Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport
Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport
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DOI：
10.1152/ajpcell.00160.2010
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发表时间：
2010-11-01
影响因子：
5.5
通讯作者：
Mao, Qingcheng
中科院分区：
文献类型：
--
作者：
Ni, Zhanglin;Bikadi, Zsolt;Mao, Qingcheng
Ni Z，Bikadi Z，Cai X，Rosenberg MF，MAO Q.人类乳腺癌耐药蛋白(BCRP/ABCG2)的跨膜螺旋1和6：识别对药物运输重要的极性残基。Am J Physiol Cell Physiol 299：C1100-C1109,2010。2010年8月25日首次出版；DOI：10.1152/ajpcell.00160.2010-人类乳腺癌耐药蛋白(bcrp/abcg2)介导药物和外源物质的外流。在这项研究中，我们研究了BCRP预测的跨膜α-螺旋1和6内或附近的极性残基在药物转运中的作用。我们用Ala替换Asn(387)、Gln(398)、Asn(629)和Thr(642)，用Ala和Arg替换Thr(402)，用Phe替换Tyr(645)，并在人胚胎肾293或FLP-in-293细胞中稳定表达。免疫印迹和共聚焦显微镜分析表明，所有突变体都得到了很好的表达，并且主要针对质膜。虽然T402A和T402R显示米托蒽醌、Hoechst 33342和联苯双唑的外排显著减少，但N629A对所有底物的外排活性均显著增加。N387A和Q398A对米托蒽醌和Hoechst 33342的外排有明显的抑制作用，但对BODIPY-哌唑嗪无明显影响。相比之下，T642A和Y645F仅显示出适度的Hoechst 33342外排减少。表达突变的人胚胎肾-293细胞的耐药性图谱通常与外排数据相关。此外，N629A与BCRP-ATPase活性显著相关，N387A和T402A与BCRP-ATPase活性显著降低相关。一些极性残基的突变可能导致构象变化，如在哌唑嗪存在下5D3抗体与BCRP结合的改变。BCRP的内向同源模型表明，跨膜1内的Thr(402)可能对螺旋相互作用起重要作用，Asn(629)可能参与BCRP与底物的相互作用。总之，我们已经证明了这些极性残基中的一些在BCRP活性中的功能重要性。
Ni Z, Bikadi Z, Cai X, Rosenberg MF, Mao Q. Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport. Am J Physiol Cell Physiol 299: C1100-C1109, 2010. First published August 25, 2010; doi:10.1152/ajpcell.00160.2010.-The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics. In this study, we investigated the role of polar residues within or near the predicted transmembrane alpha-helices 1 and 6 of BCRP in drug transport. We substituted Asn(387), Gln(398), Asn(629), and Thr(642) with Ala, Thr(402) with Ala and Arg, and Tyr(645) with Phe, and the mutants were stably expressed in human embryonic kidney-293 or Flp-In-293 cells. Immunoblotting and confocal microscopy analysis revealed that all of the mutants were well expressed and predominantly targeted to the plasma membrane. While T402A and T402R showed a significant global reduction in the efflux of mitoxantrone, Hoechst 33342, and BODIPY-prazosin, N629A exhibited significantly increased efflux activities for all of the substrates. N387A and Q398A displayed significantly impaired efflux for mitoxantrone and Hoechst 33342, but not for BODIPY-prazosin. In contrast, T642A and Y645F showed a moderate reduction in Hoechst 33342 efflux only. Drug resistance profiles of human embryonic kidney-293 cells expressing the mutants generally correlated with the efflux data. Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity. Mutations of some of the polar residues may cause conformational changes, as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin. The inward-facing homology model of BCRP indicated that Thr(402) within transmembrane 1 may be important for helical interactions, and Asn(629) may be involved in BCRP-substrate interaction. In conclusion, we have demonstrated the functional importance of some of these polar residues in BCRP activity.