Analysis of the structural mechanism of Rho guanine nucleotide exchange factor activation by G alpha 1

Heterotrimeric G proteins of the G12 subfamily regulate the Rho GTPase through Rho guanine nucleotide exchange factors that contain an amino-terminal regulator of G protein signalling (RGS) domain (RGS-RhoGEFs). It has been shown previously that Galpha13 directly stimulates the activity of RGS-RhoGEFs p115RhoGEF or LARG. The structural basis of the mechanism by which Galpha13 interacts with RGS-RhoGEFs and stimulates their RhoGEF activity was examined in these studies. Based on our results, it is likely that Galpha13 utilizes structural surfaces for RGS interaction and effector activation similar to other alpha subunits.;We found that lysine 204 within the Switch I region of Galpha 13 is important for its interaction with RGS-RhoGEFs p115RhoGEF or LARG and is critically involved in the regulation of their activity. By mutating lysine 204 of Galpha13 to alanine (Galpha13 KA), we disrupted not only its GDP-AlF4--dependent RGS interaction but also its stimulation of RhoGEF effector activity. This suggests that the interaction of Galpha13 with the RGS domain is required for RGS-RhoGEF effector activation.;Using chimeric subunits of Galpha12 and Galpha13 , we evaluated specific regions of Galpha13 for its activation of RhoA. We determined that the carboxyl-terminal region of Galpha 13 (residues 264--377) is important for activation of RhoA in HeLa cells. When the helical domain and switch regions of Galpha12 were substituted for those of Galpha13, the resulting Galpha 12/13 chimera activated RhoA to a similar extent as Galpha13 . The findings that the switch regions of Galpha12 could substitute for those of Galpha13 in terms of RhoA activation indicates that Galpha12 and Galpha13 may interact similarly with the RGS-RhoGEFs within their switch regions.;We established a novel expression and purification procedure for Galpha 13 and Galpha12 which yields milligrams of functional recombinant protein suitable for structural analysis. Substitution of the amino-terminus of Galphai1 for the corresponding region of Galpha13 or Galpha12 generated Galphai/13 or Galpha i/12 chimeric proteins which exhibit similar function to their wild-type counterparts. Crystal structures of the deactivated, GDP-bound form of Galpha i/13 and GDP-AlF4--activated Galpha i/12 demonstrated that Galpha13 and Galpha12 adopt similar three-dimensional structures to other a subunits, however they also possess some distinct differences.