Inhibitory regulation of Rac activation, membrane ruffling, and cell migration by the G protein-coupled sphingosine-1-phosphate receptor EDG5 but not EDG1 or EDG3.
Sphingosine-1-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 differentiation gene) family G protein-coupled receptors EDG1, EDG3, and EDG5 (AGR16). Among prominent activities of S1P is the regulation of cell motility; S1P stimulates or inhibits cell motility depending on cell types. In the present study, we provide evidence for EDG subtype-specific, contrasting regulation of cell motility and cellular Rac activity. In CHO cells expressing EDG1 or EDG3 (EDG1 cells or EDG3 cells, respectively) S1P as well as insulin-like growth factor I (IGF I) induced chemotaxis and membrane ruffling in phosphoinositide (PI) 3-kinase- and Rac-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 cells, 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 did in EDG1 cells but inhibited the basal Rac activity and totally abolished IGF I-induced Rac activation, which involved stimulation of Rac-GTPase-activating protein activity rather than inhibition of Rac-guanine nucleotide exchange activity. S1P induced comparable increases in the amounts of GTP-RhoA in EDG3 and EDG5 cells. Neither S1P nor IGF I increased the amount of GTP-bound Cdc42. However, expression of N(17)-Cdc42, but not N(19)-RhoA, suppressed S1P- and IGF I-directed chemotaxis, suggesting a requirement for basal Cdc42 activity for chemotaxis. Taken together, the present results demonstrate that EDG5 is the first example of a hitherto-unrecognized type of receptors that negatively regulate Rac activity, thereby inhibiting cell migration and membrane ruffling.
Okamoto
H
H
Department of Physiology, Kanazawa University School of Medicine, Kanazawa, Japan.
Takuwa
N
N
Yokomizo
T
T
Sugimoto
N
N
Sakurada
S
S
Shigematsu
H
H
Takuwa
Y
Y
D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002633:Chemotaxis / Q000187:drug effects*; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins; D017874:Immediate-Early Proteins / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008246:Lysophospholipids; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D022502:Stress Fibers / Q000378:metabolism; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D017874:Immediate-Early Proteins; D007334:Insulin-Like Growth Factor I; D008246:Lysophospholipids; D000072000:NF-KappaB Inhibitor alpha; D019869:Phosphatidylinositol 3-Kinases; D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D011994:Recombinant Proteins; D013110:Sphingosine; D020764:cdc42 GTP-Binding Protein; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; C060506:sphingosine 1-phosphate