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Originally published In Press as doi:10.1074/jbc.M105160200 on August 10, 2001

J. Biol. Chem., Vol. 276, Issue 41, 38108-38114, October 12, 2001
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Mutation of Trp1254 in the Multispecific Organic Anion Transporter, Multidrug Resistance Protein 2 (MRP2) (ABCC2), Alters Substrate Specificity and Results in Loss of Methotrexate Transport Activity*

Ken-ichi ItoDagger §, Curtis J. OleschukDagger §||**, Chris WestlakeDagger , Monika Z. VasaDagger , Roger G. DeeleyDagger , and Susan P. C. ColeDagger ||Dagger Dagger

From the Dagger  Cancer Research Laboratories and the || Department of Pharmacology & Toxicology, Queen's University, Kingston, Ontario K7L 3N6, Canada

The ATP-binding cassette (ABC) proteins comprise a large superfamily of transmembrane transporters that utilize the energy of ATP hydrolysis to translocate their substrates across biological membranes. Multidrug resistance protein (MRP) 2 (ABCC2) belongs to subfamily C of the ABC superfamily and, when overexpressed in tumor cells, confers resistance to a wide variety of anticancer chemotherapeutic agents. MRP2 is also an active transporter of organic anions such as methotrexate (MTX), estradiol glucuronide (E217beta G), and leukotriene C4 and is located on the apical membrane of polarized cells including hepatocytes where it acts as a biliary transporter. We recently identified a highly conserved tryptophan residue in the related MRP1 that is critical for the substrate specificity of this protein. In the present study, we have examined the effect of replacing the analogous tryptophan residue at position 1254 of MRP2. We found that only nonconservative substitutions (Ala and Cys) of Trp1254 eliminated [3H]E217beta G transport by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effect. In addition, only the most conservatively substituted mutant (W1254Y) transported [3H]leukotriene C4, whereas all other substitutions eliminated transport of this substrate. On the other hand, all substitutions of Trp1254 eliminated transport of [3H]MTX. Finally, we found that sulfinpyrazone stimulated [3H]E217beta G transport by wild-type MRP2 4-fold, whereas transport by the Trp1254 substituted mutants was enhanced 6-10-fold. In contrast, sulfinpyrazone failed to stimulate [3H]MTX transport by either wild-type MRP2 or the MRP2-Trp1254 mutants. Taken together, our results demonstrate that Trp1254 plays an important role in the ability of MRP2 to transport conjugated organic anions and identify this amino acid in the putative last transmembrane segment (TM17) of this ABC protein as being critical for transport of MTX.

* This work was supported by Grant MOP-10519 from the Canadian Institutes for Health Research.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.

§ These two authors contributed equally to this work.

Present address: Dept. of Surgery (II), Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto Nagamo 390-8621, Japan.

** Recipient of a Medical Research Council of Canada/Canadian Institutes for Health Research Doctoral Award and an Ontario Graduate Scholarship.

Dagger Dagger Senior Scientist of Cancer Care Ontario. To whom correspondence should be addressed: Cancer Research Laboratories, Rm. 328, Botterell Hall, Queen's University, Kingston, ON K7L 3N6, Canada. Tel.: 613-533-2636; Fax: 613-533-6830; E-mail: coles@post.queensu.ca.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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