P2Y2 receptor-Gq/11 signaling at lipid rafts is required for UTP-induced cell migration in NG 108-15 cells

TitleP2Y2 receptor-Gq/11 signaling at lipid rafts is required for UTP-induced cell migration in NG 108-15 cells
Publication TypeJournal Article
Year of PublicationSubmitted
AuthorsAndo, K, Obara Y, Sugama J, Kotani A, Koike N, Ohkubo S, Nakahata N
JournalJ Pharmacol Exp Ther
Volume334
Pagination809-19
Date PublishedSep 1
ISBN Number1521-0103 (Electronic)0022-3565 (Linking)
Accession Number20511347
KeywordsActin Depolymerizing Factors/metabolism, Blotting, Western, Cell Line, Cell Movement/*drug effects, Cholesterol/metabolism, Coloring Agents, GTP-Binding Protein alpha Subunits, Gq-G11/antagonists &, Humans, inhibitors/*physiology, L-Lactate Dehydrogenase/metabolism, Membrane Microdomains/drug effects/*physiology, Microfilaments/physiology, Peptides, Cyclic/pharmacology, Phosphatidylinositols/metabolism, Phosphorylation, Receptors, Purinergic P2/*physiology, Receptors, Purinergic P2Y2, rho GTP-Binding Proteins/metabolism, Signal Transduction/drug effects/physiology, Tetrazolium Salts, Thiazoles, Uridine Triphosphate/*pharmacology
Abstract

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-metho xyethyl]-4,9,10,12,16, 22-hexamethyl-15-methylene-2,5,8,11,14,17,-20-heptaoxo-1,19-dioxa-4,7,10,1 3,16-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.
URLhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20511347