BCRP/ABCG2 confers anticancer drug resistance without covalent dimerization
BCRP/ABCG2 confers anticancer drug resistance without covalent dimerization
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DOI：
10.1111/j.1349-7006.2010.01605.x
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发表时间：
2010-08
期刊：
影响因子：
5.7
通讯作者：
J. Shigeta;K. Katayama;J. Mitsuhashi;K. Noguchi;Y. Sugimoto
中科院分区：
文献类型：
--
作者：
J. Shigeta;K. Katayama;J. Mitsuhashi;K. Noguchi;Y. Sugimoto
在以前的研究中，我们证明了乳腺癌耐药蛋白(BCRPABCG2)形成S-S同源二聚体。BCRP-C603S突变体取代了Cys-603的胞外区，形成了70-75 kDa的单体和140-150 kDa的二聚体，表明Cys-603是共价桥上的一个重要残基。这些结果还表明其他半胱氨酸残基参与了二聚体的形成。在本研究中，我们使用双Cys和三Cys突变的BCRP转染体，研究了其他胞外Cys残基Cys-592和Cys-608可能参与BCRP的二聚化和转运蛋白功能。在非还原条件下的SDS-PAGE检测到BCRP-C592S·C603S和BCRP-C592S·C608S为二聚体，而BCRP-C603S·C608S和BCRP-C592S·C603S·C608S仅为单体。这一发现表明，除了三个胞外半胱氨酸外，没有任何半胱氨酸残基是形成二聚体的原因。BCRP-C592S·C603S二聚体的形成提示Cys-608参与了该突变BCRP的共价连接。PA/C592S·C603S·C608S-cl.7细胞表现出明显的多重耐药和低水平的米托蒽醌蓄积。这些结果清楚地表明，BCRP是一种不存在共价二聚体的耐药蛋白。在耐药的Cys突变型BCRP转染体中，PA/C603S、PA/C592S·C608S和PA/C592S·C603S·C608S对fumitremorgin C的逆转作用比PA/WT更具抵抗力，提示Cys突变型BCRP的底物识别能力发生了变化。总之，半胱氨酸介导的共价二聚化不是BCRP作为转运蛋白发挥作用所必需的。除Cys-603外，Cys-608还可能参与BCRP二聚体的形成。(癌症科学2010)
In previous studies, we demonstrated that the breast cancer resistance protein (BCRP, ABCG2) forms an S–S homodimer. The BCRP‐C603S mutant substituting Ser for Cys‐603 in the third extracellular domain formed both a 70–75‐kDa monomer and 140–150‐kDa dimer, suggesting that Cys‐603 is an important residue in the covalent bridge. These results also suggested the involvement of other Cys residues in dimer formation. In the present study, we examined the possible involvement of the other extracellular Cys residues, Cys‐592 and Cys‐608, in the dimerization and transporter functions of BCRP using double and triple Cys‐mutant BCRP transfectants. In SDS–PAGE under non‐reducing conditions, BCRP‐C592S·C603S and BCRP‐C592S·C608S were detected as dimers whereas BCRP‐C603S·C608S and BCRP‐C592S·C603S·C608S were found only as monomers. This finding indicated that no Cys residues other than the three extracellular Cys are responsible for the dimer formation. The formation of BCRP‐C592S·C603S dimer suggested the involvement of Cys‐608 in the covalent linkage of this mutant BCRP. PA/C592S·C603S·C608S‐cl.7 cells showed a significant level of multiple drug resistance and low‐level accumulation of mitoxantrone. These results clearly demonstrate that BCRP functions as a drug resistance protein without covalent dimerization. Among drug‐resistant Cys‐mutant BCRP transfectants, PA/C603S, PA/C592S·C608S, and PA/C592S·C603S·C608S were found to be more resistant to the reversal effects of fumitremorgin C than PA/WT, suggesting some alteration in the substrate recognition in Cys‐mutant BCRPs. In conclusion, Cys‐mediated covalent dimerization is not required for BCRP to function as a transporter. In addition to Cys‐603, Cys‐608 may also be involved in BCRP dimer formation. (Cancer Sci 2010)