Activation of p115-RhoGEF Requires Direct Association of Gα13 and the Dbl Homology Domain

RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G(12) class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated alpha subunits of G(12) and G(13). Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by G alpha(13), the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, we identify an additional binding site for activated G alpha(13) in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of G alpha(13) docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the alpha 3b helix of DH reduces binding to activated G alpha(13) and ablates the stimulation of p115 by G alpha(13). Complementary mutations at the predicted DH-binding site in the alpha B-alpha C loop of the helical domain of G alpha(13) also affect stimulation of p115 by G alpha(13). Although the GAP activity of p115 is not required for stimulation by G alpha(13), two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of G alpha(13) to the RH domain facilitates direct association of G alpha(13) to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling.