Abstract
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 NG108-15 cells. When NG108-15 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. Importantly, UTP-induced cell migration was inhibited by CD or the Gq/11-selective inhibitor YM254890. Moreover CD and YM254890 completely inhibited Rho-A activation. Downstream of Rho-A signaling, i.e., 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-RGS which inhibits G12/13- signaling pathway. This implies that UTP-induced Rho-A activation was relatively regulated by 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 NG108-15 cells.
Footnotes
- Received February 23, 2010.
- Revision received May 26, 2010.
- Accepted May 28, 2010.
- The American Society for Pharmacology and Experimental Therapeutics