Ren Xiao-Qin, Furukawa Tatsuhiko, Aoki Shunji, Sumizawa Tomoyuki, Haraguchi Misako, Nakajima Yuichi, Ikeda Ryuji, Kobayashi Motomasa, Akiyama Shin-ichi
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520, Japan.
Biochemistry. 2002 Dec 3;41(48):14132-40. doi: 10.1021/bi026443s.
MRP1 is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. Our recent study demonstrated that GSH is required for the labeling of MRP1(932)(-)(1531) with a photoanalogue of agosterol A (AG-A) and suggested that GSH interacts with the L(0) region of MRP1. In this study, we further characterized the GSH-dependent binding site of azido AG-A on MRP1. Coexpression of the N- and C-terminal halves of MRP1 (residues 1-1222, TM1-16, and 1223-1531, TM17, respectively) in Sf21 insect cells reconstituted a functional drug transporter with a K(m) for LTC(4) (97 nM) similar to that of intact MRP1. In membrane vesicles from those cells, GSH-dependent photolabeling of the MRP1 fragment (1-1222) required the coexpression of the C-terminal MRP1 fragment (1223-1531). An MRP1 fragment extending from residue 1 to 1295 however could be photolabeled by azido AG-A in a GSH-dependent manner. These data indicate that amino acids 1223-1295 of MRP1 are required for AG-A binding to MRP1 in a GSH-dependent manner. However, cross-linking of the photolabel to MRP1 occurs at a more upstream site. An arginine residue at position 1249 of MRP1 was shown to be important for the GSH-dependent binding of AG-A to MRP1. Mutation of this arginine to alanine (R1249A) resulted in a decreased level of GSH-dependent azido AG-A photolabeling of MRP1. Furthermore, this mutant attenuated MRP1 function by decreasing the level of LTC(4) substrate transport and impairing resistance to the drug vincristine (VCR). In summary, this study demonstrates that a region of MRP1 (amino acids 1223-1295), which includes TM helix 17, is required for azido AG-A binding to MRP1 in a GSH-dependent manner. A GSH-dependent drug binding site may exist in this region. Furthermore, our findings suggest that the charged amino acid Arg(1249) proximal to the C-terminus of TM helix 17 is indispensable for MRP1-substrate interaction and the function of MRP1.
多药耐药相关蛋白1（MRP1）是一种190 kDa的膜糖蛋白，赋予肿瘤细胞多药耐药性（MDR）。我们最近的研究表明，谷胱甘肽（GSH）是用阿戈甾醇A（AG-A）的光类似物标记MRP1(932)(-)(1531)所必需的，并提示GSH与MRP1的L(0)区域相互作用。在本研究中，我们进一步对叠氮AG-A在MRP1上的GSH依赖性结合位点进行了表征。在Sf21昆虫细胞中共表达MRP1的N端和C端片段（分别为第1 - 1222位氨基酸、跨膜区1 - 16，以及第1223 - 1531位氨基酸、跨膜区17），重构了一种功能性药物转运体，其对白三烯C4（LTC4）的米氏常数（K(m)）（97 nM）与完整的MRP1相似。在这些细胞的膜囊泡中，MRP1片段（1 - 1222）的GSH依赖性光标记需要C端MRP1片段（1223 - 1531）的共表达。然而，从第1位氨基酸延伸至1295位氨基酸的MRP1片段能够被叠氮AG-A以GSH依赖性方式进行光标记。这些数据表明，MRP1的第1223 - 1295位氨基酸是AG-A以GSH依赖性方式与MRP1结合所必需的。然而，光标记与MRP1的交联发生在更上游的位点。已证明MRP1第1249位的精氨酸残基对于AG-A以GSH依赖性方式与MRP1结合很重要。将该精氨酸突变为丙氨酸（R1249A）导致MRP1的GSH依赖性叠氮AG-A光标记水平降低。此外，该突变体通过降低LTC4底物转运水平并损害对药物长春新碱（VCR）的耐药性来减弱MRP1功能。总之，本研究表明，MRP1的一个区域（第1223 - 1295位氨基酸），包括跨膜螺旋17，是叠氮AG-A以GSH依赖性方式与MRP1结合所必需的。该区域可能存在一个GSH依赖性药物结合位点。此外，我们的研究结果表明，靠近跨膜螺旋17 C端的带电荷氨基酸精氨酸1249对于MRP1与底物的相互作用及MRP1的功能不可或缺。
