Mayer R, Kartenbeck J, Büchler M, Jedlitschky G, Leier I, Keppler D
Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany.
J Cell Biol. 1995 Oct;131(1):137-50. doi: 10.1083/jcb.131.1.137.
We have previously shown that the multi-drug resistance protein (MRP) mediates the ATP-dependent membrane transport of glutathione S-conjugates and additional amphiphilic organic anions. In the present study we demonstrate the expression of MRP in hepatocytes where it functions in hepatobiliary excretion. Analysis by reverse transcription-PCR of human and normal rat liver mRNA resulted in two expected cDNA fragments of MRP. Four different antibodies against MRP reacted on immunoblots with the glycoprotein of about 190 kD from human canalicular as well as basolateral hepatocyte membrane preparations. A polyclonal antibody directed against the carboxy-terminal sequence of MRP detected the rat homolog of MRP in liver. Double immunofluorescence microscopy and confocal laser scanning microscopy showed the presence of human MRP and rat Mrp in the canalicular as well as in the lateral membrane domains of hepatocytes. The transport function of the mrp gene-encoded conjugate export pump was assayed in plasma membrane vesicles with leukotriene C4 as a high-affinity glutathione S-conjugate substrate. The deficient ATP-dependent conjugate transport in canalicular membranes from TR- mutant rat hepatocytes was associated with a lack of amplification of one of the mrp cDNA fragments and with a selective loss of Mrp on immunoblots of canalicular membranes. Double immunofluorescence microscopy of livers from transport-deficient TR- mutant rats localized Mrp only to the lateral but not to the canalicular membrane. Our results indicate that the absence of Mrp or an isoform of Mrp from the canalicular membrane is the basis for the hereditary defect of the hepatobiliary excretion of anionic conjugates by the transport-deficient hepatocyte.
我们之前已经表明，多药耐药蛋白（MRP）介导谷胱甘肽S-共轭物及其他两亲性有机阴离子的ATP依赖性膜转运。在本研究中，我们证明了MRP在肝细胞中的表达，其在肝胆排泄中发挥作用。通过逆转录-聚合酶链反应对人和正常大鼠肝脏mRNA进行分析，得到了MRP的两个预期cDNA片段。四种针对MRP的不同抗体在免疫印迹上与来自人胆小管以及肝细胞基底外侧膜制剂的约190 kD糖蛋白发生反应。一种针对MRP羧基末端序列的多克隆抗体在肝脏中检测到了MRP的大鼠同源物。双重免疫荧光显微镜和共聚焦激光扫描显微镜显示，人MRP和大鼠Mrp存在于肝细胞的胆小管以及侧膜区域。以白三烯C4作为高亲和力谷胱甘肽S-共轭物底物，在质膜囊泡中测定了mrp基因编码的共轭物输出泵的转运功能。TR-突变大鼠肝细胞胆小管膜中缺乏ATP依赖性共轭物转运，这与mrp cDNA片段之一缺乏扩增以及胆小管膜免疫印迹上Mrp的选择性缺失有关。对转运缺陷型TR-突变大鼠肝脏进行双重免疫荧光显微镜检查发现，Mrp仅定位于侧膜而非胆小管膜。我们的结果表明，胆小管膜中缺乏Mrp或Mrp的一种同工型是转运缺陷型肝细胞肝胆排泄阴离子共轭物遗传性缺陷的基础。
