The physiological catecholamines, epinephrine and norepinephrine, and many clinical useful pharmacological agonists and antagonists act on beta-adrenergic receptors. Although much new knowledge has recently been provided regarding beta-adrenergic receptor structure, much remains unknown regarding regulation of receptor expression and function in target cells. In this project we propose to continue studies that emphasize cellular and molecular mechanisms that regulate signal transduction by beta-adrenergic receptors in intact cells. My colleagues and I will continue to study wild-type S49 lymphoma cells and S49 variants in the pathway for beta receptor signalling via the guanine nucleotide regulatory protein, G(s) and in turn, adenylyl cyclase and cAMP-dependent protein kinase. We propose three specific aims designed to characterize the life cycles of beta-adrenergic receptors, Galpha(s), and the catalyst (C) of adenylyl cyclase. Antibodies to these components will be used to characterize biosynthesis and turnover and to assess the role of phosphorylation of beta-receptors and C. Other studies are designed to use molecular biological techniques (such as antisense technology and transfection with cDNAs) to help define the role of the beta-adrenergic receptor kinase (beta-ARK) in S49 cells and to evaluate stoichiometry between alpha(s) and C. Additional experiments will use biochemical and micrologic techniques to assess the subcellular localization of Galpha(s). Taken together, experimental approaches should provide new information regarding the key components involved in beta-adrenergic receptor signal transduction. As such, the results should be relevant to a variety of clinical settings, in particular cardiovascular disorders, in which betaadrenergic receptor function may be altered. In addition, these studies should help advance understanding of the molecular basis of pharmacological action at beta-adrenergic receptors and at other receptors that activate G(s) and adenylyl cyclase.
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