Glutathione-dependent binding of a photoaffinity analog of agosterol A to the C-terminal half of human multidrug resistance protein.
MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD(0)), which is connected to a P-glycoprotein-like core region (DeltaMRP) by a cytoplasmic linker domain zero (L(0)). It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD(0) and L(0) are not known. We synthesized [(125)I]11-azidophenyl agosterol A ([(125)I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C(932-1531)) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C(4). Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L(0) is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L(0) of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.
Ren
X Q
XQ
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520, Japan.
Furukawa
T
T
Aoki
S
S
Nakajima
T
T
Sumizawa
T
T
Haraguchi
M
M
Chen
Z S
ZS
Kobayashi
M
M
Akiyama
S
S
D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D019920:Photoaffinity Labels; D011485:Protein Binding; D013261:Sterols / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005978:Glutathione; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D019920:Photoaffinity Labels; D013261:Sterols; C078444:MSH3 protein, human; C429345:agosterol A; C421109:multidrug resistance-associated protein 1