TY - JOUR T1 - P2Y<sub>2</sub> Receptor-G<sub>q/11</sub> Signaling at Lipid Rafts Is Required for UTP-Induced Cell Migration in NG 108-15 Cells JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 809 LP - 819 DO - 10.1124/jpet.110.167528 VL - 334 IS - 3 AU - Koji Ando AU - Yutaro Obara AU - Jun Sugama AU - Atsushi Kotani AU - Nobuyuki Koike AU - Satoko Ohkubo AU - Norimichi Nakahata Y1 - 2010/09/01 UR - http://jpet.aspetjournals.org/content/334/3/809.abstract N2 - Lipid rafts, formed by sphingolipids and cholesterol within the membrane bilayer, are believed to have a critical role in signal transduction. P2Y2 receptors are known to couple with Gq family G proteins, causing the activation of phospholipase C (PLC) and an increase in intracellular Ca2+ ([Ca2+]i) levels. In the present study, we investigated the involvement of lipid rafts in P2Y2 receptor-mediated signaling and cell migration in NG 108-15 cells. When NG 108-15 cell lysates were fractionated by sucrose density gradient centrifugation, Gαq/11 and a part of P2Y2 receptors were distributed in a fraction where the lipid raft markers, cholesterol, flotillin-1, and ganglioside GM1 were abundant. Methyl-β-cyclodextrin (CD) disrupted not only lipid raft markers but also Gαq/11 and P2Y2 receptors in this fraction. In the presence of CD, P2Y2 receptor-mediated phosphoinositide hydrolysis and [Ca2+]i elevation were inhibited. It is noteworthy that UTP-induced cell migration was inhibited by CD or the Gq/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 G12/13 signaling pathway. This implies that UTP-induced Rho-A activation was relatively regulated by the Gq/11 signaling pathway. These results suggest that lipid rafts are critical for P2Y2 receptor-mediated Gq/11–PLC–Ca2+ signaling and this cascade is important for cell migration in NG 108-15 cells. ER -