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J. Biol. Chem., Vol. 276, Issue 30, 27846-27854, July 27, 2001

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Transport of the -O-Glucuronide Conjugate of the Tobacco-specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the Multidrug Resistance Protein 1 (MRP1)

REQUIREMENT FOR GLUTATHIONE OR A NON-SULFUR-CONTAINING ANALOG^*

Elaine M. Leslie^§¶, Ken-ichi Ito^§, Pramod Upadhyaya, Stephen S. Hecht, Roger G. Deeley^§, and Susan P. C. Cole^§**

From the  Department of Pharmacology & Toxicology and the ^§ Cancer Research Laboratories, Queen's University, Kingston, Ontario, K7L 3N6 Canada and the  University of Minnesota Cancer Center, Minneapolis, Minnesota 55455

Received for publication, March 19, 2001, and in revised form, April 24, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) play a crucial role in the induction of lung cancer, and NNAL-O-glucuronide formation and elimination are important steps in detoxification of these compounds. In the present study, we investigated the ATP-binding cassette (ABC) protein, MRP1 (ABCC1), as a candidate transporter responsible for NNAL-O-glucuronide export. MRP1 mediates the active transport of numerous GSH-, sulfate-, and glucuronide-conjugated organic anions and can transport certain xenobiotics by a mechanism that may involve co-transport with GSH. Using membrane vesicles prepared from transfected cells, we found that MRP1 transports [³H]NNAL-O-glucuronide but is dependent on the presence of GSH (K_m 39 µM, V_max 48 pmol mg¹ min¹). We also found that the sulfur atom in GSH was dispensable because transport was supported by the GSH analog, -glutamyl--aminobutyryl-glycine. Despite stimulation of NNAL-O-glucuronide transport by GSH, there was no detectable reciprocal stimulation of [³H]GSH transport. Moreover, whereas the MRP1 substrates leukotriene C₄ (LTC₄) and 17-estradiol 17-(D-glucuronide) (E₂17G) inhibited GSH-dependent uptake of [³H]NNAL-O-glucuronide, only [³H]LTC₄ transport was inhibited by NNAL-O-glucuronide (+GSH) and the kinetics of inhibition were complex. A mutant form of MRP1, which transports LTC₄ but not E₂17G, also did not transport NNAL-O-glucuronide suggesting a commonality in the binding elements for these two glucuronidated substrates, despite their lack of reciprocal transport inhibition. Finally, the related MRP2 transported NNAL-O-glucuronide with higher efficiency than MRP1 and unexpectedly, GSH inhibited rather than stimulated uptake. These studies provide further insight into the complex interactions of the MRP-related proteins with GSH and their conjugated organic anion substrates, and extend the range of xenotoxins transported by MRP1 and MRP2 to include metabolites of known carcinogens involved in the etiology of lung and other cancers.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The nicotine-derived tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)¹ and its major reductive metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are believed to play an important role in the induction of lung cancer in smokers (1). NNK is also a potent lung carcinogen in experimental animals, independent of the route of administration (2). In order to exert their carcinogenic effects, NNK and NNAL must first be metabolically activated by a member(s) of the cytochrome P450 family of mixed function oxidases, ultimately leading to either a detoxified product or a reactive species that forms adducts with DNA (Fig. 1) (2-4). The reduction of NNK to the (R) and (S)-enantiomers of NNAL is mediated by the carbonyl reductase 11-hydroxysteroid dehydrogenase 1 (5). The enantiomers are conjugated by members of the UDP-glucuronosyltransferase family of enzymes with UDP-glucuronic acid to produce two diastereomeric O-glucuronides, NNAL-O-glucuronide I and II, which are formed from (R)- and (S)-NNAL, respectively (2, 6). NNAL-O-glucuronide I is the predominant form of this metabolite excreted by NNK-treated rats although NNAL-O-glucuronide II is the major form found in the urine of human smokers and snuff dippers (7, 8). (S)-NNAL has been shown to be more tumorigenic in mice, consistent with the finding that this enantiomer forms less O-glucuronide conjugate than (R)-NNAL. Thus, production of NNAL-O-glucuronide, which is one of three glucuronide conjugates formed during NNK metabolism (Fig. 1), is an important step in NNK detoxification.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Biotransformation pathways of NNK as determined by studies in experimental animals (rodents and primates) and humans. UGT, UDP-glucuronosyltransferase; -gluc, -glucuronidase; HPB, 4-hydroxy-1-(3-pyridyl)-1-butanone; HPB-gluc, -O-[4-oxo-4-(3-pyridyl)-but-1-yl]-D-glucosiduronic acid; P450, cytochrome P450; HSD, 11--hydroxysteroid dehydrogenase. Adapted from Murphy et al. (9).

Once generated, the glucuronide conjugates of NNAL and NNK must be actively extruded from cells. However, the transporters responsible for the elimination of these metabolites have not yet been identified. Active efflux is generally an important aspect of detoxification because it prevents the accumulation of conjugates that have the potential to be directly toxic or to be hydrolyzed, thus regenerating the parent drug or toxin. Alternatively, conjugate formation can create stable molecules that are effluxed from the cell and then transported to distant organs where they may exert selective tissue toxicity (9, 10).

The 190 kDa multidrug resistance protein 1 (MRP1/ABCC1) belongs to the "C" branch of the ATP binding cassette (ABC) superfamily of transport proteins and was originally identified on the basis of its elevated expression in a multidrug resistant lung cancer cell line (11, 12). MRP1 is expressed constitutively in many normal tissues with relatively high levels found in lung and testes (11, 13). In addition to its ability to confer resistance to chemotherapeutic agents in tumor cells, MRP1 is a primary active transporter of GSH-, sulfate-, and glucuronide-conjugated organic anions such as leukotriene C₄ (LTC₄), an important mediator of the inflammatory response, and the cholestatic-conjugated estrogen, 17-estradiol 17-(-D-glucuronide) (E₂17G) (12, 14-17). MRP1 and the related MRP2 (ABCC2) transporter have also been shown to transport conjugates of various xenobiotics and are key components of the so-called Phase III elimination pathways of drug metabolism (18-20). However, many of the anti-cancer agents to which MRP1 and MRP2 confer resistance are not conjugated to a significant extent in vivo and much in vitro evidence exists that at least some drugs are effluxed from cells by MRP1 and MRP2 via a co-transport mechanism with free GSH (14, 21-24).

In the present study, we have investigated the ability of MRP1 to transport the O-glucuronide of NNAL. Despite the fact that it is a conjugated metabolite, we found that MRP1 did not transport NNAL-O-glucuronide except in the presence of GSH or ophthalmic acid, a non-sulfur containing analog of GSH. In contrast, stimulation of GSH transport by NNAL-O-glucuronide was not detectable. E₂17G and LTC₄ inhibited GSH-dependent [³H]NNAL-O-glucuronide uptake; however, NNAL-O-glucuronide (+GSH) only reciprocally inhibited LTC₄ uptake and kinetic analysis revealed that this inhibition was not competitive. A mutant MRP1 capable of transporting LTC₄ but which does not transport E₂17G also did not transport NNAL-O-glucuronide even in the presence of GSH. Finally, we found that NNAL-O-glucuronide was transported even more efficiently by the related MRP2 but in this instance, transport was inhibited rather than stimulated by GSH.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [14,15(n)-³H]LTC₄ (38 Ci mmol¹) was purchased from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). [³H]GSH (50 Ci mmol¹) and [6,7-³H]E₂17G (55 Ci mmol¹) were from PerkinElmer Life Science Research Products (Guelph, ON, Canada). [5-³H]NNK (3.05 Ci mmol¹) was purchased from Chemsyn Science Laboratories (Lenxa, KS) and [³H]NNAL-O-glucuronide and NNAL-O-glucuronide were synthesized as described below. NNK and NNAL were purchased from Toronto Research Chemicals (Toronto, ON, Canada). LTC₄ was obtained from Calbiochem (San Diego, CA) and DTT from ICN (Aurora, OH). Verapamil, ATP, AMP, GSH, GSSG, S-decyl GSH, E₂17G, and UDP-glucuronic acid were from Sigma. Ophthalmic acid (L--glutamyl-L--aminobutyryl-glycine) was from Bachem (Torrance, CA). Creatine kinase and creatine phosphate were from Roche Molecular Biochemicals (Laval, PQ, Canada). The murine MRP1-specific MAbs QCRL-1, QCRL-3, and QCRL-4 have been described previously (25, 26) and Fab fragments were prepared using the ImmunoPure Fab kit (Pierce, Edmonton, AB, Canada).

Cell Lines-- The HeLa cell line T5 is stably transfected with a pRc/CMV vector containing the complete coding sequence of MRP1 and has been previously described (27, 28). The C1 HeLa cell line is stably transfected with the pRc/CMV vector alone. Both HeLa cell lines were maintained in RPMI 1640 medium supplemented with 4 mM L-glutamine, 5% defined bovine calf serum and 400 µg ml¹ geneticin (G418) (Life Technologies, Inc., Burlington, ON, Canada). The SV40-transformed human embryonic kidney (HEK293T) cell line was maintained in Dulbecco's modified Eagle's medium supplemented with 4 mM L-glutamine and 10% fetal bovine serum.

Synthesis of ³H-labeled and -unlabeled NNAL-O-glucuronide-- [5-³H]NNK was converted to racemic [5-³H]NNAL by NaBH₄ reduction, and then the alcohol was purified by reverse phase HPLC as described (29). The radiochemical purity was 99% with no evidence of contamination by [5-³H]NNK. [5-³H]NNAL-O-glucuronide I was prepared by overnight incubation of purified [5-³H]NNAL with rat liver microsomes, UDP-glucuronic acid, and cofactors as described previously (30), and the product was purified by HPLC as described (31). Unlabeled NNAL-O-glucuronide was produced as described (31).

MRP1 and MRP2 Expression Vectors and Transfections in HEK293T cells-- Construction of the expression vector pcDNA3.1()-MRP1_K encoding wild-type MRP1 (WT-MRP1) and the vector encoding MRP1 with the substitution of Trp¹²⁴⁶Ala (W1246A-MRP1) have been described previously (32). A full-length MRP2 cDNA was assembled from several fragments generated by PCR as will be described elsewhere² and cloned into pcDNA3.1() to generate pcDNA3.1()-MRP2. For transient transfections, the wild-type and mutant pcDNA3.1()- MRP1_K expression vectors and the pcDNA3.1()-MRP2 vector were transfected into HEK293T cells using FuGENE^TM6 (Roche Molecular Biochemicals, Laval, PQ, Canada). Briefly, cells were seeded at 3.4 × 10⁶ cells per 175-cm² culture flask, and 24 h later, 15 µg DNA and 45 µl FuGENE^TM6 (1:3 DNA:FuGENE^TM6) were combined in serum-free medium and added to each flask. When confluent (48-72 h later), the cells were harvested, and crude membranes or inside-out membrane vesicles were prepared as described below (14). Cells transfected with empty pcDNA3.1() vector were included as controls in all experiments.

Membrane Vesicle Preparation and Immunoblotting-- Plasma membrane vesicles were prepared as described with modifications (14). Briefly, transfected HeLa or HEK293T cells were homogenized in buffer containing 250 mM sucrose/50 mM Tris pH 7.5/0.25 mM CaCl₂, and protease inhibitors and cells were disrupted by N₂ cavitation. The exploded cell suspension was centrifuged at 800 × g at 4 °C, and the supernatant was layered onto a 35% (w/w) sucrose/1 mM EDTA/50 mM Tris, pH 7.4 cushion. After centrifugation at 100 000 × g at 4 °C for 1 h, the interface was removed and placed in a 25 mM sucrose/50 mM Tris, pH 7.4 buffer solution and centrifuged at 100,000 × g at 4 °C for 30 min. The membranes were washed with buffer (250 mM sucrose, 50 mM Tris, pH 7.4), resuspended by syringing with a 27-gauge needle, aliquoted, and stored at 70 °C.

Relative levels of the MRP proteins in the membrane vesicles were determined by immunoblot analysis. Briefly, after SDS-polyacrylamide gel electrophoresis and electrotransfer to a nylon membrane, blots were blocked with 4% (w/v) skim milk powder for 1 h followed by incubation with the human MRP1-specific murine MAb QCRL-1 (25, 33) or the MRP2-specific murine MAb M₂I-4 (Alexis, San Diego, CA) for 2 h. After washing, blots were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (Pierce) followed by application of Renaissance^R chemiluminescence blotting substrate (PerkinElmer Life Science Products, Guelph, ON, Canada).

[³H]NNAL-O-glucuronide Transport Studies-- NNAL-O-glucuronide transport assays were carried out by a rapid filtration method as described (14). Membrane vesicles (10 µg of protein per time point) prepared from stably transfected HeLa cells were incubated at 37 °C in a total reaction volume of 60 µl containing ATP or AMP (4 mM), MgCl₂ (10 mM), DTT (10 mM), creatine phosphate (10 mM), creatine kinase (100 µg ml¹), ±GSH (3 mM), and [³H]NNAL-O-glucuronide (400 nM, 40 nCi). At selected time points, a 14-µl aliquot was removed and placed in 800 µl of Tris/sucrose buffer and filtered through glass fiber filters (type A/E), washed twice, and radioactivity was quantitated by liquid scintillation counting. Transport in the presence of AMP was subtracted from transport in the presence of ATP and reported as ATP-dependent [³H]NNAL-O-glucuronide uptake.

Kinetic parameters were determined by measuring the initial rate of [³H]NNAL-O-glucuronide uptake (in the presence of 3 mM GSH) at eight different substrate concentrations (0.25-25 µM, 40 nCi) at a single time point of 10 min. Experiments with potential modulators of GSH-stimulated [³H]NNAL-O-glucuronide transport [E₂17G (25 µM), LTC₄ (1 µM), verapamil (100 µM), and Fab fragments of the MRP1-specific MAbs QCRL-1, 3, and 4 (10 µg ml¹)] were also carried out for 10 min. ATP-dependent transport of [³H]NNAL-O-glucuronide in the presence of S-methyl GSH (3 mM), S-decyl GSH (0.5 mM), GSSG (0.5 mM), and ophthalmic acid (3 mM) was measured under the same conditions.

[³H]NNAL-O-glucuronide transport assays with membrane vesicles prepared from HEK293T cells transiently transfected with empty vector, wild-type MRP1, mutant W1246A-MRP1, and wild-type MRP2 cDNAs were carried out under the same conditions as described above except incubations were for 15 min. Prior to measuring NNAL-O-glucuronide transport, vesicles were tested for [³H]E₂17G and [³H]LTC₄ uptake activities as described below.

[³H]GSH Transport Studies-- T5 HeLa membrane vesicles (20 µg protein) were preincubated with 500 µM acivicin (an inhibitor of -glutamyltranspeptidase) at 37 °C for 10 min and then transport activity was measured by further incubation for 20 min at 37 °C in a total reaction volume of 60 µl containing [³H]GSH (100 µM, 300 nCi), DTT (10 mM), and components as described above for [³H]NNAL-O-glucuronide transport. NNAL-O-glucuronide was dissolved in phosphate-buffered saline, diluted in Tris (50 mM, pH 7.4) sucrose (250 mM) buffer, and added over a concentration range of 0-100 µM. Uptake in the presence of verapamil (100 µM), which has previously been demonstrated to stimulate MRP1-mediated GSH transport (34), was included as a positive control.

[³H]E₂17G Transport Studies-- T5 HeLa cell membrane vesicles (5 µg of protein) were incubated at 37 °C for 90 s in a total reaction volume of 50 µl containing [³H]E₂17G (400 nM, 40 nCi), GSH (3 mM), DTT (10 mM), NNK or NNAL or NNAL-O-glucuronide (0, 10, and 100 µM, respectively) and components as described above for [³H]NNAL-O-glucuronide transport. E₂17G transport activity of vesicles prepared from transiently transfected HEK293T cells was tested as described above except 8 µg of protein were used, and incubations were carried out for 5 min.

[³H]LTC₄ Transport Studies-- To measure LTC₄ uptake, assays were carried out at 23 °C in a 50-µl volume containing vesicle protein prepared from T5 HeLa cells or HEK293T cells transiently transfected with empty vector, wild-type, and W1246A-MRP1 cDNAs (2 µg), [³H]LTC₄ (50 nM; 40 nCi), NNK or NNAL or NNAL-O-glucuronide (0, 10, 100 µM, respectively) and components as described above for [³H]NNAL-O-glucuronide transport. After incubation for 60 s, the entire reaction mixture was removed and added to 800 µl of ice-cold Tris/sucrose buffer and filtered as described above.

The mode of LTC₄ transport inhibition by NNAL-O-glucuronide plus GSH was further examined by measuring LTC₄ uptake during the linear phase of transport (30 s) as described above except LTC₄ was added at eight different concentrations (10-1000 nM), in the presence of GSH (3 mM), and the presence or absence of NNAL-O-glucuronide (100 µM).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

[³H]NNAL-O-glucuronide Uptake in Membrane Vesicles Requires GSH-- To determine whether NNAL-O-glucuronide was a substrate for MRP1, ATP-dependent uptake into membrane vesicles prepared from stably transfected HeLa cells was determined. As shown in Fig. 2A, membrane vesicles from MRP1-transfected T5 cells contain substantial levels of MRP1 in contrast to the vesicles from vector-transfected C1 cells where MRP1 was not detectable under the immunoblotting conditions used. A time course of [³H]NNAL-O-glucuronide uptake by the T5 and C1 membrane vesicles is shown in Fig. 2B. ATP-dependent uptake of [³H]NNAL-O-glucuronide by the MRP1-enriched T5 vesicles was extremely low and similar to uptake observed with AMP or vector-transfected control C1 vesicles. However, in the presence of GSH (3 mM), ATP-dependent uptake of NNAL-O-glucuronide by the T5 vesicles but not the C1 vesicles was observed. Uptake was linear for 10 min, at which time it reached a level of 4 pmol mg¹ and then began to plateau.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   MRP1-mediated GSH-dependent [3H]NNAL-O-glucuronide uptake by inside-out membrane vesicles. A, immunoblot of membrane vesicles prepared from HeLa cells stably transfected with empty vector (C1) and MRP1 cDNA (T5). Vesicle protein (2 µg) was resolved on a 7% acrylamide gel, transferred, and probed with MAb QCRL-1 as described in "Experimental Procedures." B, ATP-dependent transport of [3H]NNAL-O-glucuronide in the presence of GSH by C1 (control) () and T5 (MRP1-enriched) () membrane vesicles, and in the absence of GSH by T5 membrane vesicles (). Membrane vesicles (10 µg of protein per time point) were incubated at 37 °C in transport buffer with [3H]NNAL-O-glucuronide (400 nM, 40 nCi) with and without 3 mM GSH. C, GSH-dependent [3H]NNAL-O-glucuronide uptake was measured after preincubation of T5 membrane vesicles with Fab fragments (10 µg ml1) prepared from the indicated MRP1-specific MAbs.

Inhibition of GSH-dependent [³H]NNAL-O-glucuronide Transport by MRP1-specific MAbs-- Several MRP1-specific MAbs have been shown previously to inhibit transport of conjugated and unconjugated MRP1 substrates including LTC₄, E₂17G, aflatoxin B₁-GS, and vincristine in the presence of GSH (14, 15, 21, 26, 35). The Fab fragments of these antibodies also block transport indicating that a direct and specific interaction with MRP1 occurs (26). When the Fab fragments of MAbs QCRL-3 and -4 (which recognize different conformation-dependent epitopes) were tested for their ability to inhibit GSH-dependent uptake of [³H]NNAL-O-glucuronide by T5 membrane vesicles, complete inhibition was observed at a concentration of 10 µg ml¹ (Fig. 2C). MAb QCRL-1, which recognizes a linear epitope in part of the linker region of MRP1 and does not inhibit transport of other MRP1 substrates (33), had no effect on NNAL-O-glucuronide transport. These results further confirm the MRP1 specificity of GSH-dependent transport of NNAL-O-glucuronide.

Kinetic Analysis of MRP1-mediated GSH-dependent [³H]NNAL-O-glucuronide Uptake-- GSH-dependent transport of NNAL-O-glucuronide by MRP1 was further characterized by determining initial rates of uptake over several concentrations of substrate in the presence of 3 mM GSH (Fig. 3). Data were analyzed with GraphPad Prism^TM Michaelis-Menten curve fit and K_m and V_max values were estimated to be 39 µM and 48 pmol mg¹ min¹, respectively (mean of two independent determinations).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Kinetic analysis of MRP1-mediated GSH-dependent [3H]NNAL-O-glucuronide uptake. ATP- and GSH-dependent uptake of [3H]NNAL-O-glucuronide by membrane vesicles prepared from MRP1-transfected T5 HeLa cells was measured at various NNAL-O-glucuronide concentrations (0.25-25 µM) in the presence of 3 mM GSH for 10 min at 37 °C. Data were analyzed with GraphPad PrismTM Michaelis-Menten curve fit and kinetic parameters determined. The graph shown represents results obtained in a single experiment and the data points represent means (± S.D.) of triplicate determinations. Similar results were obtained in a second experiment. The mean Km and Vmax values were estimated to be 39 µM and 48 pmol mg1 min1, respectively.

Effect of GSH Derivatives and Ophthalmic Acid on [³H]NNAL-O-glucuronide Transport-- [³H]NNAL-O-glucuronide uptake was also measured in the presence of S-methyl GSH as well as S-decyl GSH and GSSG. As shown previously for MRP1-mediated transport of vincristine (21), S-methylGSH (3 mM) supported NNAL-O-glucuronide uptake to levels that were 75% of those observed in the presence of GSH. In contrast, S-decylGSH and GSSG did not support the transport of NNAL-O-glucuronide at all (Fig. 4A). [³H]NNAL-O-glucuronide uptake was also measured in the presence of ophthalmic acid (L--glutamyl-L--aminobutyryl-glycine), a non-sulfur containing, endogenously formed tripeptide in which the sulfhydryl moiety of the cysteine residue of GSH has been replaced by a methyl group (Fig. 4B) (36). Ophthalmic acid supported [³H]NNAL-O-glucuronide uptake at levels that were ~60% of GSH-stimulated uptake levels, demonstrating that the sulfur atom of GSH is not essential for its interaction with MRP1.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of GSSG and non-thiol tripeptides on MRP1-mediated transport of [3H]NNAL-O-glucuronide. A, T5 HeLa cell membrane vesicles (10 µg of protein) were incubated for 10 min at 37 °C in transport buffer with [3H]NNAL-O-glucuronide (400 nM, 40 nCi) in the absence (open bar) or presence (shaded bars) of GSH (3 mM), S-methylGSH (3 mM), GSSG (0.5 mM), S-decylGSH (0.5 mM) or ophthalmic acid (3 mM) (solid bar) as indicated. Results are expressed as a percent of GSH-dependent NNAL-O-glucuronide transport. Bars represent means of triplicate determinations (± S.D.). B, chemical structures of GSH, S-methylGSH, and the non-sulfur containing GSH analog, ophthalmic acid.

NNAL-O-glucuronide Does Not Stimulate MRP1-mediated [³H]GSH Transport-- In previous studies, we have shown reciprocal stimulation of transport of GSH and certain xenobiotics (e.g. vincristine) (21). Consequently, NNAL-O-glucuronide was tested for its ability to stimulate [³H]GSH uptake in MRP1-enriched membrane vesicles (Fig. 5). Verapamil (100 µM) was included as a positive control and stimulated GSH uptake by ~4-fold, as expected based on previous observations (34). In contrast, NNAL-O-glucuronide (3-100 µM) did not detectably stimulate transport of [³H]GSH.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Effect of NNAL-O-glucuronide on MRP1-mediated [3H]GSH transport. Membrane vesicles prepared from MRP1-transfected HeLa cells (T5) were incubated in transport buffer at 37 °C for 20 min with 100 µM [3H]GSH (300 nCi), DTT (10 mM), and the indicated concentrations of NNAL-O-glucuronide (hatched bars). Results are expressed as fold-stimulation of control [3H]GSH uptake activity by T5 membrane vesicles, which was 0.26 ± 0.06 nmol mg protein1 (open bar). [3H]GSH uptake in the presence of 100 µM verapamil (VRP) was included as a positive control (solid bar). The results shown were obtained in a single experiment and bars represent means of triplicate determinations (± S.D.). Similar results were obtained in two additional experiments.

Inhibition of GSH-dependent [³H]NNAL-O-glucuronide Uptake by Various Organic Anions-- Previous studies have shown that a variety of conjugated organic anions such as E₂17G and LTC_4, as well as unconjugated drugs such as the phenylalkylamine verapamil in the presence of GSH, can act as competitive inhibitors of MRP1 transport (14, 15, 21, 34). GSH-dependent transport of [³H]NNAL-O-glucuronide (400 nM) was also inhibited by E₂17G (25 µM), LTC₄ (1 µM), and verapamil (100 µM) by 80 ± 10%, 50 ± 3%, and 80 ± 10%, respectively (Fig. 6).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Inhibition of GSH-dependent [3H]NNAL-O-glucuronide uptake. ATP- and GSH-dependent uptake of [3H]NNAL-O-glucuronide by membrane vesicles prepared from MRP1-transfected T5 HeLa cells was measured at an initial substrate concentration of 400 nM (40 nCi) for 10 min at 37 °C in the presence of the organic anions LTC4 (1 µM) and E217G (25 µM), and the phenylalkylamine verapamil (100 µM). Results are expressed as a percent of control ATP- and GSH-dependent transport activity in the absence of inhibitor which was 10.3 pmol mg protein1. Bars represent means of triplicate determinations (± S.D.) in a single experiment.

Effect of NNAL-O-glucuronide on MRP1-mediated [³H]E₂17G and [³H]LTC₄ Transport-- To determine whether NNAL-O-glucuronide could modulate the transport of other conjugated organic anion substrates of MRP1, the effect of NNAL-O-glucuronide (10 and 100 µM) on [³H]E₂17G and [³H]LTC₄ uptake was tested in the presence of GSH. NNAL-O-glucuronide had no effect on [³H]E₂17G uptake (Table I). The effect of NNK and NNAL on [³H]E₂17G and [³H]LTC₄ transport was also measured but these compounds did not modulate transport of these MRP1 substrates, even in the presence of GSH (data not shown). In contrast, NNAL-O-glucuronide (100 µM) inhibited [³H]LTC₄ transport by 45% but only in the presence of GSH.

View this table: [in this window] [in a new window]   Table I Effect of NNAL-O-glucuronide plus GSH on 17-estradiol 17-(-D-glucuronide) and leukotriene C4 uptake by MRP1-enriched membrane vesicles Membrane vesicles prepared from stably transfected HeLa cells were incubated with the indicated substrates, NNAL-O-glucuronide and 3 mM GSH as described in "Experimental Procedures." The values shown are the means (± S.D.) of triplicate determinations.

The inhibition of [³H]LTC₄ transport was further characterized by measuring the effect of NNAL-O-glucuronide (100 µM) plus GSH (3 mM) on [³H]LTC₄ uptake at different substrate concentrations (10-1000 nM) (Fig. 7). Data were analyzed with GraphPad Prism^TM Michaelis-Menten curve fit program and kinetic parameters were determined. In the absence of NNAL-O-glucuronide the K_m and V_max values for LTC₄ were 67 nM and 188 pmol mg¹ min¹ respectively; in the presence of NNAL-O-glucuronide both the apparent K_m and V_max were increased to 328 nM and 445 pmol mg¹ min¹, respectively. Similar values were also obtained from Eadie-Hofstee plots.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Modulation of [3H]LTC4 uptake by NNAL-O-glucuronide and GSH. Uptake of [3H]LTC4 (10-1000 nM) by membrane vesicles prepared from MRP1-transfected HeLa T5 cells (2 µg of protein) was measured at 23 °C for 30 s. Assays were carried out with 3 mM GSH in the absence () and in the presence () of 100 µM NNAL-O-glucuronide. Data were analyzed with GraphPad PrismTM Michaelis-Menten curve fit and kinetic parameters determined. In the absence of NNAL-O-glucuronide the Km and Vmax values of LTC4 were 67 nM and 188 pmol mg1 min1, respectively; in the presence of NNAL-O-glucuronide, the apparent Km and Vmax were 328 nM and 445 pmol mg1 min1, respectively. Data points represent means (±S.D.) of triplicate determinations in a typical experiment. Similar results were found in three additional experiments.

MRP1 With a Trp¹²⁴⁶Ala Mutation Does Not Transport [³H]NNAL-O-glucuronide-- We have previously shown that substitution of the tryptophan residue at position 1246 in the last transmembrane segment of MRP1 is crucial for its ability to transport E₂17G (32). To determine whether Trp¹²⁴⁶ was also essential for GSH-dependent transport of NNAL-O-glucuronide, transport assays were carried out using membrane vesicles prepared from HEK293T cells transfected with pcDNA3.1() (empty vector), WT-MRP1, and W1246A-MRP1 cDNAs. The W1246A-MRP1 mutant and WT-MRP1 proteins were expressed at similar levels in the HEK293T cells as determined by immunoblot analysis of the membrane vesicles (Fig. 8A). Moreover, [³H]LTC₄ uptake by the W1246A-MRP1 mutant was comparable with uptake by WT-MRP1 whereas [³H]E₂17G uptake by the mutant protein was undetectable, as reported previously (Fig. 8B) (32). In contrast, whereas the addition of GSH markedly stimulated uptake of [³H]NNAL-O-glucuronide by WT-MRP1, uptake by the MRP1-W1246A mutant membrane vesicles remained low and comparable with that of membrane vesicles from cells transfected with empty vector (Fig. 8C). As expected, [³H]NNAL-O-glucuronide uptake in membrane vesicles from the wild-type, W1246A mutant and vector control-transfected cells in the absence of GSH was extremely low and indistinguishable from one another.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   GSH-dependent uptake of [3H]NNAL-O-glucuronide by wild-type and mutant W1246A-MRP1. A, membrane vesicles prepared from HEK293T cells transfected with wild-type MRP1 (WT-MRP1), mutant MRP1 (W1246A-MRP1), or empty vector (pcDNA3.1()) were immunoblotted with the MRP1-specific MAb QCRL-1 as described in "Experimental Procedures." Five µg of vesicle protein were loaded per lane. B, ATP-dependent uptake of [3H]LTC4 (left) and [3H] E217G (right) using membrane vesicles described in A. Bars represent the mean of triplicate (± S.D.) and duplicate determinations for [3H]LTC4 and [3H] E217G, respectively. C, ATP- and GSH-dependent uptake of [3H]NNAL-O-glucuronide was measured at an initial substrate concentration of 400 nM (40 nCi) for 15 min at 37 °C in the absence (solid bars) and presence (open and hatched bars) of 3 mM GSH. Bars represent means of triplicate determinations (± S.D.).

Transport of [³H]NNAL-O-glucuronide by MRP2 Is Inhibited by GSH-- To determine whether NNAL-O-glucuronide was also a substrate for the related MRP2 transporter, membrane vesicles were prepared from HEK293T cells transfected with pcDNA3.1()-WT-MRP2 and control pcDNA3.1() vectors. The MRP2-transfected cells showed significant levels of expression as determined by immunoblotting with the MRP2-specific MAb M₂I-4 whereas expression of MRP2 in the vector control-transfected cells was not detectable (Fig. 9A). Transport assays were carried out in the presence and absence of 3 mM GSH as before and, in striking contrast to MRP1, [³H]NNAL-O-glucuronide uptake by MRP2 showed no requirement for GSH (Fig. 9B). Levels of [³H]NNAL-O-glucuronide uptake by MRP2 were 22 pmol mg protein¹ whereas uptake by MRP1 vesicles under the same conditions was the same as the vector control vesicles (1 pmol mg protein¹). Again, in marked contrast to MRP1, addition of GSH almost completely inhibited [³H]NNAL-O-glucuronide uptake by MRP2. Thus, in the presence of 3 mM GSH, uptake by MRP2 was reduced more than 7-fold from 22 pmol mg protein¹ to 3.1 pmol mg protein¹ whereas uptake by MRP1 was 6.3 pmol mg protein¹.

View larger version (11K): [in this window] [in a new window]   Fig. 9.   Uptake of [3H]NNAL-O-glucuronide by MRP2. A, membrane vesicles prepared from HEK293T cells transfected with wild-type MRP2 (WT-MRP2), and empty vector (pcDNA3.1()) were immunoblotted with the MRP2-specific MAb M2I-4 as described in "Experimental Procedures." Five µg of vesicle protein were loaded per lane. B, ATP-dependent uptake of [3H]NNAL-O-glucuronide by MRP2 and MRP1 were measured at an initial substrate concentration of 400 nM (40 nCi) for 15 min at 37 °C in the presence (open and hatched bars) and absence (solid bars) of 3 mM GSH. Bars represent means of triplicate determinations (± S.D.). The WT-MRP1 vesicles used in these experiments are those shown in Fig. 8.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Several lines of evidence from in vitro and in vivo studies indicate that MRP1 has a role in protecting tissues from toxin-induced damage (20). For example, GSH conjugates of the carcinogenic mycotoxin, aflatoxin B₁, are substrates of MRP1 (35), as is the GSH conjugate of the major membrane lipid peroxidation product, 4-hydroxynonenal (37). In addition, comparative studies of mrp1(/) knock-out mice and their wild-type mrp1(+/+) counterparts have shown that mrp1 is important for protection of the choroid plexus epithelium, the oropharyngeal layer and the testicular tubules from the toxic effects of the anti-cancer drug etoposide (38-40). The results of the present study have now extended the range of xenotoxins that can be transported by MRP1 by demonstrating ATP- and GSH-dependent uptake of the tobacco-specific metabolite NNAL-O-glucuronide by MRP1-enriched membrane vesicles (Fig. 2).

In addition, our findings provide further insight into the complex interactions of the MRP-related proteins with GSH and their conjugated substrates. Whereas transport of unconjugated drugs such as vincristine has been shown previously to be GSH-dependent (21), the GSH dependence of NNAL-O-glucuronide transport by MRP1 was unexpected because transport of other conjugated organic anions by this transporter has displayed no such requirement. Precisely how GSH facilitates or enhances transport of certain conjugated and unconjugated MRP1 substrates is not yet known but it is clearly not related to the sulfhydryl reducing capacity of this tripeptide because S-methyl GSH and other short chain alkyl derivatives of GSH also support transport (17, 21, 35). Furthermore, our demonstration that GSH can be replaced with ophthalmic acid, an endogenous tripeptide analog of GSH which contains -aminobutyrate instead of cysteine, provides direct evidence that even the sulfur atom of GSH is not required for its transport modulating interactions with MRP1 (Fig. 4). Recently MRP1-mediated transport of etoposide glucuronide was reported to be enhanced 3-fold by GSH, and the uptake of the sulfate conjugate estrone 3-sulfate was enhanced 10-fold but, unlike NNAL-O-glucuronide, transport of these conjugated substrates also occurred in the absence of the tripeptide (16, 17, 41). Interestingly, ophthalmic acid also supports MRP1-mediated vincristine transport and enhances estrone 3-sulfate uptake.³ In this respect, MRP1 bears some similarity to rat Oatp2, which mediates bidirectional transport of organic anions by a GSH-sensitive facilitative diffusion mechanism (42). Thus, recent studies have shown that GSH derivatives including ophthalmic acid may also serve as intracellular substrates for this anion exchanger.

Earlier we showed that GSH not only increases the efficiency of vincristine transport by MRP1 but also enhances the affinity of the protein for the drug (21), and it has been proposed that binding of GSH to MRP1 may facilitate transport by inducing a conformational change in the protein or by exposing key amino acids that favor binding and transport of its substrates (12, 21, 43). An alternative possibility is that GSH, S-methyl GSH and now ophthalmic acid, may actually mask a site in MRP1 that diminishes the affinity of the protein for certain substrates. Our finding that NNAL-O-glucuronide transport by the related MRP2 does not require GSH and indeed, is strongly inhibited by GSH (Fig. 9B), indicates that the amino acids that comprise this site, if it exists, are not the same in MRP1 and MRP2.

The stimulation of [³H]NNAL-O-glucuronide uptake by GSH raised the possibility that GSH might be co-transported with this compound as has been shown previously for several other MRP1 substrates including etoposide and vincristine (21, 22, 38). However, [³H]GSH uptake was not stimulated by concentrations of NNAL-O-glucuronide up to 100 µM (Fig. 5). In this respect, NNAL-O-glucuronide may be compared with estrone 3-sulfate whose transport by MRP1 was markedly enhanced by GSH but reciprocal stimulation of GSH transport was not detected (17). On the other hand, it contrasts with our previous demonstration of vincristine-stimulated [³H]GSH transport and verapamil-stimulated [³H]GSH transport, which were readily detectable under experimental conditions and substrate concentrations similar to those described here (21, 34). Interestingly, although E₂17G transport by the MRP-related AtMRP2 transporter from the vascular plant Arabidopsis thalania was enhanced by GSH, the converse was not true and indeed, in this instance, E₂17G inhibited [³H]GSH transport (44). Currently the structural and/or physical features that determine whether a conjugated or unconjugated substrate will stimulate GSH transport by MRP1 and vice versa are not well understood, although a certain degree of hydrophobicity appears essential (45).

Based on the observed ability of MRP1 to transport conjugated organic anions and unconjugated xenobiotics in the presence of GSH, it has been proposed that MRP1 contains a bipartite binding site for the hydrophobic and anionic elements of its conjugated substrates (14, 46, 47). While this may hold functionally, it is now clear that although binding of many substrates may be mutually exclusive, each substrate/inhibitor forms its own individual set of atomic contacts within a multipartite binding pocket(s) of the protein (14, 15, 21, 32, 34, 48, 49). Such a model has been proposed for the transcriptional regulator of the Bacillus subtilis multidrug transporter Bmr, which binds multiple structurally dissimilar hydrophobic cations, and is supported by considerable biochemical and structural data (50, 51). According to this model, some MRP1 substrates/modulators could bind to identical sites in the protein, while others could bind to different sites that may or may not overlap. Whether or not binding of a second substrate/modulator is influenced by binding of the first, would therefore depend on the extent of overlap or whether the binding sites interact in some allosteric fashion (51). This model could explain some of the observations of the transport inhibition studies reported here. Thus while GSH-dependent [³H]NNAL-O-glucuronide transport was inhibited by E₂17G and LTC₄ (Fig. 6), NNAL-O-glucuronide (+GSH) inhibited only [³H]LTC₄ but not [³H] E₂17G transport (Table I). The lack of reciprocity between E₂17G and NNAL-O-glucuronide (+GSH) transport inhibition indicates that the binding pockets for these two glucuronidated substrates are not identical. However, a commonality of at least some binding determinants is suggested by the observation that neither E₂17G nor NNAL-O-glucuronide (+GSH) were transported by the mutant W1246A-MRP1 (Fig. 8) (32). In addition, E₂17G is significantly bulkier than NNAL-O-glucuronide and has a higher affinity for MRP1, which might explain how this conjugated estrogen could block the interaction of NNAL-O-glucuronide with MRP1 but not the converse (14, 15, 21, 34).

In the presence of NNAL-O-glucuronide (+GSH), the apparent K_m and V_max of LTC₄ were both increased (Fig. 7). The increase in V_max indicates that the increase in K_m does not simply reflect competitive inhibition. One possible explanation for this observation is that NNAL-O-glucuronide (+GSH) interacts with MRP1 and disrupts only some, but not all, LTC₄ binding determinants in the substrate binding pocket of the protein. This would decrease the affinity of MRP1 for LTC₄ but could also increase the release of this substrate after transport across the membrane, resulting in the observed increase in transport efficiency. The increase in V_max also indicates that the NNAL-O-glucuronide (+GSH) and LTC₄ interact at non-identical sites on MRP1. Consistent with this conclusion is the observation that the W1246A mutant of MRP1 retains its ability to transport LTC₄ but does not transport the tobacco-derived glucuronide (Fig. 8) (32).

The NNAL-O-glucuronide used in this study was predominantly the NNAL-O-glucuronide I isomer because (R)-NNAL is the major reductive product of NNK formed by rat liver microsomes, which were used to prepare the radiolabeled-conjugated metabolite. However, the predominant diastereomer found in human urine is NNAL-O-glucuronide II (7, 8) and although likely, the ability of MRP1 to transport this diastereomer remains to be confirmed experimentally. Previously we have shown that MRP1 transports both the endo and exo GSH conjugates of aflatoxin B₁ with similar efficiency (35). MRP1 also transports the R and S-diastereomers of prostaglandin A₂ GSH conjugates with comparable efficiency (52). Consequently, it seems unlikely that MRP1 would exhibit stereospecificity for the NNAL-O-glucuronide enantiomers. On the other hand, we have shown that MRP1 can display remarkably different affinities for closely related structural isomers of its glucuronidated estrogen substrates. For example, the ability of conjugated estrogens to inhibit MRP1-mediated E₂17G and aflatoxin B₁-GS transport is highly dependent on the site of conjugation of the steroid nucleus. Thus, while 16, 17-estriol 17-(-D-glucuronide) was a very good inhibitor of E₂17G and aflatoxin B₁-GS transport, the 16-(-D-glucuronide) conjugate was not (15, 35). Low levels of two additional glucuronide conjugates of NNK are also formed during metabolism, -hydroxymethyl NNK-glucuronide, and HPB-glucuronide (Fig. 1), but whether or not they are MRP1 substrates remains to be determined (53).

Finally, it is important to consider the potential for other MRP-related proteins to transport NNAL-O-glucuronide and other NNK-related conjugated metabolites. Previous studies using microsomes prepared from human tissues indicate that little NNAL-O-glucuronide is formed in the lung, and that the majority of the metabolite is formed in the liver (6). In smokers, an estimated 40-100% of the NNK consumed is excreted in the urine as NNAL and NNAL-O-glucuronide (I and II) (54). This urinary pathway of glucuronide excretion is similar in several laboratory primate models but contrasts with rodent models, for which excretion into the bile is the predominant elimination pathway (2, 54). The liver expresses very low levels of MRP1 but the related transporter, MRP2, is expressed at much higher levels in this tissue (55). The organic anions transported by MRP2 are similar to those transported by MRP1, although in many instances, the affinities of the two proteins for individual substrates differ substantially (18, 19, 56). In addition, unlike MRP1 and MRP3, which are localized to basolateral membranes in the liver, MRP2 is localized to apical (canalicular) membranes and thus transports its substrates into the bile. MRP1 and MRP2 are also expressed in the kidney; however, only MRP2 is localized in the apical domain of proximal tubule epithelia where it can function to export conjugated organic anions into the urine (57). Thus, our demonstration that NNAL-O-glucuronide is a substrate of MRP2 suggests that in humans this transporter could play a role in the renal excretion of NNAL-O-glucuronide (Fig. 9). In rodents, mrp2 could be important for the secretion of NNAL-O-glucuronide into the bile and thus it would be of interest to investigate the absorption, disposition, and elimination of NNK and its conjugated metabolites in hyperbilirubinemic mutant rats, which are deficient in this protein (58, 59). Our finding that NNAL-O-glucuronide transport by MRP2, in contrast to MRP1, does not require the addition of GSH was unexpected (Fig. 9) and represents the first time such a difference between MRP1 and MRP2 mediated transport of a conjugated organic anion has been observed. It also indicates that whereas the substrate binding pockets of the two proteins may share some physical similarities, there clearly must be some significant differences.

MRP3, which has greater amino acid similarity with MRP1 than MRP2, reportedly has a higher affinity and capacity for glucuronide-conjugated anions compared with GSH-conjugated anions (60, 61). Expression of the MRP3 transporter may also be induced by a number of xenobiotics and when secretion into the bile is blocked (62-67). Thus, in certain circumstances, organic anions that would normally be excreted into the bile might be transported out of the liver and re-enter the systemic circulation via MRP3 and possibly MRP1. In this way, conjugates such as NNAL-O-glucuronide formed in the liver could conceivably be transported to other tissues where they could be cleaved to regenerate the parent toxicant and exert tissue-specific toxic effects. Of interest in this respect is the recent finding that cotinine, the major metabolite of nicotine, inhibits bile flow and biliary elimination of NNK metabolites in rodents (68). While highly speculative at this point, it is possible that this inhibition could result in induction of MRP3 expression and enhanced efflux of NNAL-O-glucuronide across basolateral membranes into the systemic circulation. Consequently, it will be of particular interest to determine whether NNAL-O-glucuronide and other nicotine metabolites are substrates and/or inducers of this basolateral transporter.

	ACKNOWLEDGEMENTS

We thank Libby Eastman and Kathy Sparks for tissue culture support, Dr. Q. Mao for preparation of Fab fragments, Dr. J. N. Reynolds for helpful kinetic discussions, and Maureen Rogers for assistance in the preparation and submission of the manuscript.

	FOOTNOTES

^* This work was supported by Grant MT-10519 from the Medical Research Council of Canada (MRCC)/Canadian Institutes of Health Research (CIHR) and Grant no. CA-81301 from the National Institutes of Health.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.

^¶ Recipient of an MRCC Doctoral Award.

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

Published, JBC Papers in Press, May 25, 2001, DOI 10.1074/jbc.M102453200

² C. W. Westlake, M. Vasa, S. P. C. Cole, and R. G. Deeley, unpublished observations.

³ E. M. Leslie, R. G. Deeley, and S. P. C. Cole, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; (S/R)-NNAL-O-glucuronide, 4-[(methylnitrosamino)-1-(3-pyridyl)- but-1-yl]--O-D-glucosiduronic acid; ABC, ATP-binding cassette; GSSG, glutathione disulfide; DTT, dithiothreitol; LTC₄, leukotriene C₄; E₂17G, 17-estradiol 17-(D-glucuronide); MAb, monoclonal antibody; HPLC, high performance liquid chromatography; WT, wild type.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				K. Koike, C. J. Oleschuk, A. Haimeur, S. L. Olsen, R. G. Deeley, and S. P. C. Cole Multiple Membrane-associated Tryptophan Residues Contribute to the Transport Activity and Substrate Specificity of the Human Multidrug Resistance Protein, MRP1 J. Biol. Chem., December 13, 2002; 277(51): 49495 - 49503. [Abstract] [Full Text] [PDF]

				D. Burg, P. Wielinga, N. Zelcer, T. Saeki, G. J. Mulder, and P. Borst Inhibition of the Multidrug Resistance Protein 1 (MRP1) by Peptidomimetic Glutathione-Conjugate Analogs Mol. Pharmacol., November 1, 2002; 62(5): 1160 - 1166. [Abstract] [Full Text] [PDF]

				A. Haimeur, R. G. Deeley, and S. P. C. Cole Charged Amino Acids in the Sixth Transmembrane Helix of Multidrug Resistance Protein 1 (MRP1/ABCC1) Are Critical Determinants of Transport Activity J. Biol. Chem., October 25, 2002; 277(44): 41326 - 41333. [Abstract] [Full Text] [PDF]

				Y.-M. Qian, C. E. Grant, C. J. Westlake, D.-W. Zhang, P. A. Lander, R. L. Shepard, A. H. Dantzig, S. P. C. Cole, and R. G. Deeley Photolabeling of Human and Murine Multidrug Resistance Protein 1 with the High Affinity Inhibitor [125I]LY475776 and Azidophenacyl-[35S]Glutathione J. Biol. Chem., September 13, 2002; 277(38): 35225 - 35231. [Abstract] [Full Text] [PDF]

				Q. Mao, W. Qiu, K. E. Weigl, P. A. Lander, L. B. Tabas, R. L. Shepard, A. H. Dantzig, R. G. Deeley, and S. P. C. Cole GSH-dependent Photolabeling of Multidrug Resistance Protein MRP1 (ABCC1) by [125I]LY475776. EVIDENCE OF A MAJOR BINDING SITE IN THE COOH-PROXIMAL MEMBRANE SPANNING DOMAIN J. Biol. Chem., August 2, 2002; 277(32): 28690 - 28699. [Abstract] [Full Text] [PDF]

				P. M. Gerk and M. Vore Regulation of Expression of the Multidrug Resistance-Associated Protein 2 (MRP2) and Its Role in Drug Disposition J. Pharmacol. Exp. Ther., August 1, 2002; 302(2): 407 - 415. [Abstract] [Full Text] [PDF]

				Y.-M. Qian, W. Qiu, M. Gao, C. J. Westlake, S. P. C. Cole, and R. G. Deeley Characterization of Binding of Leukotriene C4 by Human Multidrug Resistance Protein 1. EVIDENCE OF DIFFERENTIAL INTERACTIONS WITH NH2- AND COOH-PROXIMAL HALVES OF THE PROTEIN J. Biol. Chem., October 12, 2001; 276(42): 38636 - 38644. [Abstract] [Full Text] [PDF]

				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]

				K.-i. Ito, C. J. Oleschuk, C. Westlake, M. Z. Vasa, R. G. Deeley, and S. P. C. Cole 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 J. Biol. Chem., October 5, 2001; 276(41): 38108 - 38114. [Abstract] [Full Text] [PDF]

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