G_S activation is time-limiting in initiating receptor-mediated signaling

To analyze individual steps of G_S-linked signaling in intact cells, we used fluorescence resonance energy transfer (FRET)-based assays for receptor-G protein interaction, G protein activation, and cAMP effector activation. To do so, we developed a FRET-based sensor to directly monitor G_S activation in living cells. This was done by coexpressing a Gα_s mutant, in which a yellow fluorescent protein was inserted, together with cyan fluorescent protein-tagged Gβγ subunits and appropriate receptors in HEK293 cells. Together with assays for receptor activation and receptor-G protein interaction, it is possible to characterize large parts of the G_S signaling cascade. When A_2A- adenosine or β₁-adrenergic receptors are coexpressed with G _S in HEK293T cells, the receptor-G_S interaction was on the same time scale as A_2A receptor activation with a time constant of <50 ms. G_S activation was markedly slower and around 450 ms with similar kinetics following activation of A_2A- or β₁- receptors. Taken together, our kinetic measurements demonstrate that the rate of G_S activation limits initiation of G_S-coupled receptor signaling.