Inhibitory regulation of Rac activation, membrane ruffling, and cell migration by the G protein-coupled sphingosine-1-phosphate receptor EDG5 but notEDG1 or EDG3

Sphingosine-l-phosphate (S1P) is a bioactive lysophospholipid that induces a variety of biological responses in diverse cell types. Many, if not all, of these responses are mediated by members of the EDG (endothelial differen tiation gene) family G protein-coupled receptors EDG1, EDG3, and EDG5 (AGR1 6). Among prominent activities of S1P is the regulation of cell motility; S 1P stimulates or inhibits cell motility depending on cell types. In the pre sent study, we provide evidence for EDG subtype-specific, contrasting regul ation of cell motility and cellular Rac activity. In CHO cells expressing E DG1 or EDG3 (EDG1 cells or EDG3 cells, respectively) S1P as well as insulin -like growth factor I (IGF T) induced chemotaxis and membrane ruffling in p hosphoinositide (PI) 3-kinase- and Pac-dependent manners. Both S1P and IGF I induced a biphasic increase in the amount of the GTP-bound active form of Rac. In CHO cells expressing EDG5 (EDG5 cells), IGF I similarly stimulated cell migration; however, in contrast to what was found for EDG1 and EDG3 c ells, S1P did not stimulate migration but totally abolished IGF I-directed chemotaxis and membrane ruffling, in a manner dependent on a concentration gradient of S1P. In EDG5 cells, S1P stimulated PI 3-kinase activity as it d id in EDG1 cells but inhibited the basal Pac activity and totally abolished IGF I-induced Rac activation, which involved stimulation of Rac-GTPase-act ivating protein activity rather than inhibition of Rac-guanine nucleotide e xchange activity. S1P induced comparable increases in the amounts of GRP-Rh oA in EDG3 and EDG5 cells. Neither S1P nor IGF I increased the amount of GT P-bound Cdc42. However, expression of N-17-Cdc42, but not N-19-RhoA, suppre ssed S1P- and ICF I-directed chemotaxis, suggesting a requirement for basal Cdc42 activity for chemotaxis. Taken together, the present results demonst rate that EDG5 is the first example of a hitherto-unrecognized type of rece ptors that negatively regulate Pac activity, thereby inhibiting cell migrat ion and membrane ruffling.