Function of the ABC signature sequences in the human multidrug resistance protein 1
Function of the ABC signature sequences in the human multidrug resistance protein 1
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DOI：
10.1124/mol.65.6.1536
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发表时间：
2004-06-01
影响因子：
3.6
通讯作者：
Akiyama, S
中科院分区：
文献类型：
--
作者：
Ren, XQ;Furukawa, T;Akiyama, S
人多药耐药蛋白1 （MRP1）是一种膜atp结合盒转运蛋白，通过atp依赖的方式外排细胞内药物，赋予肿瘤细胞多药耐药。转运发生的机制以及ATP水解与药物转运耦合的机制尚未完全阐明。特别是，MRP1核苷酸结合域（nbd）中特征序列的功能尚不清楚。因此，我们研究了NBDs中特征序列（G771D和G1433D）和Walker A基序（K684M和K1333M）突变对MRP1的8-azido-[α - p -32]ATP光标记和8-azido-[α - p -32]ADP钒酸盐捕获的影响。Walker A基序的两个突变几乎完全抑制了8-叠氮-[α - p -32]ATP对突变NBD的标记，但对另一个完整NBD的标记没有影响。相比之下，NBD1特征序列中的G771D突变增强了NBD1的标记性，但略微降低了NBD2的标记性。NBD2特征基序的G1433D突变增强了NBD2的标记，但不影响NBD1的标记。这些效应都与底物无关。在8-叠氮-[α - p -32]ATP的钒酸盐捕获条件下，NBD1的光标记和NBD1的轻微光致化被检测到。K684M和K1333M突变以及K684M/K1333M双突变几乎完全抑制了NBD1和NBD2的诱捕。G771D突变完全抑制了NBD2的诱捕，并显著抑制了NBD1的诱捕。然而，G1433D突变也显著抑制NBD1的诱捕，但仅部分抑制NBD2的诱捕，白三烯C-4仍能增强诱捕作用。我们的研究结果表明，MRP1的两个特征序列都参与ATP水解，并且必须是完整的ATP水解和MRP1的运输。
Human multidrug resistance protein 1 (MRP1) is a membrane ATP-binding cassette transporter that confers multidrug resistance to tumor cells by effluxing intracellular drugs in an ATP-dependent manner. The mechanisms by which transport occurs and by which ATP hydrolysis is coupled to drug transport are not fully elucidated. In particular, the function of the signature sequences in the nucleotide binding domains (NBDs) of MRP1 is unknown. We therefore investigated the effect of mutation of the signature sequences (G771D and G1433D) and of the Walker A motifs (K684M and K1333M) in the NBDs on the 8-azido-[alpha-P-32]ATP photolabeling and 8-azido-[alpha-P-32]ADP vanadate trapping of MRP1. Both mutations in the Walker A motif almost completely inhibited the labeling of the mutated NBD with 8-azido-[alpha-P-32]ATP but not the labeling of the other intact NBD. In contrast, the G771D mutation in the signature sequence of NBD1 enhanced the labeling of NBD1 but slightly decreased the labeling of NBD2. The G1433D mutation in the signature motif of NBD2 enhanced the labeling of NBD2 but did not affect the labeling of NBD1. These effects were all substrate-independent. Photolabeling of NBD2 and a very slight photolableing of NBD1 were detectable under vanadate trapping conditions with 8-azido-[alpha-P-32]ATP. Trapping at both NBD1 and NBD2 was almost completely inhibited by K684M and K1333M mutations and by the K684M/K1333M double mutation. The G771D mutation completely inhibited trapping at NBD2 and considerably inhibited trapping at NBD1. However, whereas the G1433D mutation also considerably inhibited trapping at NBD1, it only partially inhibited trapping of NBD2, and the trapping could still be enhanced by leukotriene C-4. Our findings suggest that both signature sequences of MRP1 are involved in ATP hydrolysis and must be intact for the ATP hydrolysis and the transport by MRP1.