Abstract
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 α subunits of G(12) and G(13). Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by Gα(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 Gα(13) in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of Gα(13) docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the α3b helix of DH reduces binding to activated Gα(13) and ablates the stimulation of p115 by Gα(13). Complementary mutations at the predicted DH-binding site in the αB-αC loop of the helical domain of Gα(13) also affect stimulation of p115 by Gα(13). Although the GAP activity of p115 is not required for stimulation by Gα(13), two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of Gα(13) to the RH domain facilitates direct association of Gα(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.
Highlights
regulator of G protein signaling (RGS)-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G12 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 possess RGS homology (RH) domains that interact with activated ␣ subunits of G12 and G13
RGS Box in p115-RhoGEF Are Critical for Stimulation of GEF Activity by G␣13—The GEF activity of RGS-RhoGEFs can be stimulated by activated G12 class G␣ subunits in vivo, and some of the RGS-RhoGEFs can be stimulated in vitro by activated G␣13 [1]
Summary
RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G12 class of heterotrimeric G proteins and the monomeric GTPases. The RH domains of p115 and LARG function as GTPase-activating proteins (GAPs) for G␣13 and G␣12 subunits, and binding of G␣ subunits to their respective RhoGEFs stimulates their guanine nucleotide exchange activity toward RhoA (6 –9). In addition to the RH domain, PRG and LARG contain an N-terminal PDZ domain that has been shown to mediate interaction of the RGS-RhoGEFs with regulatory proteins (10 –12).
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