Multidrug Resistance Protein
IDENTIFICATION OF REGIONS REQUIRED FOR ACTIVE TRANSPORT OF LEUKOTRIENE C4*
- Mian Gaoद,
- Masayo Yamazaki‡‖,
- Douglas W. Loe‡,
- Christopher J. Westlake‡**,
- Caroline E. Grant‡,
- Susan P. C. Cole‡§‡ and
- Roger G. Deeley‡§¶¶
- From the ‡Cancer Research Laboratories and the Departments of §Pathology and **Biochemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada
Abstract
Multidrug resistance protein (MRP) is a broad specificity, primary active transporter of organic anion conjugates that confers a multidrug resistance phenotype when transfected into drug-sensitive cells. The protein was the first example of a subgroup of the ATP-binding cassette superfamily whose members have three membrane-spanning domains (MSDs) and two nucleotide binding domains. The role(s) of the third MSD of MRP and its related transporters is not known. To begin to address this question, we examined the ability of various MRP fragments, expressed individually and in combination, to transport the MRP substrate, leukotriene C4 (LTC4). We found that elimination of the entire NH2-terminal MSD or just the first putative transmembrane helix, or substitution of the MSD with the comparable region of the functionally and structurally related transporter, the canalicular multispecific organic anion transporter (cMOAT/MRP2), had little effect on protein accumulation in the membrane. However, all three modifications decreased LTC4 transport activity by at least 90%. Transport activity could be reconstituted by co-expression of the NH2-terminal MSD with a fragment corresponding to the remainder of the MRP molecule, but this required both the region encoding the transmembrane helices of the NH2-terminal MSD and the cytoplasmic region linking it to the next MSD. In contrast, a major part of the cytoplasmic region linking the NH2-proximal nucleotide binding domain of the protein to the COOH-proximal MSD was not required for active transport of LTC4.
Footnotes
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↵* This work was supported by grants from the National Cancer Institute of Canada (No. 4570) with funds from the Canadian Cancer Society and the Medical Research Council of Canada (MT-10519).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ Supported in part by a Queen’s University graduate award.
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↵‖ Present address: Dept. of Pharmacokinetics and Biopharmaceutics, Toho University, School of Pharmaceutical Sciences, 2-2-1, Miyama, Funabashi, Chiba, 274, Japan.
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↵‡ Senior Career Scientist of Cancer Care Ontario.
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↵¶¶ Stauffer Research Professor of Queen’s University. To whom correspondence and reprint requests should be addressed: Cancer Research Laboratories, Rm. A315, Botterell Hall, Queen’s University, Kingston, Ontario, Canada, K7L 3N6. Tel.: 613-545-2981; Fax: 613-545-6830.
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↵1 The abbreviations used are: MRP, multidrug resistance protein; ABC, ATP-binding cassette; P-gp, P-glycoprotein; LTC4, leukotriene C4; CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide binding domain; MSD, membrane spanning domain; TM, transmembrane; cMOAT/MRP2, canalicular multispecific organic anion transporter; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody.
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↵2 D. R. Hipfner, M. Gao, G. L. Scheffer, R. J. Scheper, R. G. Deeley, and S. P. C. Cole (1998) Br. J. Cancer, in press.
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- Received December 1, 1997.
- Revision received February 18, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
