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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS018479-10
Application #
3398515
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1981-12-01
Project End
1993-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
10
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Cohen, J A; Baggott, L A; Romano, C et al. (1993) Characterization of a mouse beta 1-adrenergic receptor genomic clone. DNA Cell Biol 12:537-47
Luedtke, R R; Artymyshyn, R P; Monks, B R et al. (1992) Comparison of the expression, transcription and genomic organization of D2 dopamine receptors in outbred and inbred strains of rat. Brain Res 584:45-54
Gonzales, J M; O'Donnell, J K; Stadel, J M et al. (1992) Down-regulation of beta-adrenergic receptors by pindolol in Gs alpha-transfected S49 cyc- murine lymphoma cells. J Neurochem 58:1093-1103
Artymyshyn, R P; Ivins, K J; Monks, B R et al. (1992) Quantitation of isotypes of D2 receptors using solution hybridization. Neurochem Int 20 Suppl:189S-195S
Ebersole, B J; Molinoff, P B (1992) Identification of ascorbate as an endogenous substance that irreversibly inhibits binding of dihydropyridine calcium channel blockers. J Neurochem 58:1300-7
Molinoff, P B (1992) Evolving properties of beta-adrenergic receptor antagonists. Pharmacotherapy 12:144-53
Unsworth, C D; Molinoff, P B (1992) Regulation of the 5-hydroxytryptamine1B receptor in opossum kidney cells after exposure to agonists. Mol Pharmacol 42:464-70
Marzo, K P; Frey, M J; Wilson, J R et al. (1991) Beta-adrenergic receptor-G protein-adenylate cyclase complex in experimental canine congestive heart failure produced by rapid ventricular pacing. Circ Res 69:1546-56
Ivins, K J; Luedtke, R R; Artymyshyn, R P et al. (1991) Regulation of dopamine D2 receptors in a novel cell line (SUP1). Mol Pharmacol 39:531-9
Ivins, K J; Molinoff, P B (1991) Desensitization and down-regulation of 5-HT2 receptors in P11 cells. J Pharmacol Exp Ther 259:423-9

Showing the most recent 10 out of 41 publications