G protein β4 as a structural determinant of enhanced nucleotide exchange in the A2AAR-Gs complex

Abstract

Adenosine A_2A receptors (A_2AAR) evoke pleiotropic intracellular signaling events via activation of the stimulatory heterotrimeric G protein, Gs. Here, we used cryoEM to solve the agonist-bound structure of A_2AAR in a complex with full-length Gs α and Gβ₄γ₂ (A_2AAR-Gs α:β₄γ₂). The orthosteric binding site of A_2AAR-Gs α:β₄γ₂ was similar to other structures of agonist-bound A_2AAR, with or without Gs. Unexpectedly, the solvent accessible surface area within the interior of the complex was substantially larger for the complex with Gβ₄ versus the closest analog, A_2AAR-miniGs α:β₁γ₂. Consequently, there are fewer interactions between the switch II in Gs α and the Gβ₄ torus. In reconstitution experiments Gβ₄γ₂ displayed a ten-fold higher efficiency over Gβ₁γ₂ in catalyzing A_2AAR dependent GTPγS binding to Gs α. We propose that the less constrained switch II in A_2AAR-Gs α:β₄γ₂ accounts for this increased efficiency. These results suggest that Gβ₄ functions as a positive allosteric enhancer versus Gβ₁.