Molecular analysis of β2-adrenoceptor coupling to Gs-, Gi-, and Gq-proteins

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(x alpha)), we expressed fusion proteins of the beta(2)AR with the long (G(s alpha L)) and short (G(s alpha S)) splice variants of Gs alpha, the Gi-proteins G(i alpha 2) and G(i alpha 3), and the G(q)-proteins G(q alpha) and G(16 alpha) in Sf9 cells. Fusion proteins provide a rigorous approach for comparing the coupling of a given receptor to G(x alpha) 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) (GTP gamma S) binding. The combined analysis of ternary complex formation, GTP gamma S 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(i alpha 2) revealed receptor-specific differences in the kinetics of GTP gamma S binding. We also detected highly efficient stimulation of GTP gamma S dissociation from G(s alpha L), but not from G(q alpha) and G(16 alpha),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.