Abstract

The β₂-adrenoceptor (β₂AR) couples to the G-protein G_s to activate adenylyl cyclase. Intriguingly, several studies have demonstrated that the β₂AR can also interact with G-proteins of the G_i- and G_q-family. To assess the efficiency of β₂AR interaction with various G-protein α-subunits (G_xα), we expressed fusion proteins of the β₂AR with the long (G_{sα L}) and short (G_{sα S}) splice variants of G_sα, the G_i-proteins G_{iα 2} and G_{iα 3}, and the G_q-proteins G_qα and G_16α in Sf9 cells. Fusion proteins provide a rigorous approach for comparing the coupling of a given receptor to G_xα because of the defined 1:1 stoichiometry of receptor and G-protein and the efficient coupling. Here, we show that the β₂AR couples to G_s-, G_i-, and G_q-proteins as assessed by ternary complex formation and ligand-regulated guanosine 5′-O-(3-thiotriphosphate) (GTPγS) binding. The combined analysis of ternary complex formation, GTPγS binding, agonist efficacies, and agonist potencies revealed substantial differences in the interaction of the β₂AR with the various classes of G-proteins. Comparison of the coupling of the β₂AR and formyl peptide receptor to G_{iα 2} revealed receptor-specific differences in the kinetics of GTPγS binding. We also detected highly efficient stimulation of GTPγS dissociation from G_{sα L}, but not from G_qα and G_16α, by a β₂AR 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 β₂AR couples differentially to G_s-, G_i-, and G_q-proteins, 2) that there is ligand-specific coupling of the β₂AR 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.