Iwabuchi K, Zhang Y, Handa K, Withers D A, Sinaÿ P, Hakomori S
Pacific Northwest Research Institute, Seattle, Washington 98122-4327, USA.
J Biol Chem. 2000 May 19;275(20):15174-81. doi: 10.1074/jbc.275.20.15174.
GM3 ganglioside at the surface of mouse melanoma B16 cells is clustered and organized with signal transducer molecules c-Src, Rho A, and focal adhesion kinase (FAK) to form a membrane unit separable from caveolae, which are enriched in cholesterol and caveolin but do not contain GM3 or the above three signal transducers. The GM3-enriched membrane units are involved in GM3-dependent cell adhesion coupled with activation of c-Src, Rho A, and FAK and are termed the "glycosphingolipid signaling domain" or the "glycosignaling domain" (GSD). In order to assess the essential components that display GSD function, membranes with properties similar to those of GSD were reconstituted using GM3, sphingomyelin, and c-Src, with or without other lipid components. The reconstituted membrane thus prepared displayed GM3-dependent adhesion to plates coated with Gg3 or anti-GM3 antibody, resulting in enhanced c-Src phosphorylation (c-Src phosphorylation response). This response in reconstituted membrane depends on GM3 concentration and was not observed when GM3 was absent or replaced with other gangliosides GM1 or GD1a, or with LacCer. The GM3-dependent c-Src phosphorylation response was enhanced when cholesterol and phosphatidylcholine were added. Although GM3, sphingomyelin, and c-Src are essential for GSD function, a small quantity of cholesterol and phosphatidylcholine may act as an auxiliary factor to stabilize membrane. GSD function in terms of GM3-dependent adhesion and signaling was blocked in the presence of lyso-GM3 or its analogue but not psychosine, lactosyl-sphingosine, or lyso-phosphatidylcholine. Such susceptibility of reconstituted GSD to lyso-GM3 and other lyso compounds is the same as GSD of original B16 cells. Thus, functional organization of the reconstituted membrane closely simulates that of GSD in B16 cells, which is based on clustered GM3 organized with c-Src as the essential components.
小鼠黑色素瘤B16细胞表面的GM3神经节苷脂与信号转导分子c-Src、Rho A和粘着斑激酶（FAK）聚集并组织在一起，形成一个可与小窝分离的膜单元，小窝富含胆固醇和小窝蛋白，但不包含GM3或上述三种信号转导分子。富含GM3的膜单元参与依赖GM3的细胞粘附，并伴有c-Src、Rho A和FAK的激活，被称为“糖鞘脂信号结构域”或“糖信号结构域”（GSD）。为了评估发挥GSD功能的必需成分，使用GM3、鞘磷脂和c-Src，添加或不添加其他脂质成分，重建了具有与GSD相似性质的膜。由此制备的重建膜表现出对包被有Gg3或抗GM3抗体的平板的GM3依赖性粘附，导致c-Src磷酸化增强（c-Src磷酸化反应）。重建膜中的这种反应取决于GM3浓度，当GM3不存在或被其他神经节苷脂GM1或GD1a或乳糖神经酰胺取代时未观察到这种反应。当添加胆固醇和磷脂酰胆碱时，GM3依赖性c-Src磷酸化反应增强。虽然GM3、鞘磷脂和c-Src对GSD功能至关重要，但少量的胆固醇和磷脂酰胆碱可能作为辅助因子来稳定膜。在溶血GM3或其类似物存在的情况下，但在鞘氨醇、乳糖基鞘氨醇或溶血磷脂酰胆碱不存在的情况下，依赖GM3的粘附和信号传导方面的GSD功能被阻断。重建的GSD对溶血GM3和其他溶血化合物的这种敏感性与原始B16细胞的GSD相同。因此，重建膜的功能组织紧密模拟了B16细胞中GSD的功能组织，其基于以c-Src为必需成分组织的聚集GM3。
