GSH-dependent photolabeling of multidrug resistance protein MRP1 (ABCC1) by [125I]LY475776. Evidence of a major binding site in the COOH-proximal membrane spanning domain.
Substrates transported by the 190-kDa multidrug resistance protein 1 (MRP1) (ABCC1) include endogenous organic anions such as the cysteinyl leukotriene C(4). In addition, MRP1 confers resistance against various anticancer drugs by reducing intracellular accumulation by co-export of drug with reduced GSH. We have examined the properties of LY475776, an intrinsically photoactivable MRP1-specific tricyclic isoxazole modulator that inhibits leukotriene C(4) transport by this protein in a GSH-dependent manner. We show that [125I]LY475776 photolabeling of MRP1 requires GSH but is also supported by several non-reducing GSH derivatives and peptide analogs. Limited proteolysis revealed that [(125)I]LY475776 labeling was confined to the 75-kDa COOH-proximal half of MRP1. More extensive proteolysis generated two major 125I-labeled fragments of approximately 56 and approximately 41 kDa, and immunoblotting with regionally directed antibodies showed that these fragments correspond to amino acids approximately 1045-1531 and approximately 1150-1531, respectively. However, an approximately 33-kDa COOH-terminal immunoreactive fragment was not labeled, inferring that the major [125I]LY475776-labeling site resides approximately between amino acids 1150-1250. This region encompasses transmembrane (TM) segments 16 and 17 at the COOH-proximal end of the third membrane spanning domain of the protein. [125I]LY475776 labeling of mutant MRP1 molecules with substitutions of Trp(1246) in TM17 were reduced >80% compared with wild-type MRP1, confirming that TM17 is important for LY475776 binding. Finally, vanadate-induced trapping of ADP inhibited [125I]LY475776 labeling, suggesting that ATP hydrolysis causes a conformational change in MRP1 that reduces the affinity of the protein for this inhibitor.
Mao
Qingcheng
Q
Cancer Research Laboratories and Department of Pathology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Qiu
Wei
W
Weigl
Kevin E
KE
Lander
Peter A
PA
Tabas
Linda B
LB
Shepard
Robert L
RL
Dantzig
Anne H
AH
Deeley
Roger G
RG
Cole
Susan P C
SP
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001386:Azides / Q000494:pharmacology*; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007555:Isoxazoles / Q000494:pharmacology*; D017997:Leukotriene C4 / Q000378:metabolism; D008027:Light; D008956:Models, Chemical; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D009154:Mutation; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D014162:Transfection; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D001386:Azides; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005978:Glutathione; D007555:Isoxazoles; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D014638:Vanadates; C464935:LY 475776; C078444:MSH3 protein, human; C421109:multidrug resistance-associated protein 1