Basigin (CD147) Is the Target for Organomercurial Inhibition of Monocarboxylate Transporter Isoforms 1 and 4
Basigin (CD147) Is the Target for Organomercurial Inhibition of Monocarboxylate Transporter Isoforms 1 and 4
复制标题
DOI：
10.1074/jbc.m411950200
复制
发表时间：
2005-07
影响因子：
4.8
通讯作者：
Marieangela C Wilson;D. Meredith;Jocelyn E. Manning Fox;C. Manoharan;A. Davies;A. Halestrap
中科院分区：
文献类型：
--
作者：
Marieangela C Wilson;D. Meredith;Jocelyn E. Manning Fox;C. Manoharan;A. Davies;A. Halestrap
单羧酸转运蛋白 MCT1 和 MCT4 易位至质膜需要 CD147（basigin），它们与 CD147 保持紧密相关。然而，CD147 对于 MCT 活性的重要性尚不清楚。 MCT1 和 MCT4 均受到细胞不可渗透的有机汞试剂对氯汞苯磺酸盐 (pCMBS) 的抑制。在这里，我们通过定点诱变证明，去除 MCT4 上所有可接近的半胱氨酸残基并不能阻止这种抑制。 pCMBS 处理细胞消除了 MCT1 和 MCT4 与 CD147 的免疫共沉淀，并用生物素化硫醇试剂增强了 CD147 的标记。这表明CD147可能是pCMBS的靶标，并且通过用双功能有机汞试剂荧光素二汞乙酸酯处理细胞导致CD147寡聚化获得了进一步的证据。 CD147 的定点诱变表明 CD147 的 Ig 样 C2 结构域中的二硫键是 pCMBS 攻击的目标。 MCT2 对 pCMBS 不敏感，被发现与 gp70 而不是 CD147 发生共免疫沉淀。 gp70 和 MCT2 之间的相互作用通过青色荧光蛋白和黄色荧光蛋白标记的 MCT2 和 gp70 之间的荧光共振能量转移得到证实。 pCMBS 强烈抑制乳酸转运到兔红细胞中，其中 MCT1 与 CD147 相互作用，但不抑制乳酸转运到大鼠红细胞中，在那里它与 gp70 相互作用。这些数据表明，pCMBS 对 MCT1 和 MCT4 活性的抑制是通过其与 CD147 的结合介导的，而与 gp70 结合的 MCT2 对 pCMBS 不敏感。我们得出的结论是，需要辅助蛋白来维持 MCT 的催化活性以及它们易位到质膜。
Translocation of monocarboxylate transporters MCT1 and MCT4 to the plasma membrane requires CD147 (basigin) with which they remain tightly associated. However, the importance of CD147 for MCT activity is unclear. MCT1 and MCT4 are both inhibited by the cell-impermeant organomercurial reagent p-chloromercuribenzene sulfonate (pCMBS). Here we demonstrate by site-directed mutagenesis that removal of all accessible cysteine residues on MCT4 does not prevent this inhibition. pCMBS treatment of cells abolished co-immunoprecipitation of MCT1 and MCT4 with CD147 and enhanced labeling of CD147 with a biotinylated-thiol reagent. This suggested that CD147 might be the target of pCMBS, and further evidence for this was obtained by treatment of cells with the bifunctional organomercurial reagent fluorescein dimercury acetate that caused oligomerization of CD147. Site-directed mutagenesis of CD147 implicated the disulfide bridge in the Ig-like C2 domain of CD147 as the target of pCMBS attack. MCT2, which is pCMBS-insensitive, was found to co-immunoprecipitate with gp70 rather than CD147. The interaction between gp70 and MCT2 was confirmed using fluorescence resonance energy transfer between the cyan fluorescent protein- and yellow fluorescent protein-tagged MCT2 and gp70. pCMBS strongly inhibited lactate transport into rabbit erythrocytes, where MCT1 interacts with CD147, but not into rat erythrocytes where it interacts with gp70. These data imply that inhibition of MCT1 and MCT4 activity by pCMBS is mediated through its binding to CD147, whereas MCT2, which associates with gp70, is insensitive to pCMBS. We conclude that ancillary proteins are required to maintain the catalytic activity of MCTs as well as for their translocation to the plasma membrane.