Gαs-specific structural elements attenuate interactions with RGS proteins

Abstract

AbstractHeterotrimeric (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 Gαsdoes not interact with known RGS proteins. To understand the molecular basis for this divergent phenomenon, we combined a comparative structural analysis of experimental and modelled structures with functional biochemical assays. This analysis showed that Gαscontains unique structural elements in both the helical and the GTPase domains. Modeling identified helical domain insertions, missing in experimental structures, that project towards the interface with RGS proteins, and residues in the GTPase domain that might interfere with RGS binding. Mutagenesis of Gαsand measurements of RGS GAP activity showed that three residues in the GαsGTPase domain are both necessary and sufficient to prevent Gαsinactivation by RGSs. Indeed, substitution of all three Gαsresidues with the corresponding residues from Gαi1enabled efficient inactivation by RGS proteins. These results shed new light on the mechanistic bases for G protein specificity towards RGS proteins.