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 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.
Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA. Zhe.Chen@UTsouthwestern.edu
Zhe
Z
Chen
Liang
L
Guo
Jana
J
Hadas
Stephen
S
Gutowski
Stephen R
SR
Sprang
Paul C
PC
Sternweis
D020816:Amino Acid Motifs; D001665:Binding Sites; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D016296:Mutagenesis; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction / Q000502:physiology; D013329:Structure-Activity Relationship; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C576600:ARHGEF1 protein, human; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; C066737:RHOA protein, human; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020742:rhoA GTP-Binding Protein