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