Identification of proline residues in or near the transmembrane helices of the human breast cancer resistance protein (BCRP/ABCG2) that are important for transport activity and substrate specificity.
Identification of proline residues in or near the transmembrane helices of the human breast cancer resistance protein (BCRP/ABCG2) that are important for transport activity and substrate specificity.
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DOI：
10.1021/bi200573t
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发表时间：
2011-09-20
期刊：
影响因子：
2.9
通讯作者：
Mao, Qingcheng
中科院分区：
文献类型：
--
作者：
Ni, Zhanglin;Bikadi, Zsolt;Shuster, Diana L.;Zhao, Chunsheng;Rosenberg, Mark F.;Mao, Qingcheng
人类乳腺癌耐药蛋白(BCRP/ABCG2)具有多药耐药性，并介导药物和外源物质的主动外排。BCRP由一个核苷酸结合区(NBD)和一个膜跨区(MSD)组成。我们研究了跨膜螺旋内或其附近的脯氨酸是否对BCRP功能是必需的。在™-293细胞中，突变株在FLP-中稳定表达，其表达水平与野生型BCRP值相当，且主要定位于细胞膜上。P392A显示米托蒽醌、BODIPY-哌唑嗪和Hoechst33342的外排显著减少35-50%，而P485A仅BODIPY-哌唑嗪的外排显著减少约70%。其他突变体在这些底物的外流方面没有显著变化。表达突变体的细胞的耐药性曲线与外排数据有很好的相关性。与野生型BCRP相比，P392A或P485A的ATPase活性没有显著影响。这些结果有力地表明Pro392和Pro485分别在决定BCRP的整体转运活性和底物选择性方面是重要的。哌唑嗪以浓度依赖的方式不同地影响构象敏感抗体5D3与野生型BCRP、P392A或P485A的结合。相反，米托蒽醌对5D3结合无明显影响。同源模拟表明，Pro392可能在MSD和NBD之间的通讯中发挥重要作用，因为它被预测位于两个功能结构域之间的界面上，而Pro485诱导了灵活的铰链，这可能是BCRP广泛底物特异性所必需的。
The human breast cancer resistance protein (BCRP/ABCG2) confers multidrug resistance and mediates the active efflux of drugs and xenobiotics. BCRP contains one nucleotide-binding domain (NBD) followed by one membrane-spanning domain (MSD). We investigated whether prolines in or near the transmembrane helices are essential for BCRP function. Six proline residues were substituted with alanine individually, and the mutants were stably expressed in Flp-In™-293 cells at levels comparable to wild-type BCRP and predominantly localized on the plasma membrane of the cells. While P392A showed a significant reduction in the efflux of mitoxantrone, BODIPY-prazosin, and Hoechst33342 by 35–50%, P485A exhibited a significant decrease by approximately 70% in the efflux of only BODIPY-prazosin. Other mutants had no significant changes in efflux of these substrates. Drug resistance profiles of the cells expressing the mutants correlated well with the efflux data. ATPase activity was not substantially affected for P392A or P485A compared to wild-type BCRP. These results strongly suggest Pro392 and Pro485 are important in determining the overall transport activity and substrate selectivity of BCRP, respectively. Prazosin differentially affected the binding of 5D3, a conformation-sensitive antibody, to wild-type BCRP, P392A or P485A in a concentration-dependent manner. In contrast, mitoxantrone had no significant effect on 5D3 binding. Homology modeling indicates that Pro392 may play an important role in the communication between the MSD and NBD as it is predicted to be located at the interface between the two functional domains, and Pro485 induces flexible hinges that may be essential for the broad substrate specificity of BCRP.
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影响因子：
2.9
作者：
CHANG, CH;ELKABBANI, O;SCHIFFER, M
通讯作者：
SCHIFFER, M
影响因子：
3.4
作者：
Özvegy-Lacka, C;Köblös, G;Váradi, A
通讯作者：
Váradi, A
影响因子：
16.1
作者：
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通讯作者：
Bates, Susan E.
影响因子：
4.8
作者：
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通讯作者：
Sarkadi, Balazs
影响因子：
5.5
作者：
Ni, Zhanglin;Bikadi, Zsolt;Mao, Qingcheng
通讯作者：
Mao, Qingcheng