title: The multidrug resistance protein is photoaffinity labeled by a quinoline-based drug at multiple sites.
abstract: Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP has been shown to mediate the transport of cytotoxic natural products, in addition to glutathione-, glucuronidate-, and sulfate-conjugated cell metabolites. However, the mechanism of MRP drug binding and transport is at present not clear. In this study, we have used a photoreactive quinoline-based drug, N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide (IACI), to show the photoaffinity labeling of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein as the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1. Furthermore, a molar excess of leukotriene C(4), doxorubicin, colchicine, and other quinoline-based drugs, including MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI accumulation in MRP-expressing cells which was reversed by depleting ATP levels in H69/AR cells. Mild digestion of the purified IACI-photolabeled MRP with trypsin showed two large polypeptides ( approximately 111 and approximately 85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids approximately 900-1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1-900) containing the MSD1 and MSD2 plus the first nucleotide binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and approximately 6 plus 4 kDa photolabeled peptides, respectively. In addition, resolution of the exhaustively digested IACI-photolabeled MRP by HPLC showed two major and one minor radiolabeled peaks that eluted late in the gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying MRP-drug interactions.
authors.affiliation: Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue, Quebec, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Daoud
authors.forename: J
authors.initials: J
authors.lastname: Desneves
authors.forename: L W
authors.initials: LW
authors.lastname: Deady
authors.forename: L
authors.initials: L
authors.lastname: Tilley
authors.forename: R J
authors.initials: RJ
authors.lastname: Scheper
authors.forename: P
authors.initials: P
authors.lastname: Gros
authors.forename: E
authors.initials: E
authors.lastname: Georges
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D001386:Azides / Q000737:chemistry / Q000378:metabolism*; D001549:Benzamides / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D002930:Cinchona Alkaloids / Q000737:chemistry / Q000378:metabolism*; D018432:Drug Resistance, Multiple; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D011485:Protein Binding; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D001386:Azides; D001549:Benzamides; D002930:Cinchona Alkaloids; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D019920:Photoaffinity Labels; C409368:N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide; C041622:cinchonidine