The overall goal of this research program is to investigate, at a molecular level, the regulation of receptors for neurotransmitters and hormones. These experiments will emphasize studies of beta- adrenergic receptors. Most, but not all, of the affects mediated by these receptors result from increases in adenylate cyclase activity and involve formation of a ternary complex composed of agonist, receptor, and a guanine nucleotide-binding protein (G). An understanding of agonist/receptor and receptor/G protein interactions, and of the factors that modulate these interactions, is central to an understanding of information transfer across cell membranes. The properties of the binding of radiolabeled agonists and antagonists will be assessed in L6 myoblasts that have a high density of receptors, C6 cells that express both beta 1- and beta 2-adrenergic receptors, and S49 lymphoma cells, variants of which have missing or defective G proteins. Many of the proposed experiments will utilize anti-receptor antibodies that will be elicited against peptides corresponding to specific regions of the molecule whose sequence has been established in several systems. Anti-receptor antibodies will be used to investigate the orientation of the receptor in the membrane and to define specific domains involved in such functions as interactions with agonists or with G proteins. Antibodies will also be used to study the process of agonist-induced receptor sequestration and receptor down-regulation. Finally, antibodies will be used for the immunocytochemical localization of beta- adrenergic receptors. In addition to studies with anti-receptor antibodies, experiments will be carried out with antibodies raised against the subunits of Gs, Gj, and Go. These antibodies will be used to define the nature of the ternary complex in wild-type and cyc S49 lymphoma cells. Sympathomimetic effects of atypical agonists, including pindolol and celiprolol, are not associated with receptor sequestration or the formation of a ternary complex, and do not result in increases in intracellular levels of cyclic AMP. Atypical agonists do not cause receptor down-regulation in cyc- S49 lymphoma cells. These results suggest that there may be multiple mechanisms responsible for the control of receptor density by agonists. Effects of atypical agonists on the rate constants of receptor synthesis and degradation will be determined. The role of Gs in down-regulation induced by pindolol will be assessed using cyc- S49 lymphoma cells transfected with cDNA coding for the alpha subunit of Gs. The possibility that alternative second-messenger systems, involving phospholipases or intracellular calcium, are mediating the effects of atypical agonists will be investigated. Receptor synthesis and degradaton will also be investigated during down-regulation by a variety of full and partial agonists. The kinetics of receptor synthesis and degradation will be studied using irreversible alkylating agents, by measuring the approach to and recovery from desensitization, and by immunoprecipitation of receptors labeled by growing cells in the presence of 35S-methionine.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Neurological Sciences Subcommittee 1 (NLS)
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University of Pennsylvania
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