phospholipase C-? exhibits anomalous affinity. J Biol Chem 292:16787-16801
Kadamur, Ganesh; Ross, Elliott M (2016) Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-? Exposes a G?? Protein Binding Site. J Biol Chem 291:11394-406
Dyachok, Julia; Earnest, Svetlana; Iturraran, Erica N et al. (2016) Amino Acids Regulate mTORC1 by an Obligate Two-step Mechanism. J Biol Chem 291:22414-22426
Wauson, Eric M; Guerra, Marcy L; Dyachok, Julia et al. (2015) Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells. Mol Endocrinol 29:1114-22
Ross, Elliott M (2014) G Protein-coupled receptors: Multi-turnover GDP/GTP exchange catalysis on heterotrimeric G proteins. Cell Logist 4:e29391
Kadamur, Ganesh; Ross, Elliott M (2013) Mammalian phospholipase C. Annu Rev Physiol 75:127-54
Chang, Seungwoo; Ross, Elliott M (2012) Activation biosensor for G protein-coupled receptors: a FRET-based m1 muscarinic activation sensor that regulates G(q). PLoS One 7:e45651
An, Sung-Wan; Cha, Seung-Kuy; Yoon, Joonho et al. (2011) WNK1 promotes PIP? synthesis to coordinate growth factor and GPCR-Gq signaling. Curr Biol 21:1979-87
Rebres, Robert A; Roach, Tamara I A; Fraser, Iain D C et al. (2011) Synergistic Ca2+ responses by G{alpha}i- and G{alpha}q-coupled G-protein-coupled receptors require a single PLC{beta} isoform that is sensitive to both G{beta}{gamma} and G{alpha}q. J Biol Chem 286:942-51
Philip, Finly; Kadamur, Ganesh; Silos, Rosa Gonzalez et al. (2010) Synergistic activation of phospholipase C-beta3 by Galpha(q) and Gbetagamma describes a simple two-state coincidence detector. Curr Biol 20:1327-35

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