Buschman E, Gros P
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Mol Cell Biol. 1991 Feb;11(2):595-603. doi: 10.1128/mcb.11.2.595-603.1991.
A full-length cDNA clone for the mouse mdr1 gene can confer multidrug resistance when introduced by transfection into otherwise drug-sensitive cells. In the same assay, a full-length cDNA clone for a closely related member of the mouse mdr gene family, mdr2, fails to confer multidrug resistance. To identify the domains of mdr1 which are essential for multidrug resistance and which may be functionally distinct in mdr2, we have constructed chimeric cDNA molecules in which discrete domains of mdr2 have been introduced into the homologous region of mdr1 and analyzed these chimeras for their capacity to transfer drug resistance. The two predicted ATP-binding domains of mdr2 were found to be functional, as either could complement the biological activity of mdr1. Likewise, a chimeric molecule in which the highly sequence divergent linker domain of mdr2 had been introduced in mdr1 could also confer drug resistance. However, the replacement of either the amino- or carboxy-terminus transmembrane (TM) domain regions of mdr1 by the homologous segments of mdr2 resulted in inactive chimeras. The replacement of as few as two TM domains from either the amino (TM5-6) or the carboxy (TM7-8) half of mdr1 by the homologous mdr2 regions was sufficient to destroy the activity of mdr1. These results suggest that the functional differences detected between mdr1 and mdr2 in our transfection assay reside within the predicted TM domains.
小鼠mdr1基因的全长cDNA克隆转染至原本对药物敏感的细胞中时，可赋予多药耐药性。在同一实验中，小鼠mdr基因家族中一个密切相关成员mdr2的全长cDNA克隆却无法赋予多药耐药性。为了确定mdr1中对多药耐药性至关重要且可能在mdr2中功能不同的结构域，我们构建了嵌合cDNA分子，即将mdr2的离散结构域导入mdr1的同源区域，并分析这些嵌合体转移耐药性的能力。发现mdr2的两个预测的ATP结合结构域具有功能，因为任何一个都可以补充mdr1的生物学活性。同样，在mdr1中引入mdr2高度序列不同的连接子结构域的嵌合分子也能赋予耐药性。然而，用mdr2的同源片段替换mdr1的氨基末端或羧基末端跨膜（TM）结构域区域会导致嵌合体无活性。从mdr1的氨基（TM5 - 6）或羧基（TM7 - 8）半段中仅替换两个TM结构域为同源的mdr2区域就足以破坏mdr1的活性。这些结果表明，在我们的转染实验中检测到的mdr1和mdr2之间的功能差异存在于预测的TM结构域内。
