Bakos E, Evers R, Szakács G, Tusnády G E, Welker E, Szabó K, de Haas M, van Deemter L, Borst P, Váradi A, Sarkadi B
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1113 Budapest, Hungary.
J Biol Chem. 1998 Nov 27;273(48):32167-75. doi: 10.1074/jbc.273.48.32167.
The human multidrug resistance protein (MRP1) causes drug resistance by extruding drugs from tumor cells. In addition to an MDR-like core, MRP1 contains an N-terminal membrane-bound region (TMD0) connected to the core by a cytoplasmic linker (L0). We have studied truncated MRP1 versions containing either the MDR-like core alone or the core plus linker L0, produced in the baculovirus-insect (Sf9) cell system. Their function was examined in isolated membrane vesicles. Full-length MRP1 showed ATP-dependent, vanadate-sensitive accumulation of leukotriene C4 and N-ethylmaleimide glutathione. In addition, leukotriene C4-stimulated, vanadate-dependent nucleotide occlusion was detected. The MDR-like core was virtually inactive. Co-expression of the core with the N-terminal region including L0 fully restored MRP1 function. Unexpectedly, a truncated MRP1 mutant lacking the entire TMD0 region but still containing L0 behaved like wild-type MRP1 in vesicle uptake and nucleotide trapping experiments. We also expressed the MRP1 constructs in polarized canine kidney derived MDCKII cells. Like wild-type MRP1, the MRP1 protein without the TMD0 region was routed to the lateral plasma membrane and transported dinitrophenyl glutathione and daunorubicin. The TMD0L0 and the MRP1 minus TMD0L0 remained in an intracellular compartment. Taken together, these experiments strongly suggest that the TMD0 region is neither required for the transport function of MRP1 nor for its proper routing to the plasma membrane.
人类多药耐药蛋白（MRP1）通过将药物排出肿瘤细胞而导致耐药性。除了类似多药耐药（MDR）的核心区域外，MRP1还包含一个通过细胞质连接子（L0）与核心区域相连的N端膜结合区域（TMD0）。我们研究了在杆状病毒-昆虫（Sf9）细胞系统中产生的截短型MRP1版本，其要么仅包含类似MDR的核心区域，要么包含核心区域加连接子L0。在分离的膜囊泡中检测了它们的功能。全长MRP1显示出白三烯C4和N-乙基马来酰亚胺谷胱甘肽的ATP依赖性、钒酸盐敏感性积累。此外，还检测到白三烯C4刺激的、钒酸盐依赖性的核苷酸封闭。类似MDR的核心区域几乎没有活性。核心区域与包括L0的N端区域共表达可完全恢复MRP1功能。出乎意料的是，在囊泡摄取和核苷酸捕获实验中，一个缺少整个TMD0区域但仍包含L0的截短型MRP1突变体表现得与野生型MRP1一样。我们还在极化的犬肾来源的MDCKII细胞中表达了MRP1构建体。与野生型MRP1一样，没有TMD0区域的MRP1蛋白被转运至侧质膜，并转运二硝基苯基谷胱甘肽和柔红霉素。TMD0L0和缺少TMD0L0的MRP1保留在细胞内区室中。综上所述，这些实验强烈表明，TMD0区域对于MRP1的转运功能及其向质膜的正确定位均非必需。
