Abstract
Fluorescence studies with purified human β2-adrenoceptor (β2AR) revealed that the endogenous catecholamines, (-)-epinephrine (EPI), (-)-norepinephrine (NE), and dopamine (DOP), stabilize distinct active receptor conformations. However, the functional relevance of these ligand-specific conformations is as yet poorly understood. We addressed this question by studying fusion proteins of the β1-adrenoceptor (β1AR) and β2AR with the short and long splice variants of Gsα (GsαS and GsαL), respectively. Fusion proteins ensure efficient receptor/G-protein coupling and defined stoichiometry of the coupling partners. EPI, NE, DOP, and the prototypical synthetic βAR agonist, (-)-isoproterenol (ISO), showed marked differences in their efficacies at stabilizing the high-affinity ternary complex at β1AR-Gsα and β2AR-Gsα fusion proteins. Ternary complex formation was more sensitive to disruption by GTP with the β2AR than with the β1AR. Generally, in steady-state GTPase assays, ISO, EPI, and NE were full agonists, and DOP was a partial agonist. Exceptionally, at β1AR-GsαL, NE was only a partial agonist. Generally, in adenylyl cyclase assays, ISO, EPI, and NE were full agonists, and DOP was a partial agonist. At β2AR-GsαL, NE was only a partial agonist. There was no correlation between efficacy at stabilizing the ternary complex and activating GTPase, and there were also dissociations between Ki values for high-affinity agonist binding and EC50 values for GTPase activation. In contrast to synthetic partial agonists, DOP did not exhibit increased efficacy at βAR-GsαL versus βAR-GsαS fusion proteins. In conclusion, our data with βAR-Gsα fusion proteins show that endogenous catecholamines and ISO stabilize distinct conformations in the β1AR and β2AR.
Footnotes
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This study was supported by the Research Training Program (Graduiertenkolleg) GRK760 “Medicinal Chemistry: Molecular Recognition: Ligand-Receptor Interactions” of the Deutsche Forschungsgemeinschaft.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.143412.
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ABBREVIATIONS: β1AR, β1-adrenoceptor; β2AR, human β2-adrenoceptor; βAR, nonspecified human β-adrenoceptor; Gα, nonspecified G-protein α-subunit; GPCR, G-protein-coupled receptor; EPI, (-)-epinephrine; NE, (-)-norepinephrine; DOP, dopamine; ISO, (-)-isoproterenol; GsαS, short splice variant of Gsα;GsαL, long splice variant of Gsα; AC, adenylyl cyclase; β2ARCAM, constitutively active mutant of the β2AR; DHA, dihydroalprenolol; ANOVA, analysis of variance; CHO, Chinese hamster ovary.
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↵1 Current affiliation: Institute of Pharmacology, Medical School of Hannover, Hannover, Germany.
- Received July 10, 2008.
- Accepted September 3, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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