Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
J Pharmacol Exp Ther. 2010 Sep 1;334(3):809-19. doi: 10.1124/jpet.110.167528. Epub 2010 May 28.
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+)) 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+) 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.
脂质筏是由膜双层中的鞘脂和胆固醇形成的，被认为在信号转导中具有关键作用。已知 P2Y(2)受体与 G(q)家族 G 蛋白偶联，导致磷脂酶 C (PLC)的激活和细胞内 Ca(2+)(Ca(2+))水平的增加。在本研究中，我们研究了脂质筏在 NG 108-15 细胞中 P2Y(2)受体介导的信号转导和细胞迁移中的作用。当 NG 108-15 细胞裂解物通过蔗糖密度梯度离心分离时，Galpha(q/11)和一部分 P2Y(2)受体分布在富含脂质筏标记物胆固醇、 flotillin-1 和神经节苷脂 GM1 的部分。甲基-β-环糊精 (CD)不仅破坏了脂质筏标记物，还破坏了该部分的 Galpha(q/11)和 P2Y(2)受体。在 CD 存在的情况下，P2Y(2)受体介导的磷酸肌醇水解和 Ca(2+)升高被抑制。值得注意的是，CD 或 G(q/11)-选择性抑制剂 YM254890[(1R)-1-{(3S,6S,9S,12S,18R,21S,22R)-21-乙酰氨基-18-苄基-3-[(1R)-1-甲氧基乙基]-4,9,10,12,16,22-十六甲基-15-亚甲基-2,5,8,11,14,17,20-十七氧代-1,19-二氧杂-4,7,10,13,16-五氮杂环二十二烷-6-基}-2-甲基丙基 rel-(2S,3R)-2-乙酰氨基-3-羟基-4-甲基戊酸酯]抑制 UTP 诱导的细胞迁移。此外，CD 和 YM254890 完全抑制了 Rho-A 的激活。Rho-A 信号转导的下游，应激纤维的形成和 cofilin 的磷酸化也被 CD 或 YM254890 抑制。然而，CD 或 YM254890 并不影响 UTP 诱导的 cofilin 磷酸化，这表明 p115-调节 G 蛋白信号的信号转导调节剂并不影响 G(12/13)信号通路。这表明 UTP 诱导的 Rho-A 激活是由 G(q/11)信号通路相对调节的。这些结果表明，脂质筏对于 P2Y(2)受体介导的 G(q/11)-PLC-Ca(2+)信号转导至关重要，该级联反应对于 NG 108-15 细胞的迁移至关重要。
