The regulation of cellular processes in mammals by epinephrine stimulation of beta2-adrenergic receptors (betaAR) is extensive and includes important effects on the nervous, cardiovascular, and pulmonary systems. BetaAR antagonists are used as therapy for several diseases of the cardiovascular system such as hypertension, and betaAR agonists are a mainstay in the treatment of asthma. One of the most important factors that govern the effectiveness of betaAR agonists is the rate of development of tolerance, which is thought to be caused in part by desensitization of the betaAR. Desensitization, the loss of betaAR stimulation of adenylyl cyclase (AC), appears to be caused by phosphorylation of the betaAR by various protein kinases, by internalization or by downregulation. Our long-term goal is to determine the roles these mechanisms play in the regulation of betaAR responsivity in mammalian tissue. The major goal of this proposal is to identify the specific amino acids in the betaAR that are phosphorylated in response to stimuli that activate PKA-, PKC- or betaARK-mediated mechanisms of desensitization in intact human embryonic kidney (HEK 293) cells, and the consequences of the phosphorylations on betaAR activation of AC. The primary focus will be on the putative betaARK consensus phosphorylation sites. Site-directed mutagenesis of proposed protein kinase consensus sites in the betaAR and the characterization of the mutations' effects on betaAR stimulation of AC following expression in HEK 293 cells will localize the domains involved in the kinase-mediated desensitizations. This analysis will be coupled with peptide mapping and microsequencing of highly purified betaAR phosphopeptides to determine the specific amino acids phosphorylated by the various kinases. Purification of the betaAR for peptide mapping will be based on the use of hemagglutinin and 6-histidine epitopes which have been introduced into the betaAR. Secondary aims include: analysis of the relative roles of betaARK and internalization in homologous desensitization and their interdependence; exploration of internalization domains by site-directed mutagenesis; investigation of the role of the C-terminal PKA/PKC consensus site; tests of our proposal that partial agonists induce less homologous desensitization than do full agonists; and an exact quantitation of the effect of receptor number on desensitization.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Pharmacology A Study Section (PHRA)
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Cole, Alison E
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University of Texas Health Science Center Houston
Schools of Medicine
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