Gβγ signaling to the chemotactic effector P-REX1 and mammalian cell migration is directly regulated by Gαq and Gα13 proteins

G protein-coupled receptors stimulate Rho guanine nucleotide exchange factors that promote mammalian cell migration. Rac and Rho GTPases exert opposing effects on cell morphology and are stimulated downstream of G and G 12/13 or G q , respectively. These G subunits might in turn favor Rho pathways by preventing G signaling to Rac. Here, we investigated whether G signaling to phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-REX1), a key G chemotactic effector, is directly controlled by Rho-activating G subunits. We show that pharmacological inhibition of G q makes P-REX1 activation by G q /G i -coupled lysophosphatidic acid receptors more effective. Moreover, chemogenetic control of G i and G q by designer receptors exclusively activated by designer drugs (DREADDs) confirmed that G i differentially activates P-REX1. GTPase-deficient G q QL and G 13 QL variants formed stable complexes with G, impairing its interaction with P-REX1. The N-terminal regions of these variants were essential for stable interaction with G. Pulldown assays revealed that chimeric G 13-i2 QL interacts with G unlike to G i2-13 QL, the reciprocal chimera, which similarly to G i2 QL could not interact with G. Moreover, G was part of tetrameric G-G q QL-RGS2 and G-G 13-i2 QL-RGS4 complexes, whereas G 13 QL dissociated from G to interact with the PDZ-RhoGEF-RGS domain. Consistent with an integrated response, G and AKT kinase were associated with active SDF-1/ CXCL12-stimulated P-REX1. This pathway was inhibited by G q QL and G 13 QL, which also prevented CXCR4-dependent cell migration. We conclude that a coordinated mechanism prioritizes G q -and G 13 -mediated signaling to Rho over a G-dependent Rac pathway, attributed to heterotrimeric G i proteins.