Substitution of Trp1242 of TM17 alters substrate specificity of human multidrug resistance protein 3

Curtis J. Oleschuk1,2, Roger G. Deeley2, and Susan P. C. Cole1,2

1 Department of Pharmacology and Toxicology and 2 Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada K7L 3N6


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Multidrug resistance protein 3 (MRP3) is an ATP-dependent transporter of 17beta -estradiol 17beta (D-glucuronide) (E217beta G), leukotriene C4 (LTC4), methotrexate, and the bile salts taurocholate and glycocholate. In the present study, the role of a highly conserved Trp residue at position 1242 on MRP3 transport function was examined by expressing wild-type MRP3 and Ala-, Cys-, Phe-, Tyr-, and Pro-substituted mutants in human embryonic kidney 293T cells. Four MRP3-Trp1242 mutants showed significantly increased E217beta G uptake, whereas transport by the Pro mutant was undetectable. Similarly, the Pro mutant did not transport LTC4. By comparison, LTC4 transport by the Ala, Cys, Phe, and Tyr mutants was reduced by ~35%. The Ala, Cys, Phe, and Tyr mutants all showed greatly reduced methotrexate and leucovorin transport, except the Tyr mutant, which transported leucovorin at levels comparable with wild-type MRP3. In contrast, the MRP3-Trp1242 substitutions did not significantly affect taurocholate transport or taurocholate and glycocholate inhibition of E217beta G uptake. Thus Trp1242 substitutions markedly alter the substrate specificity of MRP3 but leave bile salt binding and transport intact.

bile salt transport; methotrexate; estradiol glucuronide transport; adenosine 5'-triphosphate-binding cassette; leukotriene C4; site-directed mutagenesis


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THE ATP-BINDING CASSETTE (ABC) proteins comprise a large superfamily of transmembrane proteins that use the energy of ATP hydrolysis to translocate their substrates across biological membranes (23). One branch of the superfamily, known as subfamily C, presently consists of 12 human proteins, 7 of which are designated as multidrug resistance proteins (MRPs) 1-7 (4, 9, 29). MRP1 (ABCC1) was the first of the MRP-related transporters to be cloned (7), and increased expression of this protein in tumor cells results in resistance to a remarkably diverse spectrum of anticancer drugs. MRP1 is also a primary active transporter of a variety of organic anions that include both endo- and xenobiotic molecules conjugated to GSH, glucuronide, and sulfate (9).

The ability of MRP1 to confer resistance in tumor cells and to transport conjugated organic anions is shared by the structurally related MRP2 (ABCC2) and MRP3 (ABCC3). All three proteins have an extracytosolic NH2 terminus and are predicted to have 17 transmembrane (TM) helices, which are organized as three membrane-spanning domains (MSD) (4, 28) (Fig. 1A). The first, MSD1, contains five TM segments, whereas MSD2 and MSD3 are each predicted to contain six TM segments in most topological models (12, 21, 24). MSD2 and MSD3 are each followed by a nucleotide-binding domain (NBD1 and NBD2), both of which are required for transport activity (9).


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Fig. 1.   Location of Trp1242 in transmembrane (TM) segment 17 in multidrug resistance protein 3 (MRP3) and amino acid similarities with MRP1 and MRP3. A: predicted secondary structure of MRP3 with the position of Trp1242 (W1242) in the third membrane-spanning domain (MSD3) highlighted. The amino acids in TM17 are those predicted by several algorithms. NBD, nucleotide-binding domain. B: helical wheel projections of the amino acid sequences of the predicted amphipathic, highly conserved TM17 of the third MSD of ATP-binding cassette (ABC) C subfamily proteins MRP3, MRP1, and MRP2. Open circles indicate amino acids that can participate in H-bonding interactions, whereas shaded circles indicate amino acids that cannot. Aromatic amino acids are indicated by asterisks. Shown below the helical wheel projections is an alignment of the amino acid sequences of predicted TM17 of MSD3 of human MRP3 and related ABCC proteins showing conservation of MRP3 Trp1242 indicated by boldface print and an asterisk. 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 (1224-1246) in human MRP3. Hum, human; SUR, sulfonylurea receptor; MDR1, P-glycoprotein.

The partial cDNA sequence of human MRP3 (ABCC3) and its mapping to chromosome 17p21, as well as the tissue distribution of human MRP3 mRNA, were first described by Kool et al. (26). Shortly thereafter, the complete coding sequences of human MRP3 and rat Mrp3 were reported by several groups (3, 11, 22, 25, 27, 41). The 1527-amino acid MRP3 protein exhibits 58% identity with MRP1 and 48% identity with MRP2. Despite this primary sequence similarity, MRP3, like MRP1 and MRP2, has its own distinctive pattern of tissue distribution and substrate specificity.

The highest levels of MRP3 mRNA are found in the liver, colon, and small intestine, whereas lower levels have been detected in pancreas, kidney, prostate, placenta, adrenal gland, and different parts of the brain (3, 11, 22, 39). Immunohistochemical studies have for the most part corroborated the mRNA expression studies, and in all polarized epithelial cells examined to date MRP3, like MRP1, has been localized to basolateral membranes (22, 25, 27, 38). In hepatocytes and intrahepatic bile duct epithelial cells (cholangiocytes), expression of MRP3 is induced during cholestasis, when apical expression of Mrp2/MRP2 is disrupted, such as in TR- rats and in humans with Dubin-Johnson syndrome. Under these conditions, MRP3 is thought to play a compensatory role in the basolateral efflux of toxic organic anions (32). Hepatic MRP3/Mrp3 can also be induced in response to a number of different xenobiotics (5, 22, 34).

Tumor cells transfected with MRP3 cDNA have been reported to be resistant to a relatively narrow spectrum of anticancer drugs. Thus MRP3 confers resistance to the podophyllotoxins VP-16 (etoposide) and VM-26 (teniposide), the antifolate methotrexate (MTX), and possibly the Vinca alkaloid vincristine but not to anthracyclines, platinum-containing drugs, or heavy metal oxyanions that are substrates of MRP1 or MRP2 (27, 43, 44). Glutathione- and glucuronide-conjugated organic anions such as the cysteinyl leukotriene, leukotriene C4 (LTC4), and the conjugated estrogen, 17beta -estradiol 17beta (D-glucuronide) (E217beta G), are also substrates of MRP3, as they are for MRP1 and MRP2. However, glucuronide conjugates appear to be transported by MRP3 with substantially greater efficiency than glutathione conjugates (14, 43, 46). Finally, in addition to cytotoxic drugs and conjugated organic anions, MRP3 transports primary bile acids, such as cholic acid, taurocholic acid, and glycocholic acid, as well as conjugated secondary bile acids such as taurolithocholate-3-sulfate and cholate 3-O-glucuronide (14, 46). In contrast, MRP1 and MRP2 transport only conjugated bile acids (15, 24, 32, 40). Thus the substrate specificity of MRP3 overlaps but is distinct from that of either MRP1 or MRP2.

Amino acid residues in MRP3 involved in the recognition and transport of its substrates remain largely unknown. In previous studies of the related MRP1 and MRP2, we demonstrated that a highly conserved Trp residue in the highly amphipathic TM17 of MSD3 plays a critical role in the substrate specificity of these transporters (17, 18) (Fig. 1B). In the present study, we have substituted the analogous residue in MRP3, Trp1242, with both conserved and nonconserved amino acids and examined the effects on the expression and substrate specificity of this bile salt transporter.


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Materials. [6,7-3H]E217beta G (55 Ci/mmol) and [3H(G)]taurocholic acid (2 Ci/mmol) were purchased from NEN Life Science Research Products (Boston, MA). [14,15 (n)-3H]LTC4 (115.3 Ci/mmol) was from Amersham Pharmacia Biotech (Little Chalfont, UK), and [3',5',7,9-3H(n)]leucovorin diammonium salt (17 Ci/mmol) and [3',5',7'-3H(n)] MTX sodium salt (17 Ci/mmol) were from Moravek (Brea, CA). LTC4 was purchased from Calbiochem (La Jolla, CA), and nucleotides, E217beta G, taurocholic acid, and glycocholic acid were purchased from Sigma (St. Louis, MO). MTX was from Faulding (Vaudreuil, PQ, Canada).

Vector construction and site-directed mutagenesis. To generate an MRP3 expression vector, several DNA fragments containing the complete coding sequence of human MRP3 mRNA with an optimized Kozak sequence were synthesized by PCR and assembled in the vector pBluescript II KS+ (Stratagene, La Jolla, CA) as described elsewhere (C. J. Westlake, M. Vasa, S. P. C. Cole, and R. G. Deeley, unpublished observations). The coding sequence of MRP3 cDNA was then moved into the pcDNA3.1(+) expression vector (Invitrogen, Burlington, ON, Canada), and the integrity of the resulting pcDNA3.1(+) MRP3 construct was confirmed by sequencing the entire inserted sequence in both directions. The sequence obtained was in agreement with that published previously (GenBank accession no. CAA76658 gi: 3087794) (25).

Mutagenesis was carried out in the pBluescript II KS+ MRP3 plasmid using the Quik Change site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Oligonucleotides used for site-directed mutagenesis were synthesized by ACGT (Toronto, ON, Canada). Silent mutations introducing a DraI restriction site were incorporated into the mutagenic primers to aid in screening. Mutations for Trp1242 substitutions (underlined), silent DraI restriction sites (italicized), and their corresponding oligonucleotides were as follows: W1242A (5'-G CAG GTG ACA TTC GCT TTA AAC GCG ATG ATA CGA ATG ATG TCA G-3'), W1242C (5'-G CAG GTG ACA TTC GCT TTA AAC TGC ATG ATA CGA ATG ATG TCA G-3'), W1242F (5'-G CAG GTG ACA TTC GCT TTA AAC TTC ATG ATA CGA ATG ATG TCA G-3'), W1242Y (5'-G CAG GTG ACA TTC GCT TTA AAC TAC ATG ATA CGA ATG ATG TCA G-3'), and W1242P (5'-G CAG GTG ACA TTC GCT TTA AAC CCG ATG ATA CGA ATG ATG TCA G-3'). After the presence of all mutations was confirmed by a DraI diagnostic digestion, a 0.3-kb AgeI/PmaCI fragment containing the desired mutation was subcloned back into pcDNA3.1(+)MRP3 and the entire fragment in the full-length construct was sequenced.

Transient transfections of MRP3 expression vectors. Mutant pcDNA3.1(+)MRP3 expression vectors were transfected into SV40-transformed human embryonic kidney cells (HEK293T) as before (17). Briefly, ~5 × 106 cells were seeded in 150-mm dishes, and 24 h later DNA (16 µg) was added using FuGENE 6 (Roche Diagnostics, Laval, PQ, 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 (30). Empty vector pcDNA3.1(+) DNA and vector containing the wild-type MRP3 cDNA were included as controls in all transfection experiments.

Measurement of MRP3 protein levels in transfected cells. The relative levels of wild-type and Trp1242 mutant MRP3 proteins were determined by immunoblot analysis of membrane protein fractions from the transfected cells essentially as described (17). Proteins were resolved on a 6% polyacrylamide gel and electrotransferred to a nylon membrane. Membranes were blocked with 4% (wt/vol) skim milk powder in Tris-buffered saline with 0.1% Tween 20 (vol/vol) (TBST) for 1 h followed by incubation with the MRP3-specific murine MAb M3II-9 (Alexis, San Diego, CA) diluted 1:1,000 to 1:10,000 in 4% skim milk powder in TBST. After being washed, blots were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody diluted 1:10,000 (Pierce, Edmonton, AB, Canada) followed by application of Renaissance chemiluminescence blotting substrate (NEN Life Science). Relative levels of MRP3 protein expression were estimated by densitometric analysis using a ChemiImager 4000 (Alpha Innotech, San Leandro, CA).

MRP3-mediated transport of [3H]E217beta G and [3H]LTC4 by inside-out membrane vesicles. ATP-dependent transport of 3H-labeled substrates by the membrane vesicles was measured by using a rapid filtration technique as described previously (18, 30). Briefly, time courses of E217beta G uptake were performed at 37°C in a 60-µl reaction volume containing 400 nM [3H]E217beta G (120 nCi), 4 mM AMP or ATP, 10 mM MgCl2, 10 mM creatine phosphate, 100 µg/ml creatine kinase, and 6 µg of vesicle protein in transport buffer (50 mM Tris · HCl, 250 mM sucrose, pH 7.4). In some experiments, MTX, taurocholate, and glycocholate were added at the concentrations indicated. Uptake was stopped at the appropriate times by rapid dilution of an aliquot of the reaction mixture in ice-cold transport buffer and then filtered through glass fiber (type A/E) filters that had been presoaked in transport buffer. Radioactivity was quantitated by liquid scintillation counting. All data were corrected for the amount of [3H]E217beta G that remained bound to the filter, which was usually <10% of the total radioactivity. Uptake in the presence of AMP was subtracted from uptake in the presence of ATP to determine ATP-dependent [3H]E217beta G uptake. All assays were carried out in triplicate, and results were expressed as means ± SD. Uptake of [3H]LTC4 was measured in a similar fashion, except that membrane vesicles (6 µg protein) were incubated at 37°C for 5 min with [3H]LTC4 (500 nM; 80 nCi) and components as described for [3H]E217beta G transport.

MRP3-mediated transport of [3H]MTX, [3H]leucovorin, and [3H]taurocholate by inside-out membrane vesicles. [3H]MTX uptake was also measured by rapid filtration essentially as described (17). Assays were performed at 37°C in 60 µl transport buffer containing 1 µM [3H]MTX (250 nCi per reaction), 4 mM AMP or ATP, an ATP-regenerating system, and membrane vesicles (10 µg protein). Uptake was stopped at 10 min, diluted, and filtered as above for [3H]E217beta G transport. Experiments were also carried out at an initial substrate concentration of 100 µM MTX.

[3H]leucovorin and [3H]taurocholate uptake was measured by rapid filtration essentially as described (20, 45). The incubation mixtures contained 250 µM [3H]leucovorin (500 nCi per reaction) or 20 µM [3H]taurocholate (250 nCi per reaction), 4 mM AMP/ATP, an ATP-regenerating system, and membrane vesicles (10 µg or 3 µg protein, respectively). Uptake was stopped at 20 min, diluted, and filtered as above for [3H]E217beta G transport.


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MRP3-Trp1242 mutants are expressed in human embryonic kidney cells. Based on our observation that substitution of MRP1-Trp1246 and MRP2-Trp1254 at the predicted COOH-proximal cytosol-membrane interface of TM17 can dramatically affect the substrate specificity of MRP1 and MRP2, respectively, cDNA constructs were generated in which the analogous residue, Trp1242, in MRP3 was replaced with several different amino acids. These included nonconservative substitutions with a nonaromatic nonpolar amino acid (Ala; W1242A-MRP3) and a nonaromatic polar amino acid (Cys; W1242C-MRP3), as well as conservative substitutions with aromatic polar (Tyr; W1242Y-MRP3) and nonpolar (Phe; W1242F-MRP3) amino acids. Trp1242 was also replaced with a alpha -helix-disrupting residue (Pro; W1242P-MRP3). The cDNA constructs were transfected into HEK293T cells, and after 48-72 h the cells were harvested, the membrane vesicles were prepared, and relative MRP3 protein levels were determined by immunoblotting. As shown in Fig. 2, wild-type MRP3 and the four mutants (W1242A-, W1242C-, W1242F-, and W1242Y-MRP3) were expressed at similar levels. In contrast, the W1242P-MRP3 mutant was expressed at significantly lower levels, suggesting that this mutation affects the biogenesis or stability of the protein. Mean expression levels of the mutant MRP3 proteins relative to wild-type MRP3 were as follows: W1242A, 1.1 ± 0.3; W1242C, 1.0 ± 0.2; W1242Y, 1.0 ± 0.1; W1242F, 1.0 ± 0.3; and W1242P, 0.6 ± 0.2 (4-6 independent transfections).


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Fig. 2.   Expression levels of wild-type and Trp1242 mutant MRP3 molecules. Top: MRP3 expression in membrane vesicles prepared from human embryonic kidney (HEK) 293T cells transfected with empty vector [pcDNA3.1(+)], wild-type (WT-MRP3), and mutant (W1242A, W1242C, W1242F, W1242Y, and W1242P) MRP3 cDNAs was determined by immunoblotting with MAb M3II-9, and relative levels of expression were estimated by densitometry. The bars show the relative mean levels (± SD) of MRP3 protein expression in membrane vesicles from 4-6 independent transfections. Bottom: typical immunoblot with the numbers at the bottom of each lane indicating the expression levels of the mutant MRP3 proteins relative to wild-type MRP3.

Substitution of Trp1242 enhances the ability of MRP3 to transport [3H]E217beta G. Time courses of ATP-dependent [3H]E217beta G uptake were determined for the W1242A-, W1242C-, W1242F-, W1242Y-, and W1242P-MRP3 mutants by using inside-out membrane vesicles prepared from transfected HEK293T cells (Fig. 3A). Unexpectedly, four of the five mutants (W1242A-, W1242C-, W1242F-, and W1242Y-MRP3) transported this glucuronide substrate at levels that were substantially higher than those of wild-type MRP3. In contrast, transport by W1242P-MRP3 was almost undetectable. At 3 min, [3H]E217beta G uptake in membrane vesicles enriched for W1242A-, W1242C-, and W1242F-MRP3 was 2.5- to 3-fold higher than for wild-type MRP3, whereas [3H]E217beta G uptake by the most conservatively substituted mutant, W1242Y-MRP3, was ~7-fold higher (Fig. 3B).


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Fig. 3.   3H-labeled 17beta -estradiol 17beta (D-glucuronide) ([3H]E217beta G) uptake by wild-type and Trp1242 mutant MRP3 molecules into inside-out membrane vesicles. A: time course of ATP-dependent [3H]E217beta G uptake in membrane vesicles prepared from HEK293T cells transfected with empty vector (), wild-type MRP3 (), and mutant [W1242A (black-down-triangle ), W1242C (black-lozenge ), W1242F (), W1242Y (black-triangle), and W1242P (down-triangle)] cDNA expression vectors. Vesicles were incubated at 37°C with 400 nM [3H]E217beta G and ATP or AMP in transport buffer as described in MATERIALS AND METHODS. The results shown are the means ± SD of triplicate determinations in a single experiment, and similar results were obtained in a second experiment. B: relative levels of [3H]E217beta G uptake at 3 min after subtracting uptake by membrane vesicles prepared from cells transfected with the control pcDNA3.1(+) vector and normalizing mutant MRP3 protein levels to wild-type MRP3 protein levels.

Effect of Trp1242 substitutions on MRP3-mediated transport of [3H]LTC4. Previous studies of rat and human Mrp3/MRP3 have reported that glutathione conjugates such as LTC4 are relatively poor substrates for this ABC transporter (13, 43, 46). We also observed that [3H]LTC4 uptake by wild-type MRP3 was very low when measured at an initial substrate concentration of 50 nM [6.3 ± 0.5 pmol/mg protein for WT-MRP3 vs. 5.4 ± 0.6 pmol/mg protein for pcDNA3.1(+) vector control; not shown]. This concentration is commonly used in LTC4 uptake assays by MRP1 and MRP2 and is severalfold higher than the concentration used in some previous studies of MRP3/Mrp3 (13, 46). However, we found that when transport assays were carried out at a 10-fold higher initial LTC4 concentration (500 nM), in keeping with the lower affinity of this transporter for this glutathione conjugate, significant levels of uptake were observed. Thus ATP-dependent [3H]LTC4 uptake by membrane vesicles from cells transfected with wild-type MRP3 cDNA was 94 ± 1 vs. 25 ± 2 pmol/mg protein for membrane vesicles from cells transfected with the pcDNA3.1(+) vector alone (Fig. 4A). [3H]LTC4 uptake by the W1242A-, W1242F-, and W1242Y-MRP3 mutants was somewhat less than the wild-type MRP3 transport activity (Fig. 4A). At 5 min, levels of [3H]LTC4 uptake by these three mutants were ~62-70% those of wild-type MRP3, whereas uptake by the W1242C-MRP3 mutant was only 37% of wild-type levels (after subtraction of uptake by vector control membrane vesicles and normalization of mutant MRP3 protein levels to wild-type MRP3 protein levels) (Fig. 4B). [3H]LTC4 uptake by the W1242P-MRP3 mutant was undetectable (not shown). Because of the consistently lower levels of W1242P-MRP3 expression and because this mutant did not transport either E217beta G or LTC4, it was not characterized further.


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Fig. 4.   3H-labeled leukotriene C4 ([3H]LTC4) uptake by wild-type and Trp1242 mutant MRP3 proteins. A: uptake of [3H]LTC4 was measured in vesicles prepared from cells transfected with empty vector [pcDNA3.1(+)], wild-type MRP3, and mutant cDNA expression vectors with an initial substrate concentration of 500 nM [3H]LTC4. The results shown are the means ± SD of triplicate determinations in a single experiment, and similar results were obtained in a second experiment. B: relative levels of [3H]LTC4 uptake after subtracting uptake by membrane vesicles prepared from cells transfected with the control pcDNA3.1(+) vector and normalizing mutant MRP3 protein levels to wild-type MRP3 protein levels.

Effect of Trp1242 substitutions on MRP3-mediated transport of [3H]MTX. Membrane vesicles prepared from HEK293T cells transfected with wild-type MRP3 transported [3H]MTX at a rate of ~15 pmol · mg protein-1 · min-1 (Fig. 5A). This rate is more than twofold higher than those reported previously for human MRP3 expressed in stably transfected HEK293T cells, which may simply reflect higher levels of MRP3 expression in our HEK transfectants (45). However, when Trp1242 was replaced with either nonconserved or conserved amino acids, a significant reduction in ATP-dependent [3H]MTX uptake was observed in all cases (Fig. 5A). At 10 min, ATP-dependent [3H]MTX uptake levels by the W1242A, W1242C, W1242F, and W1242Y mutants were ~12-26% those of wild-type MRP3 [after the transport activity of the pcDNA3.1(+) vector control was subtracted and values normalized for relative MRP3 protein expression] (Fig. 5B). A similar reduction in MTX transport activity was observed when assays were carried out at a 100-fold higher initial substrate concentration (100 µM [3H]MTX; data not shown).


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Fig. 5.   Time course of [3H]methotrexate (MTX) uptake and MTX-mediated inhibition of [3H]E217beta G uptake by wild-type and Trp1242 mutant MRP3 proteins. A: ATP-dependent uptake of [3H]MTX was measured in membrane vesicles prepared from HEK293T cells transfected with empty pcDNA3.1(+) vector (), wild-type (), and mutant [W1242A (black-down-triangle ), W1242C (black-lozenge ), W1242F (), and W1242Y (black-triangle)] MRP3 cDNA expression vectors. Membrane vesicles were incubated at 37°C with 1 µM [3H]MTX and ATP or AMP in transport buffer. The results shown are the means ± SD of triplicate determinations in a single experiment, and similar results were obtained in a second experiment. B: relative levels of [3H]MTX uptake at 10 min after subtracting uptake by membrane vesicles prepared from cells transfected with the control pcDNA3.1(+) vector and normalizing mutant MRP3 protein levels to wild-type MRP3 protein levels. C: membrane vesicles described in A were incubated at 37°C with 400 nM [3H]E217beta G in transport buffer and other components for 3 min in the absence (open bars) or presence of MTX (1 mM, solid bar). Each bar represents the mean ± SD of triplicate determinations.

Effect of Trp1242 substitutions on MTX inhibition of MRP3-mediated transport of [3H]E217beta G. MTX has previously been reported to inhibit E217beta G transport by MRP3 (14, 43). Consequently, the effect of MTX on [3H]E217beta G uptake by the MRP3-Trp1242 mutants with reduced MTX transport activity was examined. The results in Fig. 5C show that [3H]E217beta G uptake by wild-type MRP3 was inhibited >90% by 1 mM MTX as expected. [3H]E217beta G uptake by the four Trp1242 mutant MRP3 proteins was also inhibited by MTX, although to a much lesser degree (~35%).

Only the most conservatively substituted W1242Y-MRP3 mutant transports [3H]leucovorin. In addition to MTX, MRP3 has been reported to transport folic acid and another folic acid analog, leucovorin (N5-formyltetrahydrofolic acid) (45). To determine if Trp1242 substitutions also affected MRP3-mediated transport of the latter substrate, [3H]leucovorin uptake into membrane vesicles prepared from transfected cells expressing wild-type MRP3 and W1242A-, W1242C-, W1242F-, and W1242Y-MRP3 mutants was examined. For wild-type MRP3 membrane vesicles, the levels of [3H]leucovorin uptake were 1.32 ± 0.12 nmol/mg compared with 0.18 ± 0.04 nmol/mg for the empty vector control (Fig. 6A). Of the four Trp1242 mutants tested, only the most conservatively substituted mutant, W1242Y-MRP3, transported [3H]leucovorin at levels comparable to those of wild-type MRP3. In contrast, [3H]leucovorin transport by the less conservatively substituted Trp1242 mutants was reduced by >75% compared with wild-type MRP3 after subtraction of uptake by vector control membrane vesicles and normalization of mutant MRP3 protein levels to wild-type MRP3 protein levels (Fig. 6B).


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Fig. 6.   [3H]leucovorin uptake by wild-type and Trp1242 mutant MRP3 proteins. A: uptake of [3H]leucovorin was measured in membrane vesicles prepared from HEK293T cells transfected with empty vector [pcDNA3.1(+)], wild-type, and Trp1242 mutant (W1242A, W1242C, W1242F, and W1242Y) MRP3 cDNA expression vectors. Membrane vesicles were incubated at 37°C with 250 µM [3H]leucovorin and ATP or AMP in transport buffer for 20 min. The results shown are the means ± SD of triplicate determinations in a single experiment, and similar results were obtained in 2 additional experiments. B: relative levels of [3H]leucovorin after subtracting uptake by membrane vesicles prepared from cells transfected with the control pcDNA3.1(+) vector and normalizing mutant MRP3 protein levels to wild-type MRP3 protein levels.

Substitution of Trp1242 has no significant effect on [3H]taurocholate transport by MRP3. Rat Mrp3 has been shown to transport monovalent bile acids such as taurocholate and glycocholate, whereas human MRP3 has been reported to transport only glycocholate (1, 14, 46). However, we found that ATP-dependent [3H]taurocholate uptake by wild-type MRP3 membrane vesicles was readily detectable at a level of 331 ± 90 pmol/mg protein compared with 70 ± 18 pmol/mg protein for the vector control vesicles (Fig. 7A). Taurocholate uptake by the W1242A-, W1242C-, W1242F-, and W1242Y-MRP3 mutants was then examined and, in all cases, was not significantly different from uptake by wild-type MRP3 (Fig. 7B). Consistent with this observation, [3H]E217beta G uptake by W1242A-, W1242C-, W1242F-, and W1242Y-MRP3 could still be inhibited by taurocholic acid (40-60%) at concentrations of 50 and 100 µM (Fig. 7C). Glycocholic acid also inhibited [3H]E217beta G uptake by the four MRP3-Trp1242 mutant proteins, and this inhibition was similar to that observed with wild-type MRP3. At concentrations of 50 and 100 µM, inhibition by glycocholic acid ranged from 40 to 80% (Fig. 8).


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Fig. 7.   [3H]taurocholate uptake and taurocholate-mediated inhibition of [3H]E217beta G uptake by wild-type and Trp1242 mutant MRP3 proteins. A: ATP-dependent uptake of [3H]taurocholic acid was measured in membrane vesicles prepared from HEK293T cells transfected with empty vector [pcDNA3.1(+)] and wild-type MRP3 cDNA expression vectors. Membrane vesicles were incubated at 37°C with 20 µM [3H]taurocholic acid in transport buffer and ATP or AMP for 5 min. The results shown are the means ± SD of triplicate determinations in a single experiment, and similar results were obtained in a second experiment. B: [3H]taurocholic acid uptake was measured as in A. Cells were transfected with empty vector [pcDNA3.1(+)], wild-type, and mutant (W1242A, W1242C, W1242F, and W1242Y) MRP3 cDNA expression. C: membrane vesicles from cells expressing wild-type and W1242A, W1242C, W1242F, and W1242Y mutant MRP3 were incubated at 37°C with 400 nM [3H]E217beta G in transport buffer and other components for 3 min in the absence (open bars) or presence of taurocholic acid (50 µM, grey bar; 100 µM, solid bar). Each bar represents the mean ± SD of triplicate determinations.



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Fig. 8.   Effect of glycocholic acid on [3H]E217beta G uptake by wild-type and Trp1242 mutant MRP3 proteins. Membrane vesicles from cells expressing wild-type MRP3 (WT-MRP3) and W1242A, W1242C, W1242F, and W1242Y mutant MRP3 were incubated at 37°C with 400 nM [3H]E217beta G in transport buffer and other components for 3 min in the absence (open bars) or presence of glycocholate (50 µM, grey bar; 100 µM, solid bar). Each bar represents the mean ± SD of triplicate determinations.

The transport activities of the MRP3-Trp1242 mutants for the five substrates tested are summarized in Table 1. For comparison, the effects of nonconservative (Ala) and conservative (Tyr) substitutions of MRP1-Trp1246 (18), MRP2-Trp1254 (17), and MRP3-Trp1242 on transport activity are summarized in Table 2.

                              
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Table 1.   Transport activities of variously substituted MRP3-Trp1242 mutants


                              
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Table 2.   Effects of nonconservative (Ala) and conservative (Tyr) substitutions of MRP1-Trp1246, MRP2-Trp1254, and MRP3-Trp1242 on transport activity of common substrates


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In addition to its potential role as a cause of drug resistance in tumor cells in which it is overexpressed, MRP3 is a primary active transporter of both primary and secondary bile salts (13, 20, 46). The majority of bile salt transport in the liver takes place across the apical canalicular membrane and is mediated by bile salt export protein (BSEP; ABCB11) (32). However, because of the relatively high level of MRP3 expression in the liver, its inducibility in this tissue, and its location on basolateral membranes, it has been postulated that MRP3 may contribute to the enterohepatic circulation of bile salts that is essential for the maintenance of bile acid and cholesterol homeostasis (16, 32, 34). In this respect, the substrate specificity of MRP3 differs from the related MRP1 and MRP2, which confer resistance to a much broader range of anticancer drugs than MRP3 but are limited to transporting only conjugated bile salts. Thus all three of these ABCC proteins transport conjugated organic anions, but there are marked differences in the affinity and efficiency with which they do so (2, 24, 29, 40, 46).

Factors governing the substrate specificity of the MRP-related transporters are complex, as can be expected for any relatively large polytopic membrane protein that recognizes such a variety of different substrates. To date, a number of conserved and nonconserved amino acids in different regions of MRP1 have been shown to affect the substrate specificity and transport efficiency of this protein (8, 9, 18, 47, 48). Similarly, sequence analyses of MRP2/Mrp2 in humans with Dubin-Johnson syndrome and in hyperbilirubinemic TR-/EHBR rats, respectively, as well as site-directed mutagenesis studies have identified amino acids important for the function of this transporter (17, 19, 20, 33, 37, 42). In contrast, comparatively little is known about the amino acids that determine the substrate specificity of MRP3. However, by taking advantage of amino acid sequence differences between rat Mrp3 and Mrp2, Ito et al. (20) recently identified a nonconserved Leu residue at position 1084 in predicted TM14 of rat Mrp3 as being important for the ability of this protein to transport taurocholic acid. Thus substitution of Leu1084 in Mrp3 with Lys (as it is in MRP1) eliminated both taurocholic acid and E217beta G uptake, suggesting that these two substrates share at least one common binding determinant in a substrate-binding pocket of this transporter. In the present study, we have shown that mutation of the highly conserved Trp1242 in TM17 of human MRP3, like the nonconserved Leu1084 in rat Mrp3, significantly alters the ability of this protein to transport E217beta G, but in the case of MRP3-Trp1242, transport activity was markedly increased rather than decreased. Also in contrast to the rat Mrp3-Leu1084 mutant, the MRP3-Trp1242 mutants retained their ability to transport taurocholic acid and E217beta G transport by these mutants could still be inhibited by bile acid. Thus it appears that E217beta G and taurocholic acid share an overlapping but not identical set of binding determinants in a substrate-binding pocket of MRP3/Mrp3.

Our observation that all substitutions of MRP3-Trp1242 markedly stimulated E217beta G uptake was unexpected, because both conservative and nonconservative substitutions of the analogous Trp1246 in MRP1 had the opposite effect and essentially eliminated transport of this conjugated estrogen, as did nonconservative substitutions of MRP2-Trp1254 (17, 18) (Table 2). Trp residues with their large indole (benzopyrrole) side chains have the greatest steric bulk of all of the amino acids (36). Thus all Trp substitutions examined in the present study resulted in mutant MRP3 proteins with less steric bulk at position 1242 than is found in the wild-type protein. This could allow for greater freedom of motion in the E217beta G-binding pocket of MRP3 or possibly enhance the flexibility of TM17, which might increase the transport efficiency of this substrate. However, the highest level of E217beta G uptake was observed with W1242Y-MRP3, which is the most conservatively substituted mutant with respect to steric bulk, aromaticity, and H-bonding capability of the lateral side chain. Thus only a relatively small reduction in steric bulk seems required to enhance E217beta G transport efficiency by MRP3.

In contrast to E217beta G uptake, the variously substituted MRP3-Trp1242 mutants all showed reduced LTC4 transport, but the decrease was moderate (25-50%). We showed previously that the MRP1-Trp1246 was critical for E217beta G transport as well as being essential for conferring drug resistance (18). However, LTC4 transport by the MRP1-Trp1246 mutants was similar to wild-type MRP1 with just a small decrease in Km. This contrasts with the findings with MRP2-Trp1254 mutants, where nonconservative substitutions completely eliminated the ability of MRP2 to transport this cysteinyl leukotriene (17) (Table 2).

MRP1 and MRP2 are low-affinity transporters of GSH, and, at least in the case of MRP1, GSH transport can be markedly enhanced by a variety of compounds, including vincristine, phenylalkylamines such as verapamil, and bioflavonoids such as apigenin (2, 28, 29, 31, 35). In contrast, despite the higher degree of amino acid similarity between MRP1 and MRP3 than between MRP1 and MRP2, there is no evidence that MRP3 transports GSH alone (27, 43) nor can it be stimulated by verapamil or bioflavonoids (C. J. Oleschuk, I. Letourneau, R. G. Deeley, and S. P. C. Cole, unpublished observations). Efflux of drugs such as the Vinca alkaloids vincristine and vinblastine and the anthracyclines doxorubicin and daunorubicin by MRP1 and MRP2 seems to occur in a cotransport manner with GSH. Whether the inability of MRP3 to confer resistance to these two classes of drugs is related to its inability to transport GSH is not yet known. Also uncertain is whether the apparent inability of MRP3 to transport GSH is related to the relatively poor affinity of this transporter for GSH conjugates compared with glucuronide conjugates. However, it is clear that factors other than binding of a GSH moiety contribute to GS-X and GSH transport, because LTC4 transport by MRP3 is readily detectable.

Introduction of a Pro residue at position 1242 was the only substitution that completely eliminated the conjugated organic anion transport activity of MRP3. This substitution would not only change the spatial volume occupied by the side chain and disrupt the potential H-bonding and aromatic stacking interactions of the amino acid at this position, but it would also introduce a kink into TM17 at the predicted membrane-cytosol interface (36). The loss of H-bonding and aromatic stacking interactions alone cannot fully account for the inactivity of the W1242P-MRP3 mutant, because the Ala, Cys, Phe, and Tyr mutants all retained some transport activity. Rather, it is more likely that the unique helix-modifying properties of Pro caused a more global disruption of the substrate-binding pocket of MRP3 that was sufficient to abrogate the overall transport activity of the protein. In this regard, it is also of interest that none of the substitutions of MRP3-Trp1242 significantly affected protein expression levels, with the exception of the Pro substitution. Again, it may be that the helix-modifying properties of this amino acid adversely affected the proper packing of the TM segments of MRP3 so as to reduce the stability of the protein, which could also contribute to the elimination of transport.

Previously, we reported that MTX transport was essentially eliminated by all MRP2-Trp1254 substitutions (17), and the same is true of the MRP1-Trp1246 mutants (I. Letourneau, C. J. Oleschuk, K. Ito, R. G. Deeley, and S. P. C. Cole, unpublished observations; see Table 2). However, the MRP3-Trp1242 mutants retained a low level of MTX transport activity, which is consistent with the observation that MTX was still able to partially inhibit E217beta G uptake by the MRP3-Trp1242 mutants. These findings suggest that the conserved Trp residue may be more important for maintaining the architecture of the MTX substrate-binding pocket of MRP1 and MRP2 than MRP3. We also found that, with the exception of the Tyr substitution, all MRP3-Trp1242 mutations caused a marked decrease in the ability of MRP3 to transport leucovorin. This agent is used clinically as a rescue agent to reduce bone marrow toxicity associated with antifolate treatment (6), and it has been previously reported that the affinity of MRP3 is about threefold lower for leucovorin than it is for MTX (45). The basis for this difference in affinity is unknown but is likely to be related to differences in the interactions of these drugs with a folate-binding pocket in MRP3. These drugs, like the Trp residue itself, are capable of both H-bonding and aromatic stacking interactions. It is not presently possible to know whether or not such direct interactions occur between MRP3-Trp1242 and these drugs. However, if they do, they must do so in a somewhat different way for leucovorin than for MTX, which may account for the difference in affinities of these compounds for MRP3 as well as for the ability of the W1242Y-MRP3 mutant to transport the former but not the latter drug. As a polar aromatic amino acid, Tyr is considered a conservative substitution of Trp. Nevertheless, the two amino acids differ in certain significant ways that could affect interactions with different MRP3 substrates. These differences include the spatial volumes occupied by the side chains of Trp and Tyr (174 Å3 and 143 Å3, respectively) as well as the potential H-bonding interactions of the indole ring of the Trp side chain vs. the hydroxyl group of the Tyr side chain.

In summary, our studies show that the highly conserved Trp residue in MRP3 at the membrane-cytosol interface of TM17, as shown previously for MRP1 and MRP2, plays a unique and selective role in substrate recognition by this protein. Importantly, they show that, although the transport of a variety of organic anion substrates are affected by changes in this amino acid, binding and transport of bile salts appear to remain intact. Furthermore, our findings, together with those of Ito et al. (20), who identified a critical Leu residue in TM14 of rat Mrp3, suggest that, as we have shown previously for MRP1 (47), interactions between TM14 and TM17 may be required for MRP3/Mrp3 to transport at least some of its substrates. Finally, they also reaffirm the important notion that prediction of the substrate specificity of an ABC transporter solely on the basis of its amino acid conservation, either with homologs in the same species or orthologs in different species, is not possible.


    ACKNOWLEDGEMENTS

We wish to thank Dr. James Gerlach and Dr. Ken-ichi Ito for helpful discussions; Margaret Sankey, Colleen Schick and Kathy Sparks for technical assistance; and Maureen Rogers for expert word processing and figure preparation.


    FOOTNOTES

This work was supported in part by grant MOP-10519 from the Canadian Institutes of Health Research (CIHR). C. J. Oleschuk is the recipient of a CIHR Doctoral Award and an Ontario Graduate Scholarship. R. G. Deeley is the Queen's University Stauffer Professor of Cancer Research. S. P. C. Cole is the recipient of a Canada Research Chair in Cancer Biology and Senior Scientist of Cancer Care Ontario.

Address for reprint requests and other correspondence: S. P. C. Cole, Cancer Research Laboratories, Rm. 328, Botterell Hall, Queen's Univ., Kingston, ON, Canada, K7L 3N6 (E-mail: coles{at}post.queensu.ca).

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.

First published October 9, 2002;10.1152/ajpgi.00331.2002

Received 7 August 2002; accepted in final form 3 October 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 284(2):G280-G289
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