Sphingosine 1-phosphate regulates melanoma cell motility through a receptor-coupled extracellular action and in a pertussis toxin-insensitive manner.
Sphingosine 1-phosphate regulates melanoma cell motility through a receptor-coupled extracellular action and in a pertussis toxin-insensitive manner.
复制标题
1-磷酸鞘氨醇通过受体偶联的细胞外作用并以百日咳毒素不敏感的方式调节黑色素瘤细胞的运动。
DOI：
10.1021/bi970926s
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Igarashi,Y
中科院分区：
文献类型：
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作者：
Yamamura,S;Yatomi,Y;Ruan,F;Sweeney,EA;Hakomori,S;Igarashi,Y
我们以前的工作表明，1-磷酸鞘氨醇（Sph-1-P）在10 - 100 nM浓度下抑制小鼠黑色素瘤B16/F10和其他类型细胞的细胞运动。在本文中，我们已经确定和特点的特定细胞表面结合位点的Sph-I-P在F10细胞。固定在可控孔玻璃珠上的Sph-1-P抑制了F10细胞的运动，表明Sph-1-P从外部作用于细胞。[~ 3 H]Sph-1-P的结合实验表明，F10细胞中存在Sph-1-P的特异性细胞表面结合位点。Scatchard分析表明，一个单一的类结合位点的Sph-1-P。[3 H]Sph-1-P的F10细胞的结合被抑制，通过添加过量的未标记的Sph-1-P，但不是其他天然鞘脂。特异性结合也对用蛋白酶处理敏感。使用Sph-1-P固定化亲和层析，我们，第一次，确定41-kDa和79-kDa的Sph-1-P结合蛋白的黑色素瘤细胞表面上，虽然41-kDa的蛋白是不太具体的Sph-1-P。我们证明，百日咳毒素（PTX）治疗没有取消运动抑制Sph-1-P，这表明没有PTX敏感的G-蛋白参与的信号。此外，发现Sph-1-P从小鼠BALB/3 T3克隆A31细胞和F10细胞中特异性释放。总的来说，这些结果强烈表明，Sph-I-P通过特异性结合细胞表面受体蛋白的细胞外作用来调节黑素瘤细胞运动性，这独立于PTX敏感性G蛋白。
Our previous work showed that sphingosine 1-phosphate (Sph-1-P) inhibits the cell motility of mouse melanoma B16/F10, and other types of cells at 10−100 nM concentrations. In the present paper, we have identified and characterized specific cell surface binding sites for Sph-1-P in F10 cells. Sph-1-P immobilized on controlled pore glass beads inhibited the motility of F10 cells, suggesting that Sph-1-P acts on the cells from the outside. Binding assays with [3H]Sph-1-P revealed the presence of specific cell surface binding sites for Sph-1-P in F10 cells. Scatchard analysis demonstrated a single class of binding sites for Sph-1-P. The binding of [3H]Sph-1-P to F10 cells was inhibited by the addition of excess unlabeled Sph-1-P but not other natural sphingolipids. The specific binding was also sensitive to treatment with a protease. Using Sph-1-P-immobilized affinity chromatography, we, for the first time, identified 41-kDa and 79-kDa Sph-1-P binding proteins on the melanoma cell surface, although the 41-kDa protein was less specific to Sph-1-P. We demonstrated that pertussis toxin (PTX) treatment did not abolish the motility inhibition by Sph-1-P, suggesting that no PTX-sensitive G-protein is involved in the signaling. Furthermore, Sph-1-P was found to be specifically released from mouse BALB/3T3 clone A31 cells and F10 cells. Collectively, these results strongly suggest that Sph-1-P regulates melanoma cell motility through an extracellular action by specific binding to cell surface receptor protein(s), which is independent of PTX-sensitive G-protein.