Inhibitory and Stimulatory Regulation of Rac and Cell Motility by the G_12/13-Rho and G_i Pathways Integrated Downstream of a Single G Protein-Coupled Sphingosine-1-Phosphate Receptor Isoform

Abstract

The G protein-coupled receptors S1P₂/Edg5 and S1P₃/Edg3 both mediate sphingosine-1-phosphate (S1P) stimulation of Rho, yet S1P₂ but not S1P₃ mediates downregulation of Rac activation, membrane ruffling, and cell migration in response to chemoattractants. Specific inhibition of endogenous Gα₁₂ and Gα₁₃, but not of Gα_q, by expression of respective C-terminal peptides abolished S1P₂-mediated inhibition of Rac, membrane ruffling, and migration, as well as stimulation of Rho and stress fiber formation. Fusion receptors comprising S1P₂ and either Gα₁₂ or Gα₁₃, but not Gα_q, mediated S1P stimulation of Rho and also inhibition of Rac and migration. Overexpression of Gα_i, by contrast, specifically antagonized S1P₂-mediated inhibition of Rac and migration. The S1P₂ actions were mimicked by expression of V¹⁴Rho and were abolished by C3 toxin and N¹⁹Rho, but not Rho kinase inhibitors. In contrast to S1P₂, S1P₃ mediated S1P-directed, pertussis toxin-sensitive chemotaxis and Rac activation despite concurrent stimulation of Rho via G_12/13. Upon inactivation of G_i by pertussis toxin, S1P₃ mediated inhibition of Rac and migration just like S1P₂. These results indicate that integration of counteracting signals from the G_i- and the G_12/13-Rho pathways directs either positive or negative regulation of Rac, and thus cell migration, upon activation of a single S1P receptor isoform.

ACKNOWLEDGMENTS

This work was supported by grants from the Ministry of Education, Science and Culture of Japan, the Japan Society for the Promotion of Science Research for the Future Program, the Hoh-Ansha Foundation, and the Honjin Foundation.

We thank Nobuko Yamaguchi and Yasuhiro Hiratsuka for preparing the manuscript and for technical assistance, respectively.