RGS12 and RGS14 GoLoco Motifs Are Gα_i Interaction Sites with Guanine Nucleotide Dissociation Inhibitor Activity

The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein a subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single Gα _i/o-Loco (GoLoco) motif predicted to represent a second Gα interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with Gα_i1, Gα_i2, and Gα_i3 in their GDP-bound forms. In GTPγS binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by Gα_i1. Both regions also stabilize Gα_i1 in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF₄^-. Our results indicate that both RGS12 and RGS14 harbor two distinctly different Gα interaction sites: a previously recognized N-terminal RGS box possessing Gα_i/o GAP activity and a C-terminal GoLoco region exhibiting Gα_i GDI activity. The presence of two, independent Gα interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple Gα GAP activity.