G-protein βγ-subunits contribute to the coupling specificity of the β₂-adrenergic receptor to G_s

Abstract.

Receptors and heterotrimeric G-proteins interact with a high degree of specificity, the molecular basis of which is only partially understood. In the present study, we analyzed the influence of different G-protein βγ-subunits on the coupling of the β₂-adrenergic receptor to G_s. Sf9-cells were infected with baculoviruses coding for the β₂-adrenergic receptor, α_s,Short or α_s,Long, and various β- and γ-subunits. The ability of different β- and γ-subunits to correctly dimerize was assessed by limited proteolysis of proteins expressed in Sf9-cells and additionally by analysis of β/γ-interaction in the yeast two-hybrid system. Agonist-induced GTPγS-binding to α_s,Shortβ₁γ-trimers was significantly higher than to α_s,Shortβ₂γ-combinations, when γ₄, γ₅, or γ₇ were co-expressed. Because β₅ did not support coupling of the β₂-adrenergic receptor to G_s, the 87 C-terminal amino acids of Gβ₅ assumed to encompass the β-subunit interface with the receptor were substituted by the corresponding sequence of β₁. Whereas this β₅/β₁-chimera did not promote GTPγS-binding to α_s, histamine H₁-receptor-dependent GTPγS-binding to α_q was supported by this chimeric β-subunit and by wild-type β₅. Our findings argue that the βγ-subunit composition contributes directly to the specificity of β₂-adrenergic receptor-mediated G_s-activation.