This project is based on the premise that studies of hormone and drug receptors in intact cells can provides physiologically relevant information regarding regulation of receptor expression and function. The principal focus of the proposal is on beta- adrenergic receptors of wild-type S49 lymphoma cells and S49 variants having lesions in the pathway of beta-adrenergic receptors/Gs/adenylate cyclase/cAMP dependent protein kinase. I propose to use several different approaches to define various features of the beta-adrenergic receptor """"""""life cycle in the S49 system. These approaches includes use of a new irreversible probe, BIM, to block beta-receptors selectively and then to measure recovery of receptor binding and function (cAMP generation) to control levels. Kinetic analysis of recovery data will allow us to estimate rates of receptor appearance and disappearance. Studies with S49 variants will determine the role of distal components in the beta-receptor pathway in regulation of receptor appearance and disappearance and experiments with various inhibitors will help dissect out the contribution of particular processes to calculated rates of receptor appearance and his disappearance. Further studies will involve preparation of antireceptor antibodies using synthetic peptides and chimeric proteins derived from transient expression of restriction fragments of beta2-receptor cDNA in E. Coli. Antibodies will be used to define additional aspects of beta-receptor synthesis and turnover. Methods will also be developed to quanititate beta2 receptor mRNA in S49 cells. In additional studies we will use antipeptide antibodies that we have generated to the Gs protein to examine formation and turnover of this protein in wild-type and variant S49 cells. Other experiments will be directed at defining the basis for the depletion of beta-adrenergic receptors in two S49 variants, beta p and beta d, which lack -50% and -85% of cellular beta- receptors, respectively. Taken together these studies should provide new insights into the mechanisms by which cells regulate their expression of beta-adrenergic receptors. Because of the wide distribution of those receptors, the results should be of relevance to beta-receptors in several organ systems, perhaps most importantly in the cardiovascular and pulmonary systems, and should provide new insights into the molecular pharmacology of beta- adrenergic drug responses.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Pharmacology A Study Section (PHRA)
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University of California San Diego
Schools of Medicine
La Jolla
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Buscher, R; Herrmann, V; Insel, P A (1999) Human adrenoceptor polymorphisms: evolving recognition of clinical importance. Trends Pharmacol Sci 20:94-9
Buscher, R; Herrmann, V; Insel, P A (1998) PCR-based methods for identifying genetic variations in human alpha1B- and beta2-adrenergic receptors. Mol Genet Metab 64:266-70
Post, S R; Rump, L C; Zambon, A et al. (1998) ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells. Blockade of an autocrine/paracrine pathway to define receptor preference of an agonist. J Biol Chem 273:23093-7
Hughes, R J; Anderson, K L; Kiel, D et al. (1996) Cloning of GRK2 cDNA from S49 murine lymphoma cells. Am J Physiol 270:C885-91
Post, S R; Aguila-Buhain, O; Insel, P A (1996) A key role for protein kinase A in homologous desensitization of the beta 2-adrenergic receptor pathway in S49 lymphoma cells. J Biol Chem 271:895-900
Denker, S P; McCaffery, J M; Palade, G E et al. (1996) Differential distribution of alpha subunits and beta gamma subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas. J Cell Biol 133:1027-40
Mochizuki, N; Cho, G; Wen, B et al. (1996) Identification and cDNA cloning of a novel human mosaic protein, LGN, based on interaction with G alpha i2. Gene 181:39-43
Post, S R; Jacobson, J P; Insel, P A (1996) P2 purinergic receptor agonists enhance cAMP production in Madin-Darby canine kidney epithelial cells via an autocrine/paracrine mechanism. J Biol Chem 271:2029-32
Insel, P A (1996) Seminars in medicine of the Beth Israel Hospital, Boston. Adrenergic receptors--evolving concepts and clinical implications. N Engl J Med 334:580-5
Post, S R; Hilal-Dandan, R; Urasawa, K et al. (1995) Quantification of signalling components and amplification in the beta-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes. Biochem J 311 ( Pt 1):75-80

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