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 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.
Footnotes
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Science, Sports, and Technology of Japan [Grant 13771369 (to S.O.) and 14370737, 18058002, 19659011, 20054002] (to N.N.); and a Grant-in-Aid from the Mitsubishi Foundation (to N.N.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.167528.
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ABBREVIATIONS:
- [Ca2+]i
- intracellular Ca2+
- CD
- methyl-β-cyclodextrin
- 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
- DMEM
- Dulbecco's modified Eagle's medium
- GPCR
- G protein-coupled receptor
- HRP
- horseradish peroxidase
- LPA
- lysophosphatidic acid
- PLC
- phospholipase C
- RGS
- regulator of G protein signaling
- Y27632
- (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide
- LDH
- lactate dehydrogenase
- BzATP
- benzoyl benzoic ATP
- tBuBHQ
- tert-butyl-hydroquinone
- Fura-2/AM
- fura-2/acetoxymethylester
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- PTX
- pertussis toxin
- PBS
- phosphate-buffered saline
- TBST
- 10 mM Tris, 100 mM NaCl, 0.1% Tween 20, pH 7.4
- ER
- endoplasmic reticulum
- SH3
- Src homology 3
- MOI
- multiplicity of infection
- GEF
- guanine nucleotide exchange factor.
- Received February 23, 2010.
- Accepted May 28, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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