P2Y2 receptor-Gq/11 signaling at lipid rafts is required for UTP-induced cell migration in NG 108-15 cells.
Lipid rafts, formed by sphingolipids and cholesterol within the membrane bilayer, are believed to have a critical role in signal transduction. P2Y(2) receptors are known to couple with G(q) family G proteins, causing the activation of phospholipase C (PLC) and an increase in intracellular Ca(2+) ([Ca(2+)](i)) levels. In the present study, we investigated the involvement of lipid rafts in P2Y(2) receptor-mediated signaling and cell migration in NG 108-15 cells. When NG 108-15 cell lysates were fractionated by sucrose density gradient centrifugation, Galpha(q/11) and a part of P2Y(2) receptors were distributed in a fraction where the lipid raft markers, cholesterol, flotillin-1, and ganglioside GM1 were abundant. Methyl-beta-cyclodextrin (CD) disrupted not only lipid raft markers but also Galpha(q/11) and P2Y(2) receptors in this fraction. In the presence of CD, P2Y(2) receptor-mediated phosphoinositide hydrolysis and [Ca(2+)](i) elevation were inhibited. It is noteworthy that UTP-induced cell migration was inhibited by CD or the G(q/11)-selective inhibitor YM254890 [(1R)-1-{(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16, 22-hexamethyl-15-methylene-2,5,8,11,14,17,-20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl}-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. Moreover CD and YM254890 completely inhibited Rho-A activation. Downstream of Rho-A signaling, stress fiber formation and phosphorylation of cofilin were also inhibited by CD or YM254890. However, UTP-induced phosphorylation of cofilin was not affected by the expression of p115-regulator of G protein signaling, which inhibits the G(12/13) signaling pathway. This implies that UTP-induced Rho-A activation was relatively regulated by the G(q/11) signaling pathway. These results suggest that lipid rafts are critical for P2Y(2) receptor-mediated G(q/11)-PLC-Ca(2+) signaling and this cascade is important for cell migration in NG 108-15 cells.
Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Koji
K
Ando
Yutaro
Y
Obara
Jun
J
Sugama
Atsushi
A
Kotani
Nobuyuki
N
Koike
Satoko
S
Ohkubo
Norimichi
N
Nakahata
D008841:Actin Cytoskeleton / Q000502:physiology; D051339:Actin Depolymerizing Factors / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D002784:Cholesterol / Q000378:metabolism; D004396:Coloring Agents; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000037:antagonists & inhibitors / Q000502:physiology*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D021962:Membrane Microdomains / Q000187:drug effects / Q000502:physiology*; D010456:Peptides, Cyclic / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058473:Receptors, Purinergic P2Y2; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013778:Tetrazolium Salts; D013844:Thiazoles; D014544:Uridine Triphosphate / Q000494:pharmacology*; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D051339:Actin Depolymerizing Factors; D004396:Coloring Agents; C546385:P2RY2 protein, human; D010456:Peptides, Cyclic; D010716:Phosphatidylinositols; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D013778:Tetrazolium Salts; D013844:Thiazoles; C475455:YM-254890; D002784:Cholesterol; D007770:L-Lactate Dehydrogenase; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020741:rho GTP-Binding Proteins; C022616:thiazolyl blue; D014544:Uridine Triphosphate