Karwatsky Joel, Leimanis Mara, Cai Jie, Gros Philippe, Georges Elias
Institute of Parasitology and Department of Biochemistry, McGill University, Ste-Anne-de-Bellevue, Quebec H9X 3V9, Canada.
Biochemistry. 2005 Jan 11;44(1):340-51. doi: 10.1021/bi048853h.
The multiple drug resistance protein 1 (MRP1 or ABCC1) transports anticancer drugs and normal cell metabolites. Leucotriene C(4) (LTC(4)) is one of the highest affinity substrates of MRP1. In this study, we have synthesized and characterized a novel photoreactive azido analogue of LTC(4) (AALTC(4)). The specificity of AALTC(4) binding to MRP1 was confirmed using an LTC(4)-specific monoclonal antibody. Moreover, binding with radioiodinated [(125)I]AALTC(4) (or IAALTC(4)) to MRP1 was dramatically competed with unmodified LTC(4) and to a lesser degree by glutathione (GSH). Oxidized glutathione (GSSG) slightly increased IAALTC(4) binding to MRP1, while MK571, verapamil, and vincristine inhibited IAALTC(4) binding to MRP1. Using AALTC(4) together with a panel of epitope-specific and LTC(4)-specific monoclonal antibodies, we identified LTC(4) binding sites in MRP1. Western blotting of large tryptic fragments of MRP1 with three well-characterized epitope-specific mAbs (MRPr1, QCRL1, and MRPm6) showed LTC(4) binding in both the N- and C-terminal halves of MRP1. Furthermore, a peptide corresponding to the N-terminal membrane-spanning domain of MRP1 (MSD0) was photoaffinity labeled by AALTC(4), indicating that MSD0 contains an LTC(4) binding site. Higher resolution mapping of additional LTC(4) binding sites was obtained using eight MRP1 variants with each containing hemaglutanin A (HA) epitopes at different sites (at amino acid 4, 163, 271, 574, 653, 938, 1001, or 1222). MRP1 variants were photoaffinity labeled with IAALTC(4) and digested with trypsin to isolate specific regions of MRP1 that interact with LTC(4). These results confirmed that sequences in MSD0 interact with IAALTC(4). Other regions that were photoaffinity labeled by IAALTC(4) include TM 10-11, TM 16-17, and TM 12, shown previously to encode MRP1 drug binding site(s). Together, our results show a high-resolution map of LTC(4) binding domains in MRP1 and provide the first direct evidence for LTC(4) binding within MSD0.
多药耐药蛋白1（MRP1或ABCC1）可转运抗癌药物和正常细胞代谢物。白三烯C4（LTC4）是MRP1亲和力最高的底物之一。在本研究中，我们合成并表征了一种新型的LTC4光反应性叠氮类似物（AALTC4）。使用LTC4特异性单克隆抗体证实了AALTC4与MRP1结合的特异性。此外，未修饰的LTC4能显著竞争性抑制放射性碘化的[(125)I]AALTC4（或IAALTC4）与MRP1的结合，谷胱甘肽（GSH）的抑制作用较小。氧化型谷胱甘肽（GSSG）略微增加IAALTC4与MRP1的结合，而MK571、维拉帕米和长春新碱则抑制IAALTC4与MRP1的结合。使用AALTC4与一组表位特异性和LTC4特异性单克隆抗体，我们确定了MRP1中的LTC4结合位点。用三种特征明确的表位特异性单克隆抗体（MRPr1、QCRL1和MRPm6）对MRP1的大胰蛋白酶片段进行蛋白质印迹分析，结果显示MRP1的N端和C端均有LTC4结合。此外，一个与MRP1的N端跨膜结构域相对应的肽段（MSD0）被AALTC4进行了光亲和标记，表明MSD0含有一个LTC4结合位点。使用八个MRP1变体获得了其他LTC4结合位点的更高分辨率图谱，每个变体在不同位点（氨基酸4、163、271、574、653、938、1001或1222）含有血凝素A（HA）表位。用IAALTC4对MRP1变体进行光亲和标记，并用胰蛋白酶消化，以分离MRP1中与LTC4相互作用的特定区域。这些结果证实MSD0中的序列与IAALTC4相互作用。其他被IAALTC4光亲和标记的区域包括TM 10 - 11、TM 16 - 17和TM 12，这些区域先前已被证明编码MRP1药物结合位点。总之，我们的结果展示了MRP1中LTC4结合结构域的高分辨率图谱，并为MSD0内的LTC4结合提供了首个直接证据。
