Single amino acid (482) variants of the ABCG2 multidrug transporter:: major differences in transport capacity and substrate recognition
Single amino acid (482) variants of the ABCG2 multidrug transporter:: major differences in transport capacity and substrate recognition
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DOI：
10.1016/j.bbamem.2004.11.005
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发表时间：
2005-02-01
影响因子：
3.4
通讯作者：
Váradi, A
中科院分区：
文献类型：
--
作者：
Özvegy-Lacka, C;Köblös, G;Váradi, A
人ABCG 2蛋白是一种ATP结合盒半转运蛋白，它保护我们的细胞和组织免受各种外源性物质的侵害，而ABCG 2在肿瘤细胞中的过表达赋予多药耐药性。据记载，位置482处的单个氨基酸变化导致耐药性和转运能力改变。在本研究中，我们构建了9个ABCG 2的Arg-482突变体（G，I，M，S，T，D，N，K，Y），并在昆虫细胞中表达。所有ABCG 2变体均显示细胞表面表达，并且在分离的膜中显示ABCG 2特异性ATP酶活性。当甲氨蝶呤的积累进行了测量，在由内而外的膜囊泡，这种运输只支持野生型ABCG 2。在完整细胞中，米托蒽醌被所有ABCG 2变体转运，除了R482 K。罗丹明123被大多数突变体挤出，除了R482 K，Y和野生型ABCG 2。Hoechst 33342从表达野生型和所有Arg-482变体的细胞中泵出，但不从表达R482 K和Y的细胞中泵出。我们的研究表明，精氨酸（野生型）形式的底物特异性是独特的，在位置482处的氨基酸替换诱导该蛋白质的转运活性和底物特异性的重大改变。(C)2004 Elsevier B. V.保留所有权利。
The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein. (C) 2004 Elsevier B.V. All rights reserved.