Abstract
Adenosine A2A receptors (A2AAR) 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 A2AAR in a complex with full-length Gs alpha and Gbeta4gamma2. The orthosteric binding site of A2AAR-Gs alpha:beta4gamma2 was similar to other structures of agonist-bound A2AAR, with or without Gs. Unexpectedly, the solvent accessible surface area within the interior of the complex was substantially larger for the complex with Gbeta4 versus the closest analog, A2AAR-miniGs alpha:beta1gamma2. Consequently, there are fewer interactions between the switch II in Gs alpha and the Gbeta4 torus. In reconstitution experiments Gbeta4gamma2 displayed a ten-fold higher efficiency over Gbeta1gamma2 in catalyzing A2AAR dependent GTPgammaS binding to Gs alpha. We propose that the less constrained switch II in A2AAR-Gs alpha:beta4gamma2 accounts for this increased efficiency. These results suggest that Gbeta4 functions as a positive allosteric enhancer versus Gbeta1.