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CELLULAR AND MOLECULAR

Distinct Interactions of Human β₁- and β₂-Adrenoceptors with Isoproterenol, Epinephrine, Norepinephrine, and Dopamine

Department of Pharmacology and Toxicology, School of Pharmacy, University of Regensburg, Regensburg, Germany

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 G_s (G_sS and G_sL), 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-G_s and β₂AR-G_s 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-G_sL, 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-G_sL, 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 K_i 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-G_sL versus βAR-G_sS fusion proteins. In conclusion, our data with βAR-G_s fusion proteins show that endogenous catecholamines and ISO stabilize distinct conformations in the β₁AR and β₂AR.

Address correspondence to: Dr. Roland Seifert, Institute of Pharmacology, Medical School of Hannover, Carl-Neuberg-str. 1, D-30625 Hannover, Germany. E-mail: Seifert.roland{at}mh-hannover.de

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