Daoud R, Julien M, Gros P, Georges E
Institute of Parasitology and Department of Biochemistry, McGill University, Quebec H9X 3V9, Canada.
J Biol Chem. 2001 Apr 13;276(15):12324-30. doi: 10.1074/jbc.M009782200. Epub 2001 Jan 4.
MRP1 is an ABC (or ATP binding cassette) membrane transport protein shown to confer resistance to structurally dissimilar drugs. Studies of MRP1 topology suggested the presence of a hydrophobic N-domain with five potential membrane-spanning domains linked to an MDR1-like core (MSD1-NBD1-L1-MSD2-NBD2) by an intracellular linker domain (L0). MRP1-mediated multidrug resistance is thought to be due to enhanced drug efflux. However, little is known about MRP1-drug interaction and its drug binding site(s). We previously developed several photoreactive probes to study MRP1-drug interactions. In this report, we have used eight MRP1-HA variants that were modified to have hemagglutinin A (HA) epitopes inserted at different sites in MRP1 sequence. Exhaustive in-gel digestion of all IAARh123 photoaffinity-labeled MRP1-HA variants revealed the same profile of photolabeled peptides as seen for wild type MRP1. Photolabeling of the different MRP1-HA variants followed by digestion with increasing concentrations of trypsin or Staphylococcus aureus V8 protease (1:800 to 1:5 w/w) and immunoprecipitation with anti-HA mAb identified two small photolabeled peptides ( approximately 6-7 kDa) from MRP1-HA(574) and MRP1-HA(1222). Based on the location of the HA epitopes in the latter variants together with molecular masses of the two peptides, the photolabeled amino acid residues were localized to MRP1 sequences encoding transmembranes 10 and 11 of MSD1 (Ser(542)-Arg(593)) and transmembranes 16 and 17 of MSD2 (Cys(1205)-Glu(1253)). Interestingly, the same sequences in MRP1 were also photolabeled with a structurally different photoreactive drug, IACI, confirming the significance of transmembranes 10, 11, 16 and 17 in MRP1 drug binding. Taken together, the results in this study provide the first delineation of the drug binding site(s) of MRP1. Furthermore, our findings suggest the presence of common drug binding site(s) for structurally dissimilar drugs.
多药耐药相关蛋白1（MRP1）是一种ABC（ATP结合盒）膜转运蛋白，已证明其对结构不同的药物具有耐药性。对MRP1拓扑结构的研究表明，存在一个疏水的N结构域，其具有五个潜在的跨膜结构域，通过一个细胞内连接结构域（L0）与一个类似多药耐药蛋白1（MDR1）的核心结构（MSD1-NBD1-L1-MSD2-NBD2）相连。MRP1介导的多药耐药被认为是由于药物外排增强所致。然而，关于MRP1与药物的相互作用及其药物结合位点知之甚少。我们之前开发了几种光反应性探针来研究MRP1与药物的相互作用。在本报告中，我们使用了八个MRP1-HA变体，这些变体经过修饰，在MRP1序列的不同位点插入了血凝素A（HA）表位。对所有IAARh123光亲和标记的MRP1-HA变体进行彻底的凝胶内消化，结果显示光标记肽的图谱与野生型MRP1相同。对不同的MRP1-HA变体进行光标记，然后用浓度递增的胰蛋白酶或金黄色葡萄球菌V8蛋白酶（1:800至1:5 w/w）消化，并使用抗HA单克隆抗体进行免疫沉淀，从MRP1-HA(574)和MRP1-HA(1222)中鉴定出两个小的光标记肽（约6-7 kDa）。根据后一种变体中HA表位的位置以及这两个肽的分子量，光标记的氨基酸残基定位于编码MSD1跨膜区10和11（Ser(542)-Arg(593)）以及MSD2跨膜区16和17（Cys(1205)-Glu(1253)）的MRP1序列。有趣的是，MRP1中的相同序列也被一种结构不同的光反应性药物IACI光标记，这证实了跨膜区10、11、16和17在MRP1药物结合中的重要性。综上所述，本研究结果首次描绘了MRP1的药物结合位点。此外，我们的发现表明存在针对结构不同药物的共同药物结合位点。
