G12/13 and Gq mediate S1P2-induced inhibition of Rac and migration in vascular smooth muscle in a manner dependent on Rho but not Rho kinase

Aims The lysophospholipid mediator sphingosine-1-phosphate (S1P) activates G protein-coupled receptors (GPCRs) to induce potent inhibition of platelet-derived growth factor (PDGF)-induced Rac activation and, thereby, chemotaxis in rat vascular smooth muscle cells (VSMCs). We explored the heterotrimeric G protein and the downstream mechanism that mediated SIP inhibition of Rac and cell migration in VSMCs. Methods and results SIP inhibition of PDGF-induced cell migration and Rac activation in VSMCs was abolished by the selective S1P(2) receptor antagonist JTE-013. The C-terminal peptides of G alpha subunits (G alpha-CTs) act as specific inhibitors of respective G protein-GPCR coupling. Adenovirus-mediated expression of G alpha(12)-CT, G alpha(13)-CT, and G alpha(q)-CT, but not that of G alpha(s)-CT or LacZ or pertussis toxin treatment, abrogated S1P inhibition of PDGF-induced Rac activation and migration, indicating that both G(12/13) and G(q) classes are necessary for the SIP inhibition. The expression of G alpha q center dot CT as well as G alpha(12)-CT and G alpha(13)-CT also abolished S1P-induced Rho stimulation. C3 toxin, but not a Rho kinase inhibitor or a dominant negative form of Rho kinase, abolished S1P inhibition of PDGF-induced Rac activation and cell migration. The angiotensin 11 receptor AT,, which robustly couples to Gq, did not mediate either Rho activation or inhibition of PDGF-induced Rac activation or migration, suggesting that activation of Gq atone was not sufficient for Rho activation and resultant Rac inhibition. However, the AT(1) receptor fused to G alpha(12) was able to induce not only Rho stimulation but also inhibition of PDGF-induced Rac activation and migration. Phospholipase C inhibition did not affect S1P-induced Rho activation, and protein kinase C activation by a phorbol, ester did not mimic S1P action, suggesting that S1P inhibition of migration or Rac was not dependent on the phospholipase C pathway. Conclusion These observations together suggest that S1P(2) mediates inhibition of Rac and migration through the coordinated action of G(12/13) and G(q) for Rho activation in VSMCs.