This project tests the hypothesis that multiple cardiovascular receptors coupled to G proteins are regulated by the beta-adrenergic receptor kinase-1 (BetaARK1) and its isozyme, BetaARK2. Activated-receptor- specific, or homologous desensitization of the Beta-2 adrenergic receptor is thought to involve phosphorylation of agonist-occupied receptors by BetaARK1, and subsequent binding to the receptor of the inhibitory protein beta-arrestin. That BARKs initiate short-term desensitization of multiple cardiovascular G protein-coupled receptors derives credence from structural homology among these receptors,a nd from indirect evidence implicating BetaARK 1 in the desensitization of several receptors. This hypothesis derives importance from the diversity of cardiovascular function modulated by these receptors--from thrombosis to cardiac inotropy. Increased myocardial BARK1 expression has been associated with chronic heart failure, in which B-adrenergic desensitization may contribute to disease progression. This investigation will directly test BetaARK isozymes' potential roles in the desensitization of the Beta1-and alpha1B-adrenergic, thrombin, endothelin (subtypes A & B), and angiotensin II (type 1) receptors. Expression plasmids for each receptor will be used to transfect cells, which will undergo [32P]-phosphate metabolic labeling and agonist exposure; subsequent immunoprecipitation of receptors--epitope-tagged on their N termini will be used to assess receptor phosphorylation in cells overexpressing BetaARKS and decreased phosphorylation in cells underexpressing BetaARKs. Enzyme expression will be augmented by cotransfecting cells with receptor and BetaARK isozyme expression plasmids, and attenuated by cotransfecting with BetaARK isozyme antisense plasmids. In parallel studies, phosphorylation of the receptor in these cells will be correlated with functional receptor desensitization. Transfected cells permeabilized and treated with antibodies specific for each BetaARK isozyme and Beta-arrestin will also be used to correlate receptor desensitization and phosphorylation. Separately, each receptor will be partially purified from a baculovirus expression system, and tested for BARK isozyme-mediated, agonist-induced phosphorylation. In vivo and organ models for assessing the general significance of BetaARK in G protein-coupled receptor desensitization will be created with transgenic mice deficient in BARK isozymes. Knowing the spectrum of receptor systems desensitized by BetaARK isozymes may facilitate the development of receptor and enzyme-specific therapeutic interventions for disorders like chronic heart failure, characterized by potentially excessive desensitization.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08HL003008-05
Application #
2655209
Study Section
Research Training Review Committee (RTR)
Project Start
1994-02-01
Project End
1999-01-31
Budget Start
1998-02-01
Budget End
1999-01-31
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Duke University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Bolchoz, L J; Budinsky, R A; McMillan, D C et al. (2001) Primaquine-induced hemolytic anemia: formation and hemotoxicity of the arylhydroxylamine metabolite 6-methoxy-8-hydroxylaminoquinoline. J Pharmacol Exp Ther 297:509-15
Pitcher, J A; Freedman, N J; Lefkowitz, R J (1998) G protein-coupled receptor kinases. Annu Rev Biochem 67:653-92
Freedman, N J; Ament, A S; Oppermann, M et al. (1997) Phosphorylation and desensitization of human endothelin A and B receptors. Evidence for G protein-coupled receptor kinase specificity. J Biol Chem 272:17734-43
Oppermann, M; Freedman, N J; Alexander, R W et al. (1996) Phosphorylation of the type 1A angiotensin II receptor by G protein-coupled receptor kinases and protein kinase C. J Biol Chem 271:13266-72
Freedman, N J; Lefkowitz, R J (1996) Desensitization of G protein-coupled receptors. Recent Prog Horm Res 51:319-51;discussion 352-3
Oppermann, M; Diverse-Pierluissi, M; Drazner, M H et al. (1996) Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases. Proc Natl Acad Sci U S A 93:7649-54
Pei, G; Kieffer, B L; Lefkowitz, R J et al. (1995) Agonist-dependent phosphorylation of the mouse delta-opioid receptor: involvement of G protein-coupled receptor kinases but not protein kinase C. Mol Pharmacol 48:173-7
Freedman, N J; Liggett, S B; Drachman, D E et al. (1995) Phosphorylation and desensitization of the human beta 1-adrenergic receptor. Involvement of G protein-coupled receptor kinases and cAMP-dependent protein kinase. J Biol Chem 270:17953-61