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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 Ito^§¶, Curtis J. Oleschuk^§**, Chris Westlake, Monika Z. Vasa, Roger G. Deeley, and Susan P. C. Cole

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

Received for publication, June 5, 2001, and in revised form, August 9, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (E₂17G), 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]E₂17G 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]E₂17G 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The ATP-binding cassette (ABC)¹ proteins comprise a large superfamily of transmembrane proteins that use the energy of ATP hydrolysis to translocate their substrates across biological membranes (1-3). One branch of the superfamily, known as subfamily C, presently consists of 12 human proteins, seven of which are designated as multidrug resistance proteins (MRPs) 1-7 (4, 5). MRP1 (ABCC1) was the first of the MRP-related transporters to be cloned, and increased expression of this protein in tumor cells results in resistance to a remarkably diverse spectrum of anticancer drugs and other xenobiotics (5-10). MRP1 is also a primary active transporter of a variety of organic anions that include both endo- and xenobiotics conjugated to glutathione (GSH), glucuronide, and sulfate (5, 10-16). Well characterized organic anion substrates of MRP1 include the folic acid antimetabolite methotrexate (MTX), the cysteinyl leukotriene C₄ (LTC₄), and the conjugated estrogen 17-estradiol 17-(-D-glucuronide) (E₂17G) (8, 10, 15-21).

The ability of MRP1 to confer drug resistance and transport organic anions is shared by several other proteins belonging to the ABCC subfamily, including MRP2 (ABCC2) (11, 21-24). MRP1 and MRP2 have in common a five domain structure with a core consisting of two tandemly arranged units, each containing a membrane spanning domain (MSD) and a nucleotide-binding domain that is preceded by a third MSD with an extracytosolic NH₂ terminus (see Fig. 1A) (10, 11, 25). The substrate specificities of the two proteins are also similar in many respects, although certain significant differences have been identified (11, 26-28). MRP1 and MRP2 also differ with respect to their levels of expression in normal tissues, as well as their membrane localization in polarized cells. For example, MRP1 is expressed in many tissues throughout the body and localizes to basolateral membranes of polarized cells including hepatocytes and pneumocytes (29, 30), whereas MRP2 is found predominantly in the canalicular (apical) membrane of hepatocytes where it functions as a biliary transporter (11, 26, 31).

Mutations in the mrp2 gene that create a premature termination codon have been identified in two hyperbilirubinemic rat strains, the Groningen yellow/transport-deficient Wistar rat (GY/TR) and the Eisai hyperbilirubinemic rat, and are associated with the absence of detectable MRP2 protein (32, 33). In humans, the absence of functional MRP2 protein has been associated with several different missense, nonsense, and deletion mutations in the MRP2 gene in patients with a rare disorder known as Dubin-Johnson syndrome (34-37). This disorder is characterized by chronic conjugated hyperbilirubinemia, impaired secretion of anionic conjugates from hepatocytes into bile, and deposition of a dark pigment in the liver.

The specific amino acids in MRP2 involved in the recognition and transport of its substrates remain largely unknown, although two recent reports indicate that certain charged residues in or close to TM segments of the second and third MSDs are important (38, 39). In a previous study of the related MRP1, we identified a highly conserved tryptophan residue in the last TM segment of MSD3, Trp¹²⁴⁶, that is essential for the transport of E₂17G, as well as for the ability to confer resistance to cationic and electroneutral natural product drugs (40). In contrast to the effect on drug resistance and E₂17G transport, we found that the variously substituted MRP1-Trp¹²⁴⁶ mutants were capable of transporting LTC₄ with only a modest decrease in affinity (K_m) for this GSH conjugated substrate.² In the present study, we have mutated the analogous tryptophan residue in TM17 of MRP2, Trp¹²⁵⁴ (see Fig. 1A), and show that all but the most conservative substitutions markedly reduce the ability of the protein to transport conjugated substrates in a way that differs quantitatively and qualitatively from comparable Trp¹²⁴⁶ mutants of MRP1. In addition, we have identified Trp¹²⁵⁴ as being critical for the ability of MRP2 to transport MTX.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [6,7-³H]E₂17G (55 Ci mmol¹) was purchased from PerkinElmer Life Sciences, [14,15(n)-³H]LTC₄ (115.3 Ci mmol¹) was from Amersham Pharmacia Biotech, and [3',5',7'-³H(n)]MTX sodium salt (17 Ci mmol¹) was from Moravek Inc. (Brea, CA). LTC₄ was purchased from CalBiochem (La Jolla, CA), and nucleotides, GSH, 2-mercaptoethanol, E₂17G, sulfinpyrazone, and dithiothreitol were purchased from Sigma.

Vector Construction and Site-directed Mutagenesis-- To generate an MRP2 expression vector, a DNA fragment containing the complete coding sequence of human MRP2 mRNA with an optimized Kozak sequence was synthesized from several polymerase chain reaction products obtained from human A549 and HepG2 tumor cells and assembled in the vector pBluescript II KS⁺ (Stratagene, La Jolla, CA). The coding sequence of MRP2 cDNA was then moved into pcDNA3.1() (InVitrogen, Carlsbad, CA), and the integrity of the resulting pcDNA3.1()-MRP2 vector was confirmed by sequencing the entire inserted sequence in both directions. The sequence obtained was in agreement with that published previously (GenBank^TM accession number XP005809).

The template for site-directed mutagenesis of Trp¹²⁵⁴ of MRP2 was prepared by cloning a 2-kilobase XbaI fragment of MRP2 corresponding to nucleotides 2349-4404 into pGEM-3Z®. Mutagenesis was carried out using the Transformer^TM kit (CLONTECH, Palo Alto, CA) according to the manufacturer's instructions with the following sense mutagenic primers (substituted nucleotides are underlined): W1254A (5'-CACAAACCCTGAACGCTTCTGGTGAGGATGAC-3'), W1254C (5'-CACAAACCCTGAACTGTCTGGTGAGGATGAC-3'), W1254F (5'-CACAAACCCTGAACTTTCTGGTGAGGATGAC-3'), and W1254Y (5'-CACAAACCCTGAACTATCTGGTGAGGATGAC-3'). After confirming all mutations by sequencing or diagnostic restriction enzyme digests, a 1.7-kilobase SfiI/Bsu36I fragment containing the desired mutation was subcloned back into pcDNA3.1()-MRP2, and the entire fragment in the full-length construct was sequenced again.

Transient Transfections of MRP2 Expression Vectors-- Wild-type and mutant pcDNA3.1()-MRP2 expression vectors were transfected into SV40-transformed human embryonic kidney cells (HEK293T) as follows. Approximately 5 × 10⁶ cells were seeded in 175-cm² flasks, and 24 h later, DNA (16 µg) was added using FuGENE^TM 6 (Roche Diagnostics, Laval, Canada) according to the manufacturer's instructions. After 48-72 h, the HEK293T cells were harvested, and inside-out membrane vesicles were prepared as described previously (18). Empty vector pcDNA3.1() DNA and vector containing the wild-type MRP2 cDNA were included as controls in all transfection experiments.

Measurement of MRP2 Protein Levels in Transfected Cells-- The relative levels of wild-type and Trp¹²⁵⁴ mutant MRP2 proteins were determined by immunoblot analysis of membrane protein fractions from the transfected cells, essentially as described (40, 41). The proteins were resolved on a 6-7% polyacrylamide gel and electrotransferred to a nylon membrane. Membranes were blocked with 4% (w/v) skim milk powder for 1 h followed by incubation with the MRP2-specific murine monoclonal antibody M₂I-4 (Alexis, San Diego, CA) (diluted 1:2500 or 1:5000) (41, 42). After washing, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (Pierce) followed by application of Renaissance® chemiluminescence blotting substrate (PerkinElmer Life Sciences). Relative levels of MRP2 protein expression were estimated by densitometric analysis using a ChemiImager^TM 4000 (Alpha Innotech, San Leandro, CA).

MRP2-mediated Transport of [³H]E₂17G and [³H]LTC₄ by Inside-out Membrane Vesicles-- Inside-out membrane vesicles were prepared from the transiently transfected HEK293T cells, and ATP-dependent transport of ³H-labeled substrates by the membrane vesicles was measured using a rapid filtration technique as described previously (18, 41). Briefly, E₂17G transport assays were performed at 37 °C in a 60-µl reaction containing 400 nM [³H]E₂17G (300 nCi), 4 mM AMP or ATP, 10 mM MgCl₂, creatine phosphate (10 mM), creatine kinase (100 µg ml¹), and 8 µg of vesicle protein in transport buffer (50 mM Tris-HCl, 250 mM sucrose, pH 7.4). In some experiments, sulfinpyrazone (100 µM) and MTX (500 and 1000 µM) were added. Uptake was stopped at 5 min by rapid dilution in ice-cold buffer, and then the reaction was filtered through glass fiber (Type A/E) filters that had been presoaked in transport buffer. Radioactivity was quantitated by liquid scintillation counting. All of the data were corrected for the amount of [³H]E₂17G that remained bound to the filter, which was usually <10% of the total radioactivity. Transport in the presence of AMP was subtracted from transport in the presence of ATP to determine ATP-dependent E₂17G uptake. All transport assays were carried out for time periods during which uptake was linear, based on preliminary time course studies. Thus transport activities represent initial rates. The assays were carried out in triplicate, and the results are expressed as the means ± S.D.

Uptake of [³H]LTC₄ was measured in a similar fashion except that membrane vesicles (8 µof g protein) were incubated at 23 °C for 3 min in a total reaction volume of 50 µl containing [³H]LTC₄ (50 nM; 40 nCi) and other components as described for [³H]E₂17G transport.

MRP2-mediated Transport of [³H]MTX by Inside-out Membrane Vesicles-- [³H]MTX uptake was also measured by rapid filtration essentially as described (8, 43). The assays were performed at 37 °C in a 50-µl reaction volume containing 1 µM [³H]MTX (250 nCi/reaction), 50 mM HEPES/KOH buffer (pH 7.4), 100 mM KCl, 1 mM AMP or ATP, an ATP-regenerating system, and membrane vesicles (12.5 µg of protein). Uptake was stopped at 10 min, diluted, and filtered as above for E₂17G transport.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

MRP2-Trp¹²⁵⁴ Mutants Are Expressed at Comparable Levels in Human Embryonic Kidney Cells but Only Conservatively Substituted MRP2-Trp¹²⁵⁴ Mutants Transport [³H]E₂17G-- We showed previously that both conservative and nonconservative substitutions of Trp¹²⁴⁶ in MRP1 eliminated the E₂17G transport activity of this protein (40). To determine whether MRP2-mediated E₂17G transport activity would be similarly affected, cDNA constructs were generated in which several different amino acids were introduced in place of the analogous tryptophan residue in MRP2, Trp¹²⁵⁴. These included nonconservative substitutions with a nonaromatic nonpolar amino acid (Ala; W1254A-MRP2) and a nonaromatic polar amino acid (Cys; W1254C-MRP2), as well as conservative substitutions with polar (Tyr; W1254Y-MRP2) and nonpolar (Phe; W1254F-MRP2) aromatic amino acids. The constructs were transfected into HEK293T cells, and after 48-72 h, the cells were harvested, the membrane vesicles were prepared, and relative MRP2 protein levels were determined by immunoblotting (41, 42). As shown in Fig. 1B, wild-type MRP2 and its four mutants (W1254A, W1254C, W1254Y, and W1254F) were all expressed in the HEK293T cells at comparable levels. Mean values of expression levels from four to six independent transfections relative to wild-type MRP2 were W1254A, 1.1; W1254C, 0.9; W1254F, 0.9; W1254Y, 1.0, respectively (Fig. 1B).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Location of Trp1254 in transmembrane segment 17 in MRP2 and expression levels of wild-type and mutant MRP2 molecules. A, schematic diagram of a predicted secondary structure of MRP2. The position of Trp1254 (1254) in the third membrane spanning domain (MSD3) of MRP2 is highlighted. The amino acids in the predicted seventeenth transmembrane segment (TM17) are those predicted by several algorithms, including that of Eisenberg et al. (73). NBD, nucleotide-binding domain. B, immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1()), wild-type (WT-MRP2) and mutant (W1254A, W1254C, W1254F, and W1254Y) MRP2 cDNAs. MRP2 proteins were detected with the MRP2-specific monoclonal antibody M2I-4 as described under "Experimental Procedures." The numbers below the blot refer to the levels of the mutant MRP2 proteins relative to wild-type MRP2 protein in membrane vesicles prepared from the transfected cells and were estimated by densitometry. The bars shows the relative mean levels ± S.D. of MRP2 protein expression in membrane vesicles from four to six independent transfections.

ATP-dependent transport of [³H]E₂17G was determined using the membrane vesicles prepared from the transfected HEK cells (Fig. 2A). Like the nonconservatively substituted MRP1-Trp¹²⁴⁶ mutants, the W1254A-MRP2 and W1254C-MRP2 mutants either did not transport or only very poorly transported [³H]E₂17G (Fig. 2B). However, E₂17G transport by the W1254Y-MRP2 and W1254F-MRP2 mutants was similar to that of wild-type MRP2, in marked contrast to the comparable conservatively substituted MRP1-Trp¹²⁴⁶ mutants, which were inactive for transport of this substrate (40).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Transport activity of wild-type and Trp1254 mutant MRP2 molecules in HEK293T cells. A, [3H]E217G uptake was measured in membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(); open bar), wild-type MRP2 (WT-MRP2; solid bar), and mutant (W1254A-MRP2, W1254C-MRP2, W1254F-MRP2, and W1254Y-MRP2; shaded bars) cDNA expression vectors. Membrane vesicles were incubated at 37 °C with 400 nM [3H]E217G in transport buffer and ATP or AMP for 5 min as described under "Experimental Procedures." B, [3H]LTC4 uptake was measured as above except that the membrane vesicles were incubated at 23 °C with 40 nM [3H]LTC4 for 3 min. The results shown are the means ± S.D. of triplicate determinations in a single experiment, and similar results were obtained in a second experiment.

Only the Most Conservatively Substituted MRP2-Trp¹²⁵⁴ Mutant Transports [³H]LTC₄-- Previously, we found that the LTC₄ transport capacity of the Trp¹²⁴⁶ substituted MRP1 mutants remained intact although affinity (K_m) for this glutathione conjugated substrate was modestly reduced compared with wild-type MRP1 (40).² In contrast, [³H]LTC₄ transport activity was essentially eliminated in vesicles containing three of the four MRP2-Trp¹²⁵⁴ mutants, with levels of transport being comparable with those of vesicles prepared from control cells transfected with the empty pcDNA3.1() vector (Fig. 2B). The [³H]LTC₄ transport activity of the fourth and most conservatively substituted mutant, W1254Y-MRP2, was ~40% of wild-type MRP2.

[³H]E₂17G Transport by W1254F-MRP2 Is No Longer Inhibited by LTC₄-- It has been shown previously that LTC₄ inhibits MRP2-mediated E₂17G transport (39, 44). Consequently, to determine whether E₂17G transport by a mutant MRP2 protein that no longer transports LTC₄ could still be inhibited by this cysteinyl leukotriene, [³H]E₂17G transport by the W1254F-MRP2 mutant was examined. As shown in Fig. 3, LTC₄ (1 µM) inhibited [³H]E₂17G transport by wild-type MRP2 by ~50% as expected. In contrast, LTC₄ had no significant effect on [³H]E₂17G transport by W1254F-MRP2, indicating that the loss of LTC₄ transport by this mutant is associated with a loss of binding of this substrate.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effect of LTC4 on [3H]E217G transport into membrane vesicles enriched for wild-type and Trp1254 mutant MRP2 proteins. Membrane vesicles prepared from HEK293T cells expressing wild-type (WT-MRP2) and mutant W1254F-MRP2 were incubated with [3H]E217G in the absence (open bars) and presence (solid and shaded bars) of 1 µM LTC4 for 5 min as described under "Experimental Procedures." The results shown are the means ± S.D. of triplicate determinations.

Transport of [³H]MTX by MRP2 Is Eliminated by Trp¹²⁵⁴ Substitutions-- To determine whether the conservative and nonconservative Trp¹²⁵⁴ substitutions might affect the transport of MRP2 substrates other than glutathione- and glucuronide-conjugated organic anions, [³H]MTX uptake was measured in membrane vesicles prepared from cells transfected with wild-type and mutant Trp¹²⁵⁴ MRP2 cDNAs. As shown in Fig. 4A, ATP-dependent [³H]MTX transport by wild-type MRP2 was readily detectable. In contrast, [³H]MTX transport by all four MRP2-Trp¹²⁵⁴ mutant proteins was very low. After taking into account the different levels of MRP2 protein expression and subtracting the background transport activity of the membrane vesicles from the vector transfected control cells, the relative levels of [³H]MTX transport by the W1254A, W1254C, W1254F, and W1254Y mutants were 14, 14, 11, and 1%, respectively, of wild-type MRP2. Thus both conservative and nonconservative substitutions of Trp¹²⁵⁴ essentially eliminated MTX transport by this ABCC transporter.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   [3H]MTX uptake and MTX-mediated inhibition of [3H]E217G uptake by wild-type and Trp1254 mutant MRP2 proteins. A, membrane vesicles (12.5 µg of protein) prepared from HEK293T cells transfected with empty vector (pcDNA3.1(); open bar), wild-type (WT-MRP2; solid bar), and Trp1254 mutant MRP2 (shaded bars) cDNAs were incubated at 37 °C with 1 µM [3H]MTX in transport buffer and other components for 10 min as described under "Experimental Procedures." B, membrane vesicles (8 µg of protein) from cells expressing WT-MRP2 and mutants W1254F-MRP2 and W1254Y-MRP2 were incubated at 37 °C with 400 nM [3H]E217G in transport buffer and other components for 5 min in the absence (open bars) or the presence of MTX (500 µM, shaded bar; 1000 µM, solid bar) as described under "Experimental Procedures." Each bar represents the mean ± S.D. of triplicate determinations.

[³H]E₂17G Transport by Wild-type MRP2, but Not W1254F-MRP2, Is Inhibited by MTX-- It has previously been shown that MTX inhibits MRP2-mediated transport of the GSH-conjugated substrates, dinitrophenyl-glutathione and N-ethylmaleimide-glutathione (21, 24). In the present study, we found that MTX also inhibits E₂17G transport. Thus [³H]E₂17G transport by wild-type MRP2 was inhibited by ~25 and 50% at concentrations of 500 and 1000 µM MTX, respectively (Fig. 4B). To determine whether E₂17G transport by mutant MRP2 proteins that no longer transport MTX remained inhibitable by this antifolate, [³H]E₂17G transport in membrane vesicles prepared from cells expressing W1254F-MRP2 and W1254Y-MRP2 was examined. In contrast to wild-type MRP2, MTX had no significant effect on [³H]E₂17G transport by either W1254F-MRP2 or W1254Y-MRP2 at concentrations of either 500 or 1000 µM MTX (Fig. 4B). These results indicate that the loss of MTX transport by MRP2-Trp¹²⁵⁴ mutants is associated with a loss of binding of MTX to the protein.

Sulfinpyrazone Stimulates [³H]E₂17G Transport by Wild-type and Mutant MRP2 but Has No Effect on [³H]MTX Transport-- It has been reported previously that transport of GSH and glutathione conjugates by MRP2 can be markedly stimulated by several organic anions, including sulfinpyrazone (21, 27, 45). We have now found that sulfinpyrazone (100 µM) can also stimulate [³H]E₂17G transport by MRP2 (Fig. 5A). In addition, we observed that [³H]E₂17G transport by the MRP2 mutants W1254A, W1254C, W1254F, and W1254Y was enhanced 9-, 6-, 9-, and 10-fold, respectively, by sulfinpyrazone compared with 4-fold for wild-type MRP2. These results suggest that the Trp¹²⁵⁴ mutations may augment the ability of sulfinpyrazone to stimulate transport of conjugated organic anions by MRP2. In contrast, 100 µM sulfinpyrazone had no effect on [³H]MTX transport by either wild-type MRP2 or any of the four MRP2-Trp¹²⁵⁴ mutants (Fig. 5B).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of sulfinpyrazone on [3H]E217G uptake and [3H]MTX uptake by wild-type and Trp1254 mutant MRP2 proteins. A, [3H]E217G uptake was measured in the absence (open bars) and the presence (solid and shaded bars) of sulfinpyrazone (100 µM) in membrane vesicles prepared from cells expressing wild-type (WT-MRP2) and mutant (W1254A, W1254C, W1254F, and W1254Y) MRP2 proteins as described under "Experimental Procedures." The results shown are the means ± S.D. of triplicate determinations. B, y[3H]MTX uptake was measured in the absence (open bars) and the presence (solid and shaded bars) of sulfinpyrazone (100 µM) in membrane vesicles prepared from cells as described for A. Each bar represents the mean ± S.D. of triplicate determinations.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

MRP2 was first identified as a hepatocellular (canalicular) organic anion transporter and was originally cloned from rat liver using strategies that took advantage of its structural similarity to human MRP1 (11, 32). The human, rabbit, mouse, and canine cDNAs were cloned soon thereafter, and the five orthologs show a high degree of amino acid identity with one another (77-83%) (46-49). As demonstrated for MRP1, studies of MRP2-enriched membrane vesicles and the purified reconstituted recombinant protein show that MRP2 exhibits an ATPase activity that can be stimulated by its substrates (21, 50, 51). However, MRP2 is unique among the MRP-related proteins in that it is found exclusively on the apical membrane of polarized cells, whereas the other MRP transporters are localized on the basolateral membrane. Moreover, because MRP2 is expressed predominantly in hepatocytes, renal epithelia, and intestinal enterocytes, it is particularly well situated to play a role in the elimination, as well as oral bioavailability, of drugs, xenotoxins, and their conjugated metabolites (41, 52-56).

The amino acid sequence of human MRP2 is 49% identical to MRP1, and it has been well established that both proteins can transport a similar spectrum of organic anions in vitro, although it is clear that some differences exist with respect to substrate affinity and specificity. We have recently shown that the highly conserved Trp¹²⁴⁶ in MRP1 is critical for transport of the conjugated organic anion E₂17G, as well as being essential for the ability of this protein to confer resistance to cationic and electroneutral natural product chemotherapeutic agents, including doxorubicin, vincristine, and VP-16 (etoposide) (40). Because of the primary structure similarity and shared substrate specificity of MRP1 and MRP2, we anticipated that mutation of Trp¹²⁵⁴ in MRP2, which is analogous to MRP1 Trp¹²⁴⁶, would result in phenotypic changes similar to those observed in MRP1. However, unlike MRP1, where any substitution of Trp¹²⁴⁶ eliminated E₂17G transport activity (40), we observed that only nonconservative substitutions of Trp¹²⁵⁴ (Ala and Cys) resulted in a loss of transport of this conjugated organic anion by MRP2. In contrast, levels of E₂17G transport by MRP2-Trp¹²⁵⁴ mutants with conservative substitutions (Tyr and Phe) were similar to those of wild-type MRP2. These findings suggest that the architecture of the E₂17G-binding pocket of MRP2, in contrast to MRP1 (40), can be maintained as long as the amino acid at position 1254 is capable of participating in aromatic interactions. Aromatic interactions also appear critical for LTC₄ transport activity by MRP2, again in contrast to MRP1, because transport of this conjugated organic anion was eliminated when Trp¹²⁵⁴ was replaced with Ala and Cys. However, LTC₄ transport by MRP2 was also markedly reduced when Trp¹²⁵⁴ was replaced with Phe but less so when it was replaced with Tyr. These findings suggest that, in addition to its aromaticity, the hydrogen bonding properties and possibly the size of the lateral side chain of the tryptophan residue are important for MRP2-mediated transport of this glutathione-conjugated substrate. The selective loss of LTC₄ transport activity of the W1254F-MRP2 mutant was matched by the inability of the cysteinyl leukotriene to inhibit [³H]E₂17G transport by this mutant, suggesting that loss of LTC₄ transport is caused by a loss of binding of this substrate to the protein.

In addition to conjugated metabolites, the widely used anticancer agent MTX and certain structurally related endogenous reduced folate derivatives are reported to be substrates of several MRP-related proteins (8, 21, 24, 43, 56). However, as observed with their other substrates, the overall capacity of the different MRPs to transport MTX varies considerably. In the present study, we found that MRP2 in HEK cell membranes transported MTX much more efficiently than MRP1 (data not shown), in agreement with the earlier findings of Bakos et al. (21) using membrane vesicles derived from insect cells expressing these two transporters.

As mentioned previously, it has recently been shown that certain charged residues located in or close to TM segments in MSD2 and MSD3 are important in the transport of some conjugated molecules by MRP2 (38, 39). However, in these studies, transport of MTX by MRP2 was not investigated. We found that, in contrast to the transport of the conjugated substrates E₂17G and LTC₄, which was selectively lost depending on the nature of the amino acid replacing Trp¹²⁵⁴, [³H]MTX transport activity was eliminated regardless of whether the substitution was conservative or not. These data are the first to identify a single amino acid as being critical for MRP2-mediated transport of this clinically important antifolate drug and indicate a stringent requirement for a tryptophan residue at position 1254.

Previously, MTX has been reported to inhibit the transport of the glutathione conjugates of dinitrophenyl and N-ethylmaleimide by MRP2, and a competitive mode of inhibition has been suggested (21, 24). We have now shown that transport of [³H]E₂17G by wild-type MRP2 can also be inhibited by MTX. However, although [³H]E₂17G transport by the MRP2-W1254F and MRP2-W1254Y mutants appeared unchanged despite their inability to transport MTX, transport of the conjugated estrogen was no longer inhibited by the antifolate agent. These findings suggest that loss of MTX transport by the MRP2-Trp¹²⁵⁴ mutants is matched by a loss of binding of this substrate to the protein.

The role of this highly conserved Trp residue in TM17 of the COOH-proximal MSD3 of MRP2 and the related MRP1 is not clear. Because Trp¹²⁵⁴ is predicted to be at the membrane cytosol interface, it may serve to anchor the position of TM17 (57) and in this way ensure the correct positioning of the TM segments involved in substrate recognition and transport. It is also possible that it may interact directly with at least some MRP substrates because the tryptophan indole ring could contribute to cation- bond formation with substrates containing positive charges or polarities, - stacking interactions with substrates containing aromatic rings, and hydrogen bonding interactions with polar substituents (58). Of the three MRP2 substrates examined in the present study, none were transported by the nonconservatively substituted W1254C and W1254A mutants, one was transported by the W1254F mutant, and two were transported by the W1254Y mutant. These data indicate that Trp¹²⁵⁴ interacts differently with these different MRP2 substrates and further, as proposed earlier for MRP1, that each substrate establishes its own unique set of atomic contacts in a multipartite substrate binding pocket of MRP2, although it is clear that within each set, many binding determinants are held in common.

It is of interest to note that MRP2-Trp¹²⁵⁴ and MRP1-Trp¹²⁴⁶ are positioned on the same hydrophilic side of the amphipathic helix (within one position or helix turn) as the two amino acids in the comparable TM segment of the sulfonylurea receptors SUR2A/ABCC9 (Leu¹²⁴⁹ and Thr¹²⁵³) and SUR1/ABCC8 (Thr¹²⁸⁶ and Met¹²⁹⁰) that were recently shown to be intimately involved with the activation of K_ATP channels by K⁺ channel openers (59) (Fig. 6A). MRP2-Trp¹²⁵⁴ and MRP1-Trp¹²⁴⁶ are also on the same side of TM17 as Thr¹²⁴², which we have recently determined is important for MRP1 substrate specificity (60). These observations, together with labeling studies with photoaffinity analogs (61, 62), suggest that the polar face of this last TM helix plays a common and particularly critical role in the formation of a substrate-binding pocket in transport proteins belonging to the ABCC subfamily. The exceptionally high degree of amino acid conservation of the TM17 sequences of the ABCC proteins lends further support to this conclusion (Fig. 6B) (40).

View larger version (47K): [in this window] [in a new window]   Fig. 6.   Amino acid sequences of predicted transmembrane segment 17 of MRP2 and related proteins. A, helical wheel projections of the amino acid sequences of predicted TM17 of the third MSD of ABCC subfamily proteins MRP2, MRP1, and sulfonylurea receptors 1 and 2A. Open circles indicate amino acids that can participate in hydrogen-bonding interactions, whereas shaded circles indicate amino acids that cannot. Aromatic amino acids are indicated by asterisks. B, alignment of the amino acid sequences of predicted TM17 of MSD3 in human MRP2 and related ABCC proteins showing conservation of Trp1254 indicated by an asterisk and enclosed in a box. The column of numbers to the right of the aligned sequences indicates the number of amino acids in the sequence that are identical to the 23 amino acids (1236-1258) in human MRP2. Hum, human; can, canine; mus, Mus musculus; rab, rabbit; CFTR, cystic fibrosis transmembrane conductance regulator.

Several organic anions, including the uricosuric agent sulfinpyrazone, have been shown to inhibit the transport of certain MRP1 substrates, whereas at most concentrations, MRP2-mediated transport of GSH and GSH conjugates is stimulated by these organic anions (21, 27, 45). The molecular basis for this remarkable difference between MRP1 and MRP2 is unknown. However, we have now extended these earlier observations by demonstrating that sulfinpyrazone can also stimulate transport of glucuronide conjugates by MRP2 as exemplified by E₂17G. Transport stimulation by sulfinpyrazone was maintained in all of the variously substituted MRP2-Trp¹²⁵⁴ mutants, and indeed, for those with conserved substitutions, the magnitude of [³H]E₂17G transport stimulation by sulfinpyrazone was increased. Exactly how sulfinpyrazone stimulates transport of conjugated substrates by MRP2 is unclear and appears complex (27). However, our finding that sulfinpyrazone stimulated the E₂17G transport activity of all four MRP2-Trp¹²⁵⁴ mutants suggests that the binding site for this organic anion may be distinct from the binding site of the glucuronide-conjugated estrogen. Thus it appears likely that transport stimulation is occurring through some type of positive cooperativity or allosteric activation mechanism (27). Why sulfinpyrazone neither stimulated nor inhibited MTX transport by MRP2 is unknown but again is consistent with the idea of MRP2 having more than one drug-binding site.

Another remarkable difference in the interaction of MRP1 and MRP2 with organic anions was recently revealed by our demonstration that transport of the 3-O-glucuronide conjugate of the carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol by MRP2 was inhibited by GSH, whereas transport of this substrate by MRP1 was completely dependent on the presence of this tripeptide or certain of its analogs (41). The amino acids in MRP1 and MRP2 that are responsible for their differences in substrate and inhibitor/stimulator specificity and affinity remain to be identified; however, the observations of the current and previous studies indicate that it is not possible to predict the substrate or inhibitor specificity of an ABC transporter on the basis of amino acid conservation, either with homologs in the same species or orthologs in different species (1-3).

Recently, compelling evidence that polymorphisms in the MDR1 (ABCB1) gene encoding the 170-kDa P-glycoprotein can play a clinically important role in the bioavailability and disposition of a variety of important drugs including anticancer agents, immunosuppressive drugs, cardiac drugs, and steroids has been reported (63). Based on the results presented here and elsewhere (32, 33, 38-40, 64), it seems clear that certain mutations in MRP2, and possibly MRP1, can also be expected to contribute to altered pharmacokinetics of certain anticancer agents such as MTX. This in turn could affect their efficacy and/or the incidence and severity of toxic side effects associated with their use. Such mutations might also affect the susceptibility of an individual to damage incurred by exposure to endo- and xenotoxins, carcinogens, and their metabolites whose transport is mediated by these proteins (5, 12, 41, 54, 65). Several polymorphisms in MRP2 and MRP1 have recently been described in healthy Japanese subjects, but whether or not these mutations result in differences in the substrate specificity of the encoded proteins is not yet known (66). However, none of mutations described to date affect amino acids located in TM17 of either protein.

In summary, we have shown that Trp¹²⁵⁴ at the predicted cytoplasmic face of the last transmembrane segment TM17 in the third COOH-proximal MSD of MRP2, like the analogous residue in MRP1, is critical for the recognition and transport of E₂17G. We have also shown that, unlike MRP1-Trp¹²⁴⁶, MRP2-Trp¹²⁵⁴ is important for LTC₄ transport. Finally, we have identified Trp¹²⁵⁴ as being essential for the ability of MRP2 to transport MTX. Together with previous studies, these findings demonstrate that this conserved Trp residue plays a pivotal role in the substrate specificity and transport capacity of both MRP1 and MRP2, which suggests that it is also likely to be important in other members of the ABCC subfamily. The related basolateral transporter, MRP3, like MRP1 and MRP2, can confer drug resistance and transport conjugated organic anions but has its own distinctive pattern of tissue expression and substrate specificity (67-72). Experiments are in progress to examine the effect of Trp¹²⁴² substitutions on the transport activities of MRP3.

	ACKNOWLEDGEMENTS

We thank Elaine Leslie for helpful advice and discussion and Maureen Rogers for expert word processing and assistance with preparation of the figures.

	FOOTNOTES

^* 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.

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.

Published, JBC Papers in Press, August 10, 2001, DOI 10.1074/jbc.M105160200

² K. Ito, K. E. Weigl, C. J. Oleschuk, R. G. Deeley, and S. P. C. Cole, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: ABC, ATP-binding cassette; MSD, membrane spanning domain; LTC₄, leukotriene C₄; E₂17G, 17-estradiol-17--(D-glucuronide); MTX, methotrexate; GSH, reduced glutathione; HEK, human embryonic kidney; TM, transmembrane; MRP, multidrug resistance protein.

	REFERENCES

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