1 Department of Pharmacology and Toxicology and 2 Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada K7L 3N6
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ABSTRACT |
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Multidrug
resistance protein 3 (MRP3) is an ATP-dependent transporter of
17-estradiol 17
(D-glucuronide)
(E217
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 E217
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 E217
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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INTRODUCTION |
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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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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, 17-estradiol
17
(D-glucuronide) (E217
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 AND METHODS |
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Materials.
[6,7-3H]E217G (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, E217
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]E217G
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 E217
G uptake were performed at 37°C in a 60-µl
reaction volume containing 400 nM
[3H]E217
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]E217
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]E217
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]E217
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]E217G transport. Experiments were also
carried out at an initial substrate concentration of 100 µM MTX.
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RESULTS |
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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 -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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Substitution of Trp1242 enhances the ability of MRP3 to
transport [3H]E217G.
Time courses of ATP-dependent [3H]E217
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]E217
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]E217
G uptake by the most
conservatively substituted mutant, W1242Y-MRP3, was ~7-fold higher
(Fig. 3B).
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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 E217G or
LTC4, it was not characterized further.
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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
protein1 · 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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Effect of Trp1242 substitutions on MTX inhibition of
MRP3-mediated transport of [3H]E217G.
MTX has previously been reported to inhibit E217
G
transport by MRP3 (14, 43). Consequently, the effect of
MTX on [3H]E217
G uptake by the
MRP3-Trp1242 mutants with reduced MTX transport activity
was examined. The results in Fig. 5C show that
[3H]E217
G uptake by wild-type MRP3 was
inhibited >90% by 1 mM MTX as expected.
[3H]E217
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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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]E217G 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]E217
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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DISCUSSION |
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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 E217
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 E217
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
E217
G transport by these mutants could still be
inhibited by bile acid. Thus it appears that E217
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 E217G 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
E217
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 E217
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
E217
G transport efficiency by MRP3.
In contrast to E217G 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
E217
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 E217G 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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