Molecular analysis of beta(2)-adrenoceptor coupling to G(s)-, G(i)-, and G(q)-proteins.

Abstract

The beta(2)-adrenoceptor (beta(2)AR) couples to the G-protein G(s) to activate adenylyl cyclase. Intriguingly, several studies have demonstrated that the beta(2)AR can also interact with G-proteins of the G(i)- and G(q)-family. To assess the efficiency of beta(2)AR interaction with various G-protein alpha-subunits (G(xalpha)), we expressed fusion proteins of the beta(2)AR with the long (G(salphaL)) and short (G(salphaS)) splice variants of G(salpha), the G(i)-proteins G(ialpha2) and G(ialpha3), and the G(q)-proteins G(qalpha) and G(16alpha) in Sf9 cells. Fusion proteins provide a rigorous approach for comparing the coupling of a given receptor to G(xalpha) because of the defined 1:1 stoichiometry of receptor and G-protein and the efficient coupling. Here, we show that the beta(2)AR couples to G(s)-, G(i)-, and G(q)-proteins as assessed by ternary complex formation and ligand-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding. The combined analysis of ternary complex formation, GTPgammaS binding, agonist efficacies, and agonist potencies revealed substantial differences in the interaction of the beta(2)AR with the various classes of G-proteins. Comparison of the coupling of the beta(2)AR and formyl peptide receptor to G(ialpha2) revealed receptor-specific differences in the kinetics of GTPgammaS binding. We also detected highly efficient stimulation of GTPgammaS dissociation from G(salphaL), but not from G(qalpha) and G(16alpha), by a beta(2)AR agonist. Moreover, we show that the 1:1 stoichiometry of receptor to G-protein in fusion proteins reflects the in vivo stoichiometry of receptor/G-protein coupling more closely than was previously assumed. Collectively, our data show 1) that the beta(2)AR couples differentially to G(s)-, G(i)-, and G(q)-proteins, 2) that there is ligand-specific coupling of the beta(2)AR to G-proteins, 3) that receptor-specific G-protein conformational states may exist, and 4) that nucleotide dissociation is an important mechanism for G-protein deactivation.