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

Abstract

Fluorescence studies with purified human beta(2)-adrenoceptor (beta(2)AR) 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 beta(1)-adrenoceptor (beta(1)AR) and beta(2)AR with the short and long splice variants of G(s)alpha (G(s)alpha(S) and G(s)alpha(L)), respectively. Fusion proteins ensure efficient receptor/G-protein coupling and defined stoichiometry of the coupling partners. EPI, NE, DOP, and the prototypical synthetic betaAR agonist, (-)-isoproterenol (ISO), showed marked differences in their efficacies at stabilizing the high-affinity ternary complex at beta(1)AR-G(s)alpha and beta(2)AR-G(s)alpha fusion proteins. Ternary complex formation was more sensitive to disruption by GTP with the beta(2)AR than with the beta(1)AR. Generally, in steady-state GTPase assays, ISO, EPI, and NE were full agonists, and DOP was a partial agonist. Exceptionally, at beta(1)AR-G(s)alpha(L), 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 beta(2)AR-G(s)alpha(L), 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 EC(50) values for GTPase activation. In contrast to synthetic partial agonists, DOP did not exhibit increased efficacy at betaAR-G(s)alpha(L) versus betaAR-G(s)alpha(S) fusion proteins. In conclusion, our data with betaAR-G(s)alpha fusion proteins show that endogenous catecholamines and ISO stabilize distinct conformations in the beta(1)AR and beta(2)AR.