title: GSH inhibits trypsinization of the C-terminal half of human MRP1.
abstract: MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance to tumor cells. The accumulated evidence has proved that GSH interacts with MRP1 and stimulates drug transport. However, the mechanism of GSH-dependent drug transport by MRP1 remains unclear. In this study, we used limited tryptic digestion of MRP1 in isolated membrane vesicles, in the presence and absence of GSH, to investigate the influence of GSH on MRP1 conformation. We found that GSH inhibited the generation of an approximately 35-kDa C-terminal tryptic fragment (including a C-terminal His tag) termed C2 from MRP1. This effect of GSH was not because of direct inhibition of trypsin activity, and agosterol A enhanced the inhibitory effect of GSH. The main cleavage site in MRP1 for the generation of the C2 fragment by trypsin resided between TMD2 and NBD2 of MRP1. Limited tryptic digestion of membrane vesicles expressing various truncated and co-expressed MRP1 fragments in the presence and absence of GSH revealed that GSH inhibited the production of the C2 fragment only in the presence of the L(0) region of MRP1. Thus the L(0) region is required for the inhibition of trypsinization of the C-terminal half of MRP1 by GSH. These findings, together with previous reports, suggest that GSH induces a conformational change at a site within the MRP1 that is indispensable for the interaction of MRP1 with its substrates.
authors.affiliation: Department of Molecular Oncology, Field of Oncology, Course of Advanced Therapeutics, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan.
authors.forename: Xiao-Qin
authors.initials: XQ
authors.lastname: Ren
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Furukawa
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Nakajima
authors.forename: Homare
authors.initials: H
authors.lastname: Takahashi
authors.forename: Shunji
authors.initials: S
authors.lastname: Aoki
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Sumizawa
authors.forename: Misako
authors.initials: M
authors.lastname: Haraguchi
authors.forename: Motomasa
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Kazuo
authors.initials: K
authors.lastname: Chijiiwa
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Akiyama
mesh_terms: D001665:Binding Sites; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000378:metabolism / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism*
chemical_list: D005978:Glutathione; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D014357:Trypsin; C421109:multidrug resistance-associated protein 1