Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan
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ABSTRACT |
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Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.
adenosine 5'-triphosphate-binding cassette transporter superfamily; bile acid; site-directed mutagenesis; multidrug resistance-associated protein 2
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INTRODUCTION |
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MULTIDRUG
RESISTANCE-ASSOCIATED protein 2 (MRP2) belongs to the ATP-binding
cassette (ABC) transporter family cloned from rat liver as a homologue
of human MRP1 (4, 13, 27). MRP2 is highly expressed on the
bile canalicular membrane and is able to excrete structurally diverse
organic anions into the bile as shown by comparing the behavior of
normal and MRP2-deficient rats (16, 31). A hereditary
defect in human MRP2 results in a form of hyperbilirubinemia referred
to as Dubin-Johnson syndrome (15, 28, 32, 33). Substrates
for MRP2 are very similar to those for MRP1 and include glutathione
conjugates [e.g., 2,4-dinitrophenyl-S-glutathione (DNP-SG)
and leukotriene C4 (LTC4)], glucuronide
conjugates [e.g., 17-estradiol 17-(
-D-glucuronide)
(E217
G) and bilirubin glucuronide], nonconjugated
organic anions (e.g., methotrexate), and sulfate and glucuronide
conjugates of certain bile acids (e.g., taurolithocholate-3-sulfate and
cholate 3-O-glucuronide; Refs. 19, 31). Unlike
the sulfate and glucuronide conjugates of certain bile acids,
monovalent bile acids such as taurocholate (TC), cholate, and
glycocholate (GC) are not transported by MRP2, but excreted into the
bile via the bile salt export pump/sister of P-glycoprotein (Bsep/spgp;
6), which is also a member of the ABC transporter family. The primary structure of Bsep/spgp is more similar to that of rat
multidrug-resistance protein 1b (mdr1b) and mdr2 with homology of 70%
and 69%, respectively, than to that of the MRP family proteins
(~50% homology with MRP1 and rat MRP2; 6).
MRP3, the third member of the MRP family, has been cloned as a homologue of MRP1 and MRP2 (9, 17, 20, 21, 34). Induction of MRP3 on the basolateral membrane of rat hepatocytes under cholestatic conditions (3, 20) led us to hypothesize that MRP3 compensates for the reduced ability of MRP2 to pump out common substrates from hepatocytes into blood. Indeed, MRP3 has been shown (10) to accept glucuronide conjugates as substrates although glutathione conjugates are poor substrates. In addition, it has been demonstrated (1, 11, 36) that MRP3 transports monovalent bile acids such as TC and GC. These findings clearly indicate that there is a difference in substrate specificity among MRP1, MRP2, and MRP3. Together with the localization of MRP3 on the basolateral membrane of cholangiocytes and the intestinal epithelium (22), possible involvement of MRP3 in the enterohepatic and/or cholehepatic circulation has been proposed, although further data are required.
Cumulative information on the primary structure and substrate
specificity of MRP families has allowed us to take advantage of
comparative studies on chimeric proteins and/or mutated proteins produced by site-directed mutagenesis to search for a region(s) of the
protein involved in determining the particular substrate specificity of
each of these cognate gene products. Stride et al. (30)
clearly demonstrated that the anthracycline resistance and the ability
to transport E217G by MRP1 are conferred by the COOH-terminal third of the protein, using a chimeric approach to study
human MRP1 and mouse MRP1 expressed in HEK 293 cells. Moreover, we
(14) have recently demonstrated that the cationic amino
acids in transmembrane domain (TM) 6 and TM11 of rat MRP2, the cationic
charge of which is conserved among MRP1, MRP2, and MRP3, are involved
in the recognition of glutathione conjugates. The role of cationic
amino acids in TMs in transporting glutathione conjugates by human MRP2
has also been reported (29). In the present study, the
cationic amino acids in the TMs of rat MRP2, the cationic charges of
which are fully conserved among MRP1, MRP2, and MRP3 or conserved in
MRP1 and MRP2 but not in MRP3, were substituted with neutral amino
acids to examine whether these amino acids are involved in gaining
access to monovalent bile acids. Substitution of the amino acid at
position 586 (an amino acid with a cationic charge that is conserved
among MRP1, MRP2, and MRP3) and position 1096 (an amino acid with a
cationic charge that is conserved in MRP1 and MRP2 but not in MRP3) of
rat MRP2 resulted in acquired transport activity for TC. The transport activity of mutant rat MRP3 with a mutation at position 1084, which
corresponds to the amino acid at position 1096 of rat MRP2, was also investigated.
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EXPERIMENTAL PROCEDURES |
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Chemicals.
[3H]LTC4 (165 Ci/mmol),
[3H]E217G (55 Ci/mmol), and
[3H]TC were purchased from NEN Life Science Products
(Boston, MA). [3H]DNP-SG (50 Ci/mmol) was synthesized
enzymatically using [glycine-2-3H]glutathione (NEN Life
Science Products), 1-chloro-2,4-dinitrobenzene, and glutathione
S-transferase (Sigma Chemical, St. Louis, MO) as described
previously (18), followed by purification by HPLC on
LiChrosorb RP-18 column (Kanto Chemical, Tokyo, Japan). The solvent
system used was H2O-acetnitrile (22:78) containing 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. The purity of [3H]DNP-SG was >99% throughout the experiments.
Unlabeled DNP-SG was synthesized using the method of Hinchman et al.
(7). Briefly, 2,4-dinitrofluorobenzene (0.01 mol; Sigma
Chemical) dissolved in 1 ml MeOH was added slowly to GSH (0.01 mol)
dissolved in 5 ml 1 N KHCO3. After incubation for 15 min,
the solution was filtered and acidified to pH 2 with diluted HCl. The
precipitate was collected by vacuum filtration. The recrystallization
of DNP-SG was performed from boiling H2O.
E217
G, TC, ATP, GTP, CTP, UTP, AMP, creatine phosphate,
and creatine phosphokinase were also obtained from Sigma Chemical.
Sf9 cells were maintained as a suspension culture at 27°C with
serum-free Excel 420 (Nichirei, Tokyo, Japan) supplemented with an
antibiotic-antimycotic mixture (LifeTechnologies, Tokyo, Japan).
Plasmid construction.
Rat MRP2 cDNA inserted in the recombinant donor plasmid pFASTBAC1 was
obtained as reported previously (14). A 5.2-kb
ApaLI-SalI fragment of rat MRP3 cDNA containing
the full-length coding region flanked by an untranslated sequence of 15 and 572 nt at the 5' and 3' ends, respectively, was excised from
pBluescript SK() vector (10). This fragment was ligated
with BamHI-SmaI linker and subsequently inserted
into the BamHI and SalI site of the donor plasmid
pFASTBAC1 (Life Technologies) downstream from the polyhedrin promoter.
Site-directed mutagenesis.
Site-directed mutagenesis was performed by the method of Kunkel et al.
(23). Single-strand DNAs encoding rat MRP2 and MRP3 were
rescued from CJ236 transfected with pBluescript SK() containing the
expression cassette of rat MRP2-Aor51HI-SalI
(13) and rat MRP3 cDNA (9) after they were
infected with f1 helper phage. The resulting single-strand DNAs and
respective mutagenic primers were heat denatured at 96°C for 1 min
and then slowly cooled to 40°C to allow them anneal properly. An in
vitro polymerase reaction was performed using Sequenase version 2.0 (Life Technologies) and T4 DNA ligase (Life Technologies) at 37°C for
90 min. The resulting double-stranded DNA was transfected to competent
DH5
and spread onto LB agar plate containing 50 µg/ml of
ampicillin. Mutant MRP2 and MRP3/pBluescript SK(
) were prepared from
several colonies and verified by automated sequencing. The
SmaI-SalI fragment of the wild-type MRP2 cDNA
cassette in pFASTBAC1 was replaced with the mutant MRP2 cDNA cassette
excised as an Aor51HI-SalI fragment from the
mutant MRP2/pBluescript SK(
). Similarly, the NheI-SalI fragment of the wild-type MRP3 partial
cDNA cassette (2,388 bp of coding region and 572 bp of 3'-noncoding
region) was replaced with corresponding mutant MRP3 cDNA cassette
excised as the NheI-SalI fragment from the mutant
MRP3/pBluescript SK(
).
Production and infection of recombinant baculovirus.
Recombinant baculovirus was prepared as described previously
(14). Sf9 cells were infected with an appropriate
amount of the respective virus and cultured for 60-72 h in the
presence of 5% fetal bovine serum. Sf9 cells infected with the
baculovirus carrying green fluorescent protein (GFP) cDNA were used as
a control throughout the experiment (GFP control). Cells were harvested 60-72 h after infection, and subsequently, membrane vesicles were isolated from 1 to 2 × 108 Sf9 cells using the
standard method described previously with some modifications. Briefly,
the harvested cells (1-2 × 108 cells) were
diluted 40-fold with hypotonic buffer (1 mM Tris · HCl and 0.1 mM EDTA, pH 7.4 at 4°C) and stirred gently for 1 h on ice in the
presence of 2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 µg/ml pepstatin, and 5 µg/ml aprotinin. The cell lysate was
centrifuged at 100,000 g for 30 min at 4°C, and the
resulting pellet was suspended in 10 ml of isotonic TS buffer (10 mM
Tris · HCl, pH 7.4 at 4°C, and 250 mM sucrose) and
homogenized with Dounce B homogenizer (glass/glass, tight pestle, 30 strokes). The crude membrane fraction was layered on top of a 38%
(wt/vol) sucrose solution in 5 mM Tris-HEPES (pH 7.4 at 4°C) and
centrifuged in a Beckman SW41 rotor at 280,000 g for 45 min
at 4°C. The turbid layer at the interface was collected, diluted to
23 ml with TS buffer, and centrifuged at 100,000 g for 30 min at 4°C. The resulting pellet was suspended in 400 µl of TS
buffer. Vesicles were formed by passing the suspension 30 times through
a 25-gauge needle with a syringe. The membrane vesicles were finally
frozen in liquid nitrogen and stored at 80°C until use. Protein
concentrations were determined by the Lowry method.
Transport study. The transport study was performed using a rapid filtration technique, as described previously (12). Briefly, 16 µl of transport medium [10 mM Tris, 250 mM sucrose, 10 mM MgCl2, 5 mM ATP or AMP, and ATP-regenerating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase), pH 7.4 at 37°C], containing radiolabeled compounds with or without unlabeled substrate, were preincubated at 37°C for 3 min and then rapidly mixed with 4 µl of membrane vesicle suspension (10 µg protein). The transport reaction was stopped by the addition of 1 ml ice-cold buffer containing 250 mM sucrose, 0.1 M NaCl, and 10 mM Tris · HCl (pH 7.4 at 4°C). The stopped reaction mixture was filtered through a 0.45-µm HAWP filter (Millipore, Bedford, MA) and then washed twice with 5 ml of stop solution. The radioactivity retained on the filter was determined using a liquid scintillation counter (LSC-3500, Aloka, Tokyo, Japan).
Western blot analysis. Expression of MRP2 and MRP3 protein on the Sf9 membrane was determined as described previously (12) with some modifications. Membrane vesicles from Sf9 cells were loaded onto a 8.5% polyacrylamide slab gel containing 0.1% SDS and then transferred onto a Pall Fluoro Trans W membrane filter (Ann Arbor, MI) by electroblotting. The filter was blocked with Tris-buffered saline containing 0.05% Tween 20 and 3% BSA for 10 h at 4°C and probed for 1 h at room temperature with polyclonal anti-MRP2 antibody raised against the upstream region of the COOH-terminal nucleotide-binding domain (amino acid residues 1272-1285; CP-2 antibody was supplied by Dr. J. Nakayama, Kumamoto University, Kumamoto, Japan) or anti-MRP3 antibody directed against 838-973 of the deduced rat MRP3 amino acid sequence (10) diluted with Tris-buffered saline containing 0.05% Tween 20 and 0.1% BSA (1:1,000). The primary antibody was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Uppsala, Sweden) with a horseradish peroxidase-conjugated anti-rabbit antibody (Amersham Pharmacia Biotech) as the secondary antibody that was used after 2,000-fold dilution.
Data analysis. Uptake rates were fitted to the Michaelis-Menten equation using a nonlinear least-squares program (MULTI; 35) to calculate the kinetic parameters.
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RESULTS |
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Preparation of membrane vesicles from Sf9 cells infected
with baculoviruses carrying mutated MRP2 cDNA.
Comparison of the amino acid sequence among MRP1, MRP2, and MRP3
indicated that six cationic charges (Lys-308 and -325 and Arg-586,
-1019, -1201, and -1226 in rat MRP2) are conserved among MRP1, MRP2,
and MRP3 and two cationic charges (Arg-1096 and -1206 in rat MRP2) are
conserved in MRP1 and MRP2 but not in MRP3 (Fig. 1B; 14). We have focused on
the role of these eight cationic amino acids in determining the TC
transport activity and prepared mutated rat MRP2 by substituting
neutral amino acids (Leu or Met). Membrane vesicles isolated from
Sf9 cells infected with baculoviruses carrying the respective
mutant MRP2 cDNA were analyzed. As shown in Fig. 2, Western blot analysis revealed
detectable levels of expression of wild-type MRP2 and mutant MRP2 on
the Sf9 cell membrane except for mutants with Met substituted
for Arg-1201 and -1226 (R1201M and R1226M, respectively), which were
expressed below the detection limit (data not shown) as reported
previously (14). The molecular mass of wild-type
and mutant MRP2 was also comparable (~175 kDa; Fig. 2).
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Transport of [3H]TC into mutant MRP2-expressing
membrane vesicles.
The uptake of [3H]E217G was stimulated by
ATP in mutants with Met substituted for Lys-308, -325, and Arg-1019
(K308M, K325M, and R1019M, respectively), Leu substituted for Arg-586
and -1096 (R586L and R1096L, respectively), as well as wild-type MRP2,
whereas the mutant with Met substituted for Arg-1206 (R1206M) lost the transport activity for both [3H]E217
G and
[3H]LTC4 (data not shown). The ATP-dependent
uptake of [3H]TC by R586L and R1096L was significantly
higher than the GFP control (Fig. 3). The
uptake of [3H]TC by R586L and R1096L was linear at least
for the first 5 min in the presence of ATP (Fig.
4). Moreover, to confirm that the TC
molecules associated with the membrane vesicles reflect transport into
the intravesicular space, rather than binding to the vesicle surface,
the uptake of [3H]TC was examined with media of different
osmolarities. As shown in Fig. 4B, the uptake of
[3H]TC by both R586L and R1096L was osmotically
sensitive. To confirm the nucleotide dependence of TC transport by
these mutant MRP2, the uptake of [3H]TC by R586L and
R1096L was measured in the presence of several kinds of nucleotides at
a concentration of 5 mM. Of the four nucleotide triphosphates tested,
ATP was shown to stimulate TC uptake most efficiently (Fig.
4C) for both mutant MRP2, although other nucleotides can
also stimulate the uptake by up to 20-40% of that in the presence of ATP (Fig. 4C). In contrast, AMP was unable to support TC
uptake (data not shown).
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Concentration dependence of TC, E217G, and DNP-SG
uptake by R586L and R1096L.
The initial velocity for the uptake of [3H]TC by R586L
and R1096L was determined in the presence of unlabeled TC
(0.7-500 µM; Fig. 5).
Kinetic analysis yielded Michaelis constant (Km)
values of 28.8 ± 6.9 and 41.6 ± 5.6 µM for R586L and
R1096L, respectively (Table 1). Transport
kinetics was also determined for E217
G and DNP-SG in
wild-type MRP2, R586L, and R1096L (Fig. 5 and Table 1). As shown in
Table 1, Km for E217
G were
7.46 ± 3.39, 1.46 ± 0.30, and 5.00 ± 0.16 µM for
wild-type MRP2, R586L, and R1096L, respectively.
Km values for DNP-SG were 62.6 ± 10.6, 134 ± 29, and 101 ± 13 µM for wild-type MRP2, R586L, and
R1096L, respectively (Table 1).
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Mutual effect of TC and other MRP2 substrates.
To gain insight into the nature of the acquired TC transport activity
by R586L and R1096L, the effect of unlabeled TC on the transport of
authentic substrates for MRP2, including [3H]DNP-SG and
[3H]E217G, was investigated in R586L
and R1096L compared with wild-type MRP2. The uptake of
[3H]DNP-SG and [3H]E217
G by
wild-type MRP2 was increased in the presence of unlabeled TC (Fig.
6, A and B),
whereas that by R586L and R1096L was reduced with an approximate
IC50 of 200 µM (Fig. 6, A and B).
Moreover, transport of [3H]TC by R1096L was increased
~2.4- and 1.9-fold in the presence of unlabeled DNP-SG (200 µM) and
E217
G (37.5 µM), respectively (Fig. 6, C
and D), whereas that by R586L was not affected by up to 600 µM of DNP-SG or 75 µM of E217
G (Fig. 6, C
and D).
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Transport of [3H]E217G and
[3H]TC by R586- and R1096-mutant MRP2 and L1084-mutant
MRP3.
The role of Arg at positions 586 and 1096 in rat MRP2 in the
transport of TC was further studied by substituting these amino acids
with Lys (R586K and R1096K) or other neutral amino acids [Ile for R586
(R586I) and Met for R1096 (R1096M)]. Mutant MRP2 proteins were
detected as bands of 175 kDa (Fig.
7A) at the same position as
wild-type MRP2. ATP-dependent uptake of
[3H]E217
G was observed in all R586
and R1096 mutant MRP2 (Fig. 8A). In addition, significant
ATP-dependent uptake of [3H]TC was also observed for R586
and R1096 MRP2 mutants, except for R586K (Fig.
8A).
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Concentration dependence of TC uptake by wild-type MRP3,
L1084MMRP3, and L1084VMRP3.
Transport kinetics was determined for [3H]TC uptake
by mutant MRP3 and compared with that of wild-type MRP3 to examine
whether the substitution of Leu at 1084 of rat MRP3 affects the
transport properties. The initial velocity of uptake of
[3H]TC by these MRP3 was determined in the presence of
unlabeled TC (0.2-150 µM; Fig. 9).
Kinetic analysis yielded Km of 52.2 ± 5.8, 37.3 ± 4.5, and 73.1 ± 11.7 µM for wild-type MRP3,
L1084MMRP3, and L1084VMRP3, respectively (Table
2). No statistically significant differences were observed in the Km values for
TC among wild-type and L1084-mutated MRP3.
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DISCUSSION |
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In the present study, we substituted eight cationic amino acids in
TMs of rat MRP2 with neutral amino acids (Fig. 1) to examine the
transport activity of these mutated MRP2. The ability to transport [3H]TC was acquired by substituting Arg at position 586 or 1096 with Leu (Fig. 3). The osmotic sensitivity of TC accumulation by membrane vesicles expressing R586L and R1096L (Fig. 4B)
suggests that TC molecules are indeed transported in an ATP-dependent
manner by these mutated transporters. Along with other MRP-family
proteins (10, 24), ATP was the most effective nucleotide
triphosphate in stimulating the transport of TC concerned (Fig.
4C). The transport was saturable with calculated
Km of 28.8 ± 6.9 and 41.6 ± 5.6 µM
(Table 1) for R586L and R1096L, respectively. These values are slightly
higher than that reported in rat Bsep/spgp (5.3 µM) (6)
or MRP3 (15.9 µM) (11). Although R1096L acquired the ability to transport TC, the transport characteristics of this mutant
with respect to conjugated metabolites are similar to those of
wild-type MRP2 (Table 1). In contrast, the Km
values of R586L for the transport of E217G have been
reported to be significantly reduced (by a factor of 5) compared with
wild-type MRP2 (14).
To further characterize the acquired transport site for TC in R586L and
R1096L, the mutual effects of TC and conjugated metabolites were
examined. TC exhibited an inhibitory effect on the uptake of
[3H]DNP-SG and [3H]E217G in
both R586L and R1096L, with IC50 of ~200 µM (Fig. 6,
A and B), which were approximately seven and five
times higher than the respective Km values of
R586L and R1096L for the transport of TC (Table 1). In contrast,
transport of [3H]TC was not affected by DNP-SG or
E217
G in R586L and was enhanced in R1096L by these
conjugates (Fig. 6, C and D), suggesting that the
acquired interaction sites for TC in R586L and R1096L may not be
exactly the same as those for DNP-SG or E217
G. Because mutual inhibition has been observed between DNP-SG,
E217
G, and TC in wild-type MRP3 (11), the
molecular mechanism for substrate recognition differs between MRP3 and
these mutant MRP2 (R586L and R1096L). Moreover, TC exhibited a
stimulatory effect on the wild-type MRP2-mediated transport of
[3H]DNP-SG and [3H]E217
G
(Fig. 6, A and B). Although this result suggests
that even wild-type MRP2 has a site able to interact with TC, the
molecular mechanism for the interaction with TC differs between
wild-type MRP2 and these mutant MRP2. The presence of such
modulation/allosteric sites on the transporter molecules has also been
reported for mdr1 P-glycoprotein (5). Dey et al.
(5) demonstrated that the transport activity of the F983A
mutant of P-glycoprotein was the same as that of wild-type
P-glycoprotein, whereas the susceptibility of F983A to the stimulatory
and inhibitory effect of cis- and trans-flupentixol, respectively, was markedly reduced
compared with that of wild-type P-glycoprotein. These findings suggest the presence of modulation/allosteric site(s) for flupentixol, distinct
from the transport site for fluorescence-labeled verapamil for
P-glycoprotein. Using isolated bile canalicular membrane
vesicles, we (26) have previously shown that the
MRP2-mediated transport of [3H]DNP-SG is stimulated by up
to 240% in the presence of 500 µM 4-methylumbelliferone sulfate. It
is possible that MRP2 also has a modulation site(s).
The role of Arg at positions 586 and 1096 in rat MRP2 was additionally
explored by introducing further mutations at these positions. As shown
in Fig. 8A, R586K, which also has a cationic charge, could
not significantly transport TC, whereas R586I, along with R586L,
acquired transport activity for TC. This suggests that the presence of
the cationic charge at amino acid 586 may prevent the transport of TC
in rat MRP2. The presence of a cationic amino acid at position 586 of
rat MRP2 has previously been shown to be important for the efficient
transport of glutathione conjugates, because the transport activity for
LTC4 was markedly reduced in R586L and R586I, whereas R586K
exhibited significant transport activity for LTC4
(14). Overall, a cationic amino acid at position 586 may
contribute to the formation of the recognition/transport site that is
favorable for glutathione conjugates but unfavorable for TC, whereas a
neutral amino acid at this position may contribute to the formation of
a site that is favorable for TC, but unfavorable for glutathione
conjugates. Concerning rat MRP3, we (A. Morikawa, H. Suzuki, and Y. Sugiyama, unpublished observations) recently found that the
substitution of Arg at position 578 (which corresponds to R586 in rat
MRP2) with Met results in the loss of transport activity for
E217G and TC. This result suggests that the neutral amino acids at this position may not support the transport of rat MRP3.
Our initial hypothesis was that the charge of the amino acid at the 1096 position of rat MRP2 may be an obstacle to TC transport, because MRP3 has a neutral amino acid at the corresponding position (Fig. 1B). However, transport of TC was observed not only in R1096L and R1096M, but also in R1096K (Fig. 8A). The fact that human and mouse MRP1 do not accept TC as a substrate, irrespective of the presence of Lys at the corresponding position (Lys at 1092 in human MRP1 and Lys at position 1089 in mouse MRP1; Fig. 1B), suggests that not only the amino acid at this position but also other surrounding amino acid(s) may be jointly involved in exhibiting transport activity for TC.
Next, we examined the role of Leu at position 1084 in MRP3 by preparing
several kinds of mutants. First, we substituted Leu at position
1084 with Arg (L1084RMRP3), because wild-type MRP2 has an Arg at the corresponding position (Arg-1096; Fig.
1B), expecting that the transport of TC would be selectively
reduced in L1084RMRP3. However, we could not obtain mature
L1084RMRP3 proteins, and only truncated protein with an
approximate molecular mass of 80 kDa was produced (Fig. 7B).
Although we do not have a satisfactory explanation to account for the
production of a shorter band of ~80 kDa in L1084RMRP3, it
is possible this mutant protein may have easy access to some type of
protease(s) that may be produced due to the Arg substitution at
position 1084. Loo and Clarke (25) demonstrated that the
substitution of Gly with Cys at position 341, located in the middle of
the TM6 of P-glycoprotein, resulted in cleavage of the extracellular
loop located between TM1 and TM2 to produce a truncated protein product of 130 kDa. To examine the role of the amino acid at position 1084 in
rat MRP3, we also substituted L1084 with Lys (L1084KMRP3), Met (L1084MMRP3), and Val (L1084VMRP3). Because
the molecular weight of these three mutant MRP3 is the same as that of
wild-type MRP3 (Fig. 7B), we assumed that the extent of
glycosylation of the mutant proteins is not altered by introduction of
the mutation. The transport activity for both TC and
E217G was completely abolished in L1084KMRP3
(Fig. 8B), irrespective of exhibiting the same protein expression level as wild-type MRP3 (Fig. 7B). Disruption of
the conformation required for transport activity, produced by
introducing a cationic amino acid inside the potential
-helix
region, may be one possible reason for the disappearance of TC and
E217
G transport activity in L1084KMRP3. As
far as L1084MMRP3 and L1084VMRP3 are concerned,
the Km values for TC were similar to that of
wild-type MRP3 (Fig. 9 and Table 2). In addition, although some
reduction in transport activity for TC was found for
L1084MMRP3 and L1084VMRP3, compared with
wild-type MRP3, the reduction in transport activity for
E217
G was comparable with that for TC (Fig.
8B), suggesting that the recognition/transport of TC and
E217
G cannot be distinguished by these mutants.
In conclusion, we have demonstrated that substitution of a single cationic amino acid in the predicted TM11 and TM14 provides rat MRP2 with a transport site for monovalent bile acids, which is distinguishable from the native transport sites for glutathione and glucuronide conjugates. These findings provide new insights into the multispecific and potential transport activity of MRP families.
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ACKNOWLEDGEMENTS |
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This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (ABC proteins) 10044243 from the Ministry of Education, Science, and Culture of Japan.
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FOOTNOTES |
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This study was presented in part at the 51st Annual Meeting of the American Association for Liver Diseases, in Dallas, TX, October 27-31, 2000, and has been previously published in abstract form (Hepatology 32: 442A, 2000).
Present address of K. Ito: Graduate School of Pharmaceutical Sciences, Chiba Univ., Yayoi-cho 1-33, Inage-ku, Chiba, 263-8522, Japan.
Address for reprint requests and other correspondence: H. Suzuki, Graduate School of Pharmaceutical Sciences, Univ. of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan (E-mail: seizai.suzuki{at}nifty.ne.jp).
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.
Received 26 October 2000; accepted in final form 18 June 2001.
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