Localization of the GSH-dependent photolabelling site of an agosterol A analog on human MRP1.
1. Human multidrug resistance protein 1 (MRP1) is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. We recently demonstrated that glutathione (GSH) is required for the labelling of the C-terminal half of MRP1 with a photoanalog of agosterol A (azido AG-A). In this study, we further characterized the GSH-dependent photolabelling site of azido AG-A on MRP1. 2. An epitope-inserted MRP1, MRP1 1222HA, which has two hemagglutinin A (HA) epitopes in the extracellular loop between transmembrane segment (TM) 16 and TM17 of the transporter, could bind azido AG-A in a GSH-dependent manner. 3. Protease digestion of the photolabelled MRP1 1222HA, followed by immunoprecipitation with an anti-HA antibody suggested that the GSH-dependent azido AG-A photolabelling site on MRP1 resides in the region within TM14-17 and the cytoplasmic region proximate to the C-terminus of TM17. 4. Arg(1210) in human MRP2 that corresponds to Arg(1202) in human MRP1 has an important role in the transporting activity of MRP2. Therefore, we replaced the Arg residue at position 1202 of MRP1 with Gly. Whereas photolabelling of the mutant MRP1 R1202G was greatly reduced, it retained leukotriene C(4) (LTC(4)) transport activity and conferred Vincristine resistance in LLC-PK1 cells. 5. In summary, this study demonstrated that the GSH-dependent azido AG-A photolabelling site on MRP1 resides in the region within TM14-17 and the cytoplasmic region proximate to the C-terminus of TM17. The charged amino acid Arg(1202) proximate to TM helix 16 is of critical importance for the GSH-dependent photolabelling of MRP1 with azido AG-A. Arg(1202) itself or the region nearby Arg(1202) may be involved in azido AG-A photolabelling.
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520, Japan.
Xiao-Qin
XQ
Ren
Tatsuhiko
T
Furukawa
Shunji
S
Aoki
Tomoyuki
T
Sumizawa
Misako
M
Haraguchi
Xiao-Fang
XF
Che
Motomasa
M
Kobayashi
Shin-ichi
S
Akiyama
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis* / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D005978:Glutathione / Q000032:analysis*; D007120:Immunochemistry; D007313:Insecta; D000089762:Multidrug Resistance-Associated Protein 2; D019920:Photoaffinity Labels / Q000032:analysis*; D013261:Sterols / Q000032:analysis* / Q000737:chemistry; D013552:Swine
C000715087:ABCC2 protein, human; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000089762:Multidrug Resistance-Associated Protein 2; D019920:Photoaffinity Labels; D013261:Sterols; C429345:agosterol A; D005978:Glutathione