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.
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