Abstract

The overall goal of this proposal is the elucidation of the complex mechanisms which cause the rapid desensitization and sensitization of beta2-adrenergic receptor (beta2AR) stimulation of adenylyl cyclase (AC). The knowledge gained will help in the understanding of diseases such as asthma and hypertension in which these mechanisms may be altered, and in the rationale for pharmacological intervention. This proposal has three specific aims which are focused on determining the role of phosphorylation/dephosphorylation in the regulation of the responsivity of AC to beta2AR stimulation.
The first aim i s to determine the precise domains of the beta2AR involved in the cAMP-dependent protein kinase, the protein kinase C (PKC) and the beta-adrenergic receptor kinase pathways of desensitization. beta2AR mutants will be constructed, expressed in L cells and their regulation of AC characterized in response to various treatments which elicit desensitization. To measure the stoichiometry of phosphorylation, mutant beta2AR's will be constructed containing foreign epitopes with the intent of developing a rapid high recovery procedure for purification based on epitope binding to affinity columns.
The second aim i s to assess the hypothesis that PKC-mediated phosphorylation and inactivation of either the Gi/alpha subunits of the GTP-binding proteins, or the catalytic subunit of AC is the mechanism of the 4Beta-phorbol 12-myristate 13-acetate (PMA) and purinergic receptor- induced sensitizations of AC. This will be accomplished through the use of immunoprecipitation to measure phosphorylation of Gi/alpha subunits and AC catalytic subunits, construction of mutants of these proteins to aid in purification and analysis of domains involved, and reconstitution studies to determine if Gi function can be restored. PKC isozyme involvement in the PMA and purinergic sensitizations will be identified by measurement of translocation by immunoprecipitations and phorbol dibutyrate binding.
The third aim i s to identify phosphatases involved in the desensitization/sensitization in intact cells through the use of phosphatase inhibitors.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031208-11
Application #
2176049
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1983-04-01
Project End
1996-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
11
Fiscal Year
1995
Total Cost
Indirect Cost

  Institution

Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225

  Publications

Gimenez, Luis E; Baameur, Faiza; Vayttaden, Sharat J et al. (2015) Salmeterol Efficacy and Bias in the Activation and Kinase-Mediated Desensitization of ?2-Adrenergic Receptors. Mol Pharmacol 87:954-64
Baameur, Faiza; Hammitt, Richard A; Friedman, Jacqueline et al. (2014) Biochemical and Cellular Specificity of Peptide Inhibitors of G Protein-Coupled Receptor Kinases. Int J Pept Res Ther 20:1-12
Baameur, Faiza; Morgan, Daniel H; Yao, Hui et al. (2010) Role for the regulator of G-protein signaling homology domain of G protein-coupled receptor kinases 5 and 6 in beta 2-adrenergic receptor and rhodopsin phosphorylation. Mol Pharmacol 77:405-15
Vayttaden, Sharat J; Friedman, Jacqueline; Tran, Tuan M et al. (2010) Quantitative modeling of GRK-mediated beta2AR regulation. PLoS Comput Biol 6:e1000647
Liang, Wei; Hoang, Quang; Clark, Richard B et al. (2008) Accelerated dephosphorylation of the beta2-adrenergic receptor by mutation of the C-terminal lysines: effects on ubiquitination, intracellular trafficking, and degradation. Biochemistry 47:11750-62
Xin, Wenkuan; Tran, Tuan M; Richter, Wito et al. (2008) Roles of GRK and PDE4 activities in the regulation of beta2 adrenergic signaling. J Gen Physiol 131:349-64
Tran, Tuan M; Friedman, Jacqueline; Baameur, Faiza et al. (2007) Characterization of beta2-adrenergic receptor dephosphorylation: Comparison with the rate of resensitization. Mol Pharmacol 71:47-60
Tran, Tuan M; Jorgensen, Rasmus; Clark, Richard B (2007) Phosphorylation of the beta2-adrenergic receptor in plasma membranes by intrinsic GRK5. Biochemistry 46:14438-49
Moore, Robert H; Millman, Ellen E; Godines, Veronica et al. (2007) Salmeterol stimulation dissociates beta2-adrenergic receptor phosphorylation and internalization. Am J Respir Cell Mol Biol 36:254-61
Vaughan, David J; Millman, Ellen E; Godines, Veronica et al. (2006) Role of the G protein-coupled receptor kinase site serine cluster in beta2-adrenergic receptor internalization, desensitization, and beta-arrestin translocation. J Biol Chem 281:7684-92

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