Tetraspanin CD9 is a "proteolipid," and its interaction with α3 integrin in microdomain is promoted by GM3 ganglioside, leading to inhibition of laminin-5-dependent cell motility
Tetraspanin CD9 is a "proteolipid," and its interaction with α3 integrin in microdomain is promoted by GM3 ganglioside, leading to inhibition of laminin-5-dependent cell motility
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DOI：
10.1074/jbc.m200771200
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发表时间：
2002-09-13
影响因子：
4.8
通讯作者：
Hakomori, S
中科院分区：
文献类型：
--
作者：
Kawakami, Y;Kawakami, K;Hakomori, S
GM 3神经节苷脂抑制各种细胞系中四跨膜蛋白CD 9促进的细胞运动性（Ono，M.，汉达，K.，Sonnino，S.，Withers，D.一、永井，H.，和Hakomori，S.（2001）Biochemistry 40，6414 -6421）。现报告如下。(i)CD 9具有可溶于氯仿/甲醇的新特性，可归类为“蛋白脂质”;（ii）CD 9和α（3）整联蛋白一起集中在ldlD/CD 9细胞的低密度糖脂富集微区（GEM）中，和α 3表达率（在+Gal条件下生长的细胞的值除以在-Gal条件下生长的细胞的值）在ldlD/CD 9细胞的GEM中高于对照ldlD/moc细胞，提示CD 9在+Gal条件下募集GEM中的α（3），其中存在GM 3。(iii)在+Gal与-Gal条件下生长的细胞的总提取物或膜组分中α（3）和CD 9的化学水平几乎相同，而在流式细胞术中通过抗体结合探测的细胞表面表达的α（3）在-Gal条件下高于+Gal条件。这些结果表明，在+Gal条件下合成的GM 3促进α（3）与CD 9的相互作用，这限制了α（3）与其抗体的结合。由GM 3促进的α（3）/CD 9相互作用的概念进一步得到了在+Gal条件下而不是-Gal条件下CD 9和α（3）的免疫共沉淀的支持，（ii）当GM 3在-Gal条件下外源地加入细胞时，CD 9和α（3）的免疫共沉淀增强，和（iii）在荧光激光扫描共聚焦显微镜中CD 9与α（3）和GM 3与CD 9的共定位图像。基于GM 3促进α（3）/CD 9相互作用和层粘连蛋白-5作为α（3）的真正配体的状态，发现ldlD/CD 9细胞的层粘连蛋白-5/α（3）依赖性运动在-Gal条件下大大增强，但在+Gal条件下强烈抑制。对于ldlD/moc细胞，在+Gal与-Gal条件下未观察到这种运动性差异。在+Gal条件下在ldlD/CD 9细胞中观察到的抑制作用在加入抗α（3）抗体后逆转，因此基于GEM中α（3）、CD 9和GM 3之间的相互作用。
GM3 ganglioside inhibits tetraspanin CD9-facilitated cell motility in various cell lines (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40,6414-6421). We now report the following. (i) CD9 has the novel feature of being soluble in chloroform/methanol, and classifiable as "proteolipid"; (ii) CD9 and alpha(3) integrin were concentrated together in the low-density glycolipid-enriched microdomain (GEM) of ldlD/CD9 cells, and the alpha(3) expression ratio (value for cells grown under +Gal condition divided by the value for cells grown under -Gal condition) in GEM of ldlD/CD9 cells was higher than that in control ldlD/moc cells, suggesting that CD9 recruits alpha(3) in GEM under +Gal condition, whereby GM3 is present. (iii) Chemical levels of alpha(3) and CD9 in the total extract or membrane fractions from cells grown under +Gal versus -Gal condition were nearly identical, whereas alpha(3) expressed at the cell surface, probed by antibody binding in flow cytometry, was higher under -Gal than +Gal condition. These results suggest that GM3 synthesized under +Gal condition promotes interaction of alpha(3) with CD9, which restricts alpha(3) binding to its antibody. A concept of the alpha(3)/CD9 interaction promoted by GM3 was further supported by W co-immunoprecipitation of CD9 and alpha(3) under +Gal but not -Gal condition, (ii) enhanced co-immunoprecipitation of CD9 and alpha(3) when GM3 was added exogenously to cells under -Gal condition, and (iii) the co-localization images of CD9 with alpha(3) and of GM3 with CD9 in fluorescence laser scanning confocal microscopy. Based on the promotion of alpha(3)/CD9 interaction by GM3 and the status of laminin-5 as a true ligand for alpha(3), the laminin-5/alpha(3)-dependent motility of ldlD/CD9 cells was found to be greatly enhanced under -Gal condition, but strongly inhibited under +Gal condition. Such a motility difference under +Gal versus -Gal condition was not observed for ldlD/moc cells. The inhibitory effect observed in ldlD/CD9 cells under +Gal condition was reversed upon addition of anti-alpha(3) antibody and is therefore based on interaction between alpha(3), CD9, and GM3 in GEM.