Abstract
Heterotrimeric (αβγ) G proteins are molecular switches that are activated by G protein-coupled receptors (GPCRs) and regulate numerous intracellular signaling cascades. Most active Gα subunits are inactivated by regulators of G protein signaling (RGS) proteins, which determine the duration of G protein-mediated signaling by accelerating the catalytic turn-off of the Gα subunit. However, the G protein Gα(s) does not interact with known RGS proteins. To understand the molecular basis for this divergent phenomenon, we combined a comparative structural analysis of experimental and modeled structures with functional biochemical assays. This analysis showed that Gα(s) contains unique structural elements in both the helical and the GTPase domains. Modeling suggested that helical domain insertions, which were missing in experimental structures, might project toward the interface with RGS proteins. Alternatively, residues in the Gα(s) GTPase domain might lead to direct interference with RGS binding. Mutagenesis of Gα(s) and measurements of RGS GTPase-activating protein (GAP) activity showed that three residues in the Gα(s) GTPase domain are both necessary and sufficient to prevent Gα(s) inactivation by RGSs. Indeed, substitution of all three Gα(s) residues with the corresponding residues from Gα(i1) enabled efficient inactivation by RGS proteins. These results shed new light on the mechanistic bases for G protein specificity toward RGS proteins.