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Cancer Research Laboratories and Departments of Pathology [D. R. H., K. C. A., R. G. D., S. P. C. C.] and Biochemistry [B. D. S., R. G. D.], Queen's University, Third Floor, Botterell Hall, Kingston, Ontario, K7L 3N6 Canada
Multidrug resistance protein (MRP) is a Mr 190,000 integral membrane phosphoglycoprotein which has been shown by transfection studies to confer multidrug resistance. We have previously raised and characterized a panel of MRP-specific monoclonal antibodies (MAbs) which detect distinct epitopes in the MRP molecule (D. R. Hipfner et al., Cancer Res., 54: 5788–5792, 1994), and, in the present study, we have identified the epitope of one of these, MAb QCRL-1. Immunoblot analysis of MRP fragments generated by digestion with formic acid or trypsin suggested that the MAb QCRL-1 epitope was located in the region connecting the two halves of MRP. Subsequent analyses of a series of truncated bacterial glutathione S-transferase fusion proteins containing segments of human MRP further localized the MAb QCRL-1 epitope to a region encompassing amino acids 903–956. Similar experiments with an analogous segment of murine MRP demonstrated that MAb QCRL-1 was highly specific for the human protein. The reactivity of MAb QCRL-1 with a series of overlapping hexapeptides and heptapeptides within this region identified the human MRP-specific heptapeptide SSYSGDI (corresponding to amino acids 918–924) as the epitope, and this peptide was shown to specifically inhibit MAb QCRL-1 binding to MRP. The results of these studies confirm that this epitope has a cytoplasmic location consistent with the topology of MRP predicted from hydrophobicity analyses. These experiments also revealed the presence of a number of protease-sensitive sites on either side of the MAb QCRL-1 epitope in the cytoplasmic domain connecting the two halves of MRP. Future epitope-mapping studies with other MRP-specific MAbs will provide additional insights into the topology of MRP, and may help to identify functionally important regions of this protein. Moreover, definition of the epitope recognized by MAb QCRL-1 as well as the other MAbs will facilitate the use of these reagents for immunohistological studies of MRP expression in drug-resistant tumors.
1 This work was supported by a grant from the Medical Research Council of Canada. D. R. H. and K. C. A. are recipients of studentships from the Medical Research Council of Canada, and B. D. S. is the recipient of an Ontario Graduate Scholarship. R. G. D. is the Stauffer Research Professor of Queen's University, and S. P. C. C. is a Career Scientist of the Ontario Cancer Foundation.
2 To whom requests for reprints should be addressed. Phone: (613) 545-6507; Fax: (613) 545-6830.
Received 2/26/96. Accepted 5/15/96.
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 V. V. Rao, J. L. Dahlheimer, M. E. Bardgett, A. Z. Snyder, R. A. Finch, A. C. Sartorelli, and D. Piwnica-Worms Choroid plexus epithelial expression of MDR1 P glycoprotein and multidrug resistance-associated protein contribute to the blood-cerebrospinal-fluid drug-permeability barrier PNAS, March 30, 1999; 96(7): 3900 - 3905. [Abstract] [Full Text] [PDF]
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C. Kast and P. Gros Topology Mapping of the Amino-terminal Half of Multidrug Resistance-associated Protein by Epitope Insertion and Immunofluorescence J. Biol. Chem., October 17, 1997; 272(42): 26479 - 26487. [Abstract] [Full Text] [PDF]
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D. R. Hipfner, K. C. Almquist, E. M. Leslie, J. H. Gerlach, C. E. Grant, R. G. Deeley, and S. P. C. Cole Membrane Topology of the Multidrug Resistance Protein (MRP). A STUDY OF GLYCOSYLATION-SITE MUTANTS REVEALS AN EXTRACYTOSOLIC NH2 TERMINUS J. Biol. Chem., September 19, 1997; 272(38): 23623 - 23630. [Abstract] [Full Text] [PDF]
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B. D. Stride, C. E. Grant, D. W. Loe, D. R. Hipfner, S. P. C. Cole, and R. G. Deeley Pharmacological Characterization of the Murine and Human Orthologs of Multidrug-Resistance Protein in Transfected Human Embryonic Kidney Cells Mol. Pharmacol., September 1, 1997; 52(3): 344 - 353. [Abstract] [Full Text]
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D. W. Loe, R. K. Stewart, T. E. Massey, R. G. Deeley, and S. P. C. Cole ATP-Dependent Transport of Aflatoxin B1 and Its Glutathione Conjugates by the Product of the Multidrug Resistance Protein (MRP) Gene Mol. Pharmacol., June 1, 1997; 51(6): 1034 - 1041. [Abstract] [Full Text]
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M. Gao, D. W. Loe, C. E. Grant, S. P.C. Cole, and R. G. Deeley Reconstitution of ATP-dependent Leukotriene C4 Transport by Co-expression of Both Half-molecules of Human Multidrug Resistance Protein in Insect Cells J. Biol. Chem., November 1, 1996; 271(44): 27782 - 27787. [Abstract] [Full Text] [PDF]
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Y.-x. Hou, L. Cui, J. R. Riordan, and X.-b. Chang Allosteric Interactions between the Two Non-equivalent Nucleotide Binding Domains of Multidrug Resistance Protein MRP1 J. Biol. Chem., June 30, 2000; 275(27): 20280 - 20287. [Abstract] [Full Text] [PDF]
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Q. Mao, R. G. Deeley, and S. P. C. Cole Functional Reconstitution of Substrate Transport by Purified Multidrug Resistance Protein MRP1 (ABCC1) in Phospholipid Vesicles J. Biol. Chem., October 27, 2000; 275(44): 34166 - 34172. [Abstract] [Full Text] [PDF]
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Y.-M. Qian, W.-C. Song, H. Cui, S. P. C. Cole, and R. G. Deeley Glutathione Stimulates Sulfated Estrogen Transport by Multidrug Resistance Protein 1 J. Biol. Chem., February 23, 2001; 276(9): 6404 - 6411. [Abstract] [Full Text] [PDF]
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R. Daoud, M. Julien, P. Gros, and E. Georges Major Photoaffinity Drug Binding Sites in Multidrug Resistance Protein 1 (MRP1) Are within Transmembrane Domains 10-11 and 16-17 J. Biol. Chem., April 6, 2001; 276(15): 12324 - 12330. [Abstract] [Full Text] [PDF]
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K.-i. Ito, S. L. Olsen, W. Qiu, R. G. Deeley, and S. P. C. Cole Mutation of a Single Conserved Tryptophan in Multidrug Resistance Protein 1 (MRP1/ABCC1) Results in Loss of Drug Resistance and Selective Loss of Organic Anion Transport J. Biol. Chem., May 4, 2001; 276(19): 15616 - 15624. [Abstract] [Full Text] [PDF]
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M. F. Rosenberg, Q. Mao, A. Holzenburg, R. C. Ford, R. G. Deeley, and S. P. C. Cole The Structure of the Multidrug Resistance Protein 1 (MRP1/ABCC1). CRYSTALLIZATION AND SINGLE-PARTICLE ANALYSIS J. Biol. Chem., May 4, 2001; 276(19): 16076 - 16082. [Abstract] [Full Text] [PDF]
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D.-W. Zhang, S. P. C. Cole, and R. G. Deeley Identification of a Nonconserved Amino Acid Residue in Multidrug Resistance Protein 1 Important for Determining Substrate Specificity. EVIDENCE FOR FUNCTIONAL INTERACTION BETWEEN TRANSMEMBRANE HELICES 14 AND 17 J. Biol. Chem., September 7, 2001; 276(37): 34966 - 34974. [Abstract] [Full Text] [PDF]
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