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J. Biol. Chem., Vol. 278, Issue 26, 23538-23544, June 27, 2003
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Evidence for Two Interacting Ligand Binding Sites in Human Multidrug Resistance Protein 2 (ATP Binding Cassette C2)*
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From the
Division of Molecular Biology and Center
of Biomedical Genetics, and the Divisions of
Experimental Therapy and
¶Molecular Carcinogenesis, Netherlands Cancer
Institute, Amsterdam 1066 CX, The Netherlands
Received for publication, April 4, 2003 , and in revised form, April 15, 2003.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Multidrug resistance protein 2 (MRP2) belongs to the ATP binding cassette family of transporters. Its substrates include organic anions and anticancer drugs. We have used transport assays with vesicles derived from Sf9 insect cells overproducing MRP2 to study the interactions of drugs, organic anions, and bile acids with three MRP2 substrates: estradiol-17-
-D-glucuronide (E217G),
methotrexate, and glutathione-S-dinitrophenol. Complex inhibition and
stimulation patterns were obtained, different from those observed with the
related transporters MRP1 and MRP3. In contrast to a previous report, we found
that the rate of E217G transport by MRP2 increases
sigmoidally with substrate concentration indicative of homotropic
cooperativity. Half-maximal transport was obtained at 120 µM
E217G, in contrast to values < 20 µM for
MRP1 and 3. MRP2 stimulators, such as indomethacin and sulfanitran, strongly
increased the affinity of MRP2 for E217G (half-maximal
transport rates at 65 and 16 µM E217G,
respectively) and shifted the sigmoidal dependence of transport rate on
substrate concentration to a more hyperbolic one, without substantially
affecting the maximal transport rate. Sulfanitran also stimulated MRP2
activity in cells, i.e. the transport of saquinavir through
monolayers of Madin-Darby canine kidney II cells. Some compounds that
stimulate E217G transport, such as penicillin G or
pantoprazole, are not detectably transported by MRP2, suggesting that they
allosterically stimulate transport without being cotransported with
E217G. We propose that MRP2 contains two similar but
nonidentical ligand binding sites: one site from which substrate is
transported and a second site that regulates the affinity of the transport
site for the substrate. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Members of the ABC1 family of membrane transporters mediate the transport of various substrates across biological membranes at the expense of ATP hydrolysis (1, 2). The ABCC subfamily (3) contains multidrug resistance proteins 1–9 (MRP1–9) along with SUR1, SUR2, and CFTR (1, 2, 4, 5). Interest in the multidrug resistance proteins was sparked by their possible involvement in the clinical resistance of tumors to chemotherapeutic agents. The first member of this family to be cloned, MRP1, confers resistance to a broad spectrum of anticancer drugs when overproduced in cells (6). A common feature of MRPs is that they transport a wide variety of organic anions and compounds that are conjugated with sulfate, glucuronate, or glutathione (GSH) (7; for review, see Refs. 2 and 8–10).
How MRPs transport their substrates is not known in detail. MRPs are large membrane-associated proteins, and their structural analysis has proven difficult (11). Although several high resolution structures of bacterial ABC transporters have been determined (12, 13), only low resolution structures are available for the drug transporters MRP1 and MDR1 P-glycoprotein (14–16). In the absence of a detailed structure, the mechanism of transport has been inferred from a combination of transport, binding and mutational studies. Models proposed for MDR1 P-glycoprotein predict three or four drug binding sites or a single complex substrate binding site in which the binding of one compound can affect the binding of another one, the induced-fit model (17–20). Also for MRP1 evidence for more than one ligand binding site was obtained (for review, see Refs. 2 and 21).
The major canalicular organic anion transporter, MRP2 (ABCC2), is closely related to MRP1 (2, 8). The substrate specificities of MRP1 and 2 overlap to a large extent (9, 22–24), but their tissue localizations differ. MRP1 is localized in the basolateral membranes of polarized cells and is present in all tissues, whereas MRP2 is found in the apical membranes of polarized cells and is expressed mainly in the liver, kidney, and intestine. Bakos et al. (25) demonstrated in vesicular transport assays that transport of the GSH conjugate of N-ethylmaleimide by MRP2 is stimulated by several organic anions. Experiments with polarized cells led to a model in which MRP2 cotransports drugs from two distinct drug binding sites (26). Cotransport cannot account for recent observations on MRP1, however (27).
In vectorial transport assays with MDCKII/MRP2 cells we recently observed
that the transport of saquinavir is stimulated by probenecid
(28). Such drug interactions
could potentially affect the oral bioavailability and pharmacokinetics of
drugs transported by MRP2. We have therefore studied drug interactions with
MRP2 more in detail using transport assays with membrane vesicles from
Spodoptera frugiperda (Sf9) insect cells that were infected with a
baculovirus construct containing MRP2
(25). Using
estradiol-17--D-glucuronide (E217G),
methotrexate (MTX), and glutathione-S-dinitrophenol (GS-DNP) as model
substrates, we found stimulation of substrate transport by a range of
compounds. We propose that MRP2 contains two distinguishable binding sites:
one site from which drug is transported and a second site that allosterically
regulates the former. Analogous results have been independently obtained by
Bodo and colleagues and are presented in the accompanying manuscript.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Materials—40.5 Ci/mmol [3H]E217
G, 21 Ci/mmol [3H]penicillin G,
and 20 mCi/mmol [14C]indomethacin were obtained from PerkinElmer
Life Sciences. 9 Ci/mmol [3H]MTX was obtained from Amersham
Biosciences. 13.6 µCi/mg [14C]saquinavir was from Roche Applied
Science. Vials of omeprazole (Losec®, AstraZeneca) and pantoprazole
(Pantozol, Altana Pharma BV) were obtained from the pharmacy of The
Netherlands Cancer Institute and dissolved in saline according to the
manufacturers' instructions. Creatine phosphate and creatine kinase were
obtained from Roche, and RC-L55 and OE-67 filters were from Schleicher &
Schuell. All other chemicals and reagents were purchased from Sigma. Cell Lines and Culture Conditions—Sf9 insect cells in suspension were grown in Sf-900 II SFM medium in the absence of serum (Invitrogen). The MDCKII control and MRP2-overproducing lines were described previously (24) and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 units of penicillin/streptomycin/ml. Cells were grown at 37 °C with 5% CO2 under humidifying conditions.
Protein Analysis and Immunoblotting—Membrane vesicle preparations were diluted in buffer (10 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 7.4), supplemented by a mixture of protease inhibitors used at the concentration recommended by the manufacturer (Roche). The indicated amount of protein was size fractionized on a 7.5% SDS-polyacrylamide gel and subsequently blotted overnight in a tank blotting system. MRP1, 2, and 3 were detected with the monoclonal antibodies MRP-r1 (1:1000), M2II5 (1:250) and M3II9 (1:250), respectively, as described previously (24, 29). Signals were visualized with chemiluminescence (ECL, Amersham Biosciences).
Transepithelial Transport Assays—Transepithelial transport
assays were done as described previously
(28). Briefly, Cells were
seeded on microporous polycarbonate membrane filters (Transwell 3414, Costar,
Corning, NY) at a density of 1.0 x 106 cells/well in 2 ml of
complete medium. Cells were grown for 3 days with medium replacements every
day. 2 h before the start of the experiment, complete medium was replaced from
both compartments with Opti-MEM, without serum containing 5 µM
GF120918 and the appropriate concentration of transport modulators. At
t = 0 h the experiment was started by replacing the medium from both
compartments containing 5 µM appropriate radiolabeled drug
(
3 kBq/well) and either 3H- or 14C-labeled inulin
( 3 kBq/well) in the appropriate compartment. The latter compound was
added to check for leakage through the cell layers. Cells were incubated at 37
°C in 5% CO2, and 50-µl aliquots were taken each hour. The
radioactivity in these aliquots was measured by the addition of 4 ml of
scintillation fluid (Ultima-gold; Packard, Meriden, CT) and subsequent liquid
scintillation counting. Inulin leakage did not exceed 3% over 4 h. The
percentage of radioactivity appearing in the opposite compartment, of the
total amount initially applied, was measured and plotted. The amount of
radiolabeled drug in the cell layer at the end of the experiment was
determined by liquid scintillation counting of the excised filter, after
washing with ice-cold phosphate-buffered saline.
Preparation of Membrane Vesicles—Membrane vesicles from Sf9 cells were obtained after infection with an MRP1 (25), MRP2 (25), MRP3 (29), or MRP4 (30, 31) cDNA-containing baculovirus at a multiplicity of infection of 1. After incubation at 27 °C for 3 days, cells were harvested by centrifugation at 3,000 rpm for 5 min. The pellet was resuspended in ice-cold hypotonic buffer (0.5 mM sodium phosphate, 0.1 mM EDTA, pH 7.4) supplemented with protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 10 µM pepstatin) and incubated at 4 °C for 90 min. The suspension was centrifuged at 4 °C at 100,000 x g for 40 min, and the pellet was homogenized in ice-cold TS buffer (50 mM Tris-HCl, 250 mM sucrose, pH 7.4) using a tight fitting Dounce homogenizer. After centrifugation at 500 x g at 4 °C for 10 min, the supernatant was centrifuged at 4 °C at 100,000 x g for 40 min. The pellet was resuspended in TS buffer and passed through a 27-gauge needle 25 times. The vesicles were dispensed in aliquots, frozen in liquid nitrogen, and stored at –80 °C until use.
Vesicular Transport Assays—Vesicular transport assays were performed in buffer consisting of 100 mM KCl, 50 mM HEPES/KOH, pH 7.4, in the presence or absence of 4 mM ATP (32). Similar results were obtained with a Tris/sucrose buffer (not shown). The time- and concentration-dependent uptake of substrates into membrane vesicles was studied following the rapid filtration method as described previously (29). For all results presented here, accumulation of substrate increased with time for at least 10 min and was dependent on the presence of ATP. The ATP-dependent transport was calculated by subtracting the transport in the absence of ATP from that in its presence. In experiments where the effect of GSH on transport was studied, 10 mM dithiothreitol was in the reaction mixture. Concentration-dependent uptake was analyzed using a nonlinear regression algorithm.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Effects of Drugs and Organic Anions on E217
G Transport by
MRP2—Membrane vesicles were prepared from Sf9 insect cells
transfected with a recombinant baculovirus coding for human MRP2. These
vesicles contain high levels of MRP2 (Fig.
1) and were used to study the effect of various organic anions and
commonly used drugs on the transport of 1 µM
E217G by MRP2. The compounds tested could be divided into
four classes based on their interaction with MRP2: 1) compounds that only
showed a stimulatory effect at the concentrations tested
(Fig. 2, A and
B); 2) compounds that stimulated transport at low
concentrations but showed a decrease in their stimulation capacity at higher
concentrations (Fig. 2, C and
D); 3) compounds that only inhibited transport
(Fig. 2E); and 4)
compounds that had no substantial effect on the transport of
E217G by MRP2 (Fig.
2F).
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Sulfanitran, the strongest stimulator of MRP2-mediated
E217G transport, also stimulated the vectorial transport of
saquinavir, a recently described MRP2 substrate
(28), across polarized MDCKII
monolayers demonstrating that it also stimulates MRP2 in intact cells
(Fig. 3). Comparison of
B and D of Fig.
3 shows that sulfanitran increases transport of saquinavir in the
apical direction, decreases transport in the basolateral direction, and
substantially decreases the intracellular concentration of saquinavir.
Saquinavir is too hydrophobic to study in the vesicular transport assay, but
in transepithelial transport assays, we have shown previously that in addition
to sulfanitran, other stimulators of vesicular transport also stimulate MRP2
in intact cells: transport of saquinavir is stimulated by both sulfinpyrazone
and probenecid (28), and
sulfinpyrazone and indomethacin stimulate transport of GSH
(26).
|
) and from the basolateral to apical (•) directions was determined.
Each point is the mean ± S.E. of experiments in triplicate.
Values within figures represent the percent of radioactivity
found in the monolayer after a 4-h incubation with saquinavir in the apical
(Ap) or basolateral (Bl) compartments.
The stimulation by the compounds studied was specific for MRP2. Neither
lansoprazole nor saquinavir at their maximal MRP2-stimulatory concentration
stimulated transport of E217G in wild type, MRP1, MRP3, or
MRP4 vesicles, and sulfanitran had no effect on transport of
E217G in wild type vesicles either (data not shown).
Furosemide and acetaminophen-glucuronide even inhibited MRP3-mediated
transport of E217G (Fig.
4), whereas these compounds stimulated transport of
E217G by MRP2 (Fig.
2). Sulfanitran, the compound that stimulated MRP2 transport most,
had only a minimal effect on MRP3 (Fig.
4).
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Effects of Drugs on Transport of GS-DNP by MRP2—GSH
conjugates are another class of molecules transported by MRP2. We therefore
tested whether transport of GS-DNP, a model GSH conjugate and a known
substrate of MRP2, could be stimulated like transport of
E217G. The results are summarized in
Table I. Like
E217G, GS-DNP transport is stimulated by sulfanitran and
indomethacin albeit to a lower extent. Sulfinpyrazone stimulates GS-DNP
transport in vesicular transport assays, similar to what we found previously
in MDCKII/MRP2 cells (24).
Furosemide at its maximal stimulatory concentration (500 µM) has
only a marginal effect on GS-DNP transport, in contrast to its effect on
E217G transport. Moreover, whereas probenecid strongly
stimulated the transport of E217G, it inhibited GS-DNP
transport as is the case for MTX as well.
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Effects of Stimulators of MRP2-mediated
E217G Transport on the Affinity of
MRP2 for Substrate—Evers et al.
(26) demonstrated that
sulfinpyrazone stimulates transport of GSH by MRP2 and that vinblastine
transport is accompanied by GSH transport at an approximate ratio of 1:1.
Sulfinpyrazone also stimulates transport of E217G by MRP2
(Fig. 2C). Compounds
that stimulate E217G transport by MRP2 might therefore be
cotransported with this substrate, as proposed previously
(26). We have tested this in
vesicular and transepithelial transport assays. We did not detect vesicular
transport of [3H]penicillin G at concentrations of up to 1
mM either in the absence or presence of varying concentrations of
E217G (data not shown). Similarly, In vesicular transport
assays we did not detect transport of [14C]indomethacin (at
concentrations up to 50 µM) by MRP2. Negative results in
vesicular transport assays are not conclusive, however, as the substrate may
leak out of the vesicles at high rate, preventing transport measurements. This
is not a problem in transepithelial transport assays with MDCKII/MRP2 cells.
Indeed, in these assays we detected marginal transport of indomethacin and
probenecid by MRP2 (data not shown). In the same assays, we did not detect
transport of penicillin G or pantoprazole, another stimulator of
E217G transport by MRP2
(Fig. 2, and data not shown).
These results indicate that these compounds are either not transported by MRP2
or are poor substrates, even though they strongly stimulate transport of
E217G by MRP2, making cotransport unlikely.
To investigate the mechanism of stimulation further, we determined the rate
of transport of E217G by MRP2 as a function of substrate
concentration in the absence or presence of 100 µM indomethacin
or 1 mM sulfanitran (Fig.
5). The transport of E217G by MRP2 was not
consistent with simple Michaelis-Menten kinetics, but the plot of reaction
velocity versus substrate concentration was clearly sigmoidal with an
estimated apparent half-maximal rate (S1/2) at 120
µM E217G
(Fig. 5A). In the
presence of either of the two stimulators, the curve was shifted to a more
hyperbolic shape with apparent S1/2 values of 65 and 16
µM in the presence of 100 µM indomethacin and 1
mM sulfanitran, respectively
(Fig. 5, B and
C). The maximal rate of transport remained relatively
unchanged. The degree of stimulation of E217G transport at
low substrate concentration (1 µM) by these compounds correlates
well with the increased affinity for this substrate (Figs.
2 and
5). At 200 µM
E217G transport was not stimulated by 100 µM
indomethacin and was only stimulated by 10% by 1 mM sulfanitran
(data not shown) suggesting that at this concentration of substrate MRP2 is
close to saturation. We note, however, that Bodo et al.2
found higher rates of E217G transport at 1 mM than
at 200 µM, the maximal concentration that we were able to test
because of solubility problems.
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As a control, we also determined the concentration-dependent transport of
E217G by MRP1, for which we found saturation kinetics with a
Km of 3.1 ± 0.3 µM and a
Vmax of 38 ± 1 pmol/mg/min. This further
strengthens the notion that the requirements for optimal transport of the same
substrate by MRP1 and MRP2 are different
(27), even though the
substrate specificity of these transporters largely overlaps.
Characterization of MTX Transport by MRP2—MTX is transported
by MRP2, but MRP2 has such a low affinity for this substrate that we were
unable to determine reliable kinetic parameters for this transport process
(not shown and Refs. 25 and
33). Sulfinpyrazone and
indomethacin stimulate MTX transport, but to a much lower extent than the
transport of E217G (Fig.
6A). In contrast, E217G, GSSG, and
probenecid only inhibited MTX transport by MRP2
(Fig. 6B).
E217G at a concentration of 200 µM inhibits the
transport of MTX by 80%, suggesting that these two substrates share a common
step in transport. In this light, the absence of a substantial inhibitory or
stimulatory effect of MTX (at a concentration up to 4.4 mM) on
transport of 1 µM E217G by MRP2
(Fig. 2F) is
unexpected. A possible explanation is that MTX is a weak stimulator of
E217G transport and that at low E217G
concentrations (allosteric) stimulation and inhibition (by competition) of
E217G transport by MTX balance out. Indeed, MTX did inhibit
the transport of high concentrations of E217G (200
µM) and of 1 µM E217G stimulated
by 1 mM sulfanitran (Fig.
7). Moreover, trimetrexate, a structural analog of MTX, stimulates
transport of 1 µM E217G by 320 ± 10% at
a concentration of 300 µM (data not shown). This is compatible
with the hypothesis that MTX itself might have a weak stimulatory effect as
well. Following the reasoning applied to MTX, GSH, another low affinity
substrate of MRP2 (34), should
be able to inhibit E217G and MTX transport under appropriate
conditions. However, GSH at concentrations up to 10 mM had no
effect on the transport of 200 µM E217G or 100
µM MTX by MRP2 (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Our work shows complex effects of various drugs and organic anions on MRP2. For transport of E217
G the plot of reaction velocity
versus substrate concentration is sigmoidal
(Fig. 5), indicative of at
least two drug binding sites that interact in a positively cooperative manner.
Many compounds stimulate E217G transport at low substrate
concentrations, and for two stimulators, sulfanitran and indomethacin, we have
shown that they increase the affinity of MRP2 for substrate with no
significant effect on the Vmax.
Compounds that stimulate transport of substrates by MRP2 are not
necessarily transported by MRP2. In vesicular transport assays we do not
detect transport of [3H]penicillin G and
[14C]indomethacin. Using vectorial transport assays with
MDCKII/MRP2 cells we found only marginal transport of indomethacin and no
transport of pantoprazole, another stimulator. Taurocholate is a good
stimulator of E217G transport by human
(Fig. 2D) and rat
Mrp2, but it is not transported by rat Mrp2
(35), as is the case with
furosemide (36). Taken
together, these observations indicate that transport of a compound by MRP2 is
not a prerequisite for its ability to stimulate the transport of another
compound.
The effect of a transport modulator depends on the substrate transported.
Probenecid strongly stimulates transport of E217G
(Fig. 2) but inhibits transport
of MTX (Fig. 6) and GS-DNP
(Table I). Indomethacin and
sulfinpyrazone stimulate E217G transport more than transport
of GS-DNP or MTX. Furosemide strongly stimulates E217G
transport but has no effect on GS-DNP transport. It even inhibits
GS-N-ethylmaleimide transport
(25). These observations are
compatible with the idea that each substrate-modulator pair forms unique
interactions within the complex drug binding sites of MRP2, a possibility
raised previously for MRP1
(27,
32).
Although the substrate specificities of MRP1, 2, and 3 largely overlap,
these transporters handle some substrates/inhibitors in different ways. The
affinity of MRP1 (this study and Ref.
37) and MRP3
(29) for
E217G is relatively high, and transport follows
Michaelis-Menten kinetics, in contrast to our result for MRP2. Stimulators of
MRP2-mediated E217G transport may even inhibit transport by
MRP3. Furosemide and acetaminophen glucuronide are examples. Although
glycocholic and taurocholic acid can stimulate transport of
E217G by MRP2, they only inhibit transport of etoposide
glucuronide (38) and MTX
(39) by MRP3. These results
suggest that MRPs bind a similar range of compounds, but not in the same
manner.
On the basis of our findings we propose that MRP2 has two similar but
nonidentical ligand binding sites: one site from which substrate is
transported (S site) and a second site that is able to modulate transport (M
site). Binding of a modulator to the M site induces a structural change that
results in a better fit of the substrate at the S site. Compounds that only
stimulate transport of E217G (e.g. sulfanitran) bind
only to the M site. Compounds that display a "bell-shaped"
stimulation of MRP2-mediated E217G and MTX transport
(e.g. indomethacin) bind at a low concentration predominantly to the
M site. At a higher concentration they compete for binding with
E217G at the S site as well. Whether such a stimulating
compound is also transported by MRP2 must depend on its interactions with the
substrate site. The stimulators penicillin G and pantoprazole are not
detectably transported by MRP2, but sulfinpyrazone and saquinavir are
(26,
28). The GSH conjugates we
tested (GS-DNP and GSSG) only inhibited the transport of both
E217G and MTX. This suggests that these GSH conjugates, both
known to be transported by MRP2
(25,
34), bind MRP2 predominantly
at the S site in a way that competes with binding of other transported
substrates. Some of the compounds that stimulate E217G
transport also stimulate the transport of GS-DNP, indicating that binding of
GS-DNP to the S site leaves the M site accessible to modulators that are able
to fit together with GS-DNP and stimulate its transport (e.g.
sulfanitran). We surmise that probenecid, in contrast, cannot fit together
with GS-DNP to form a stimulator-substrate pair and therefore only inhibits
transport of GS-DNP.
MTX represents another class of MRP2 substrates
(25,
33). Ito et al.
(40) reported that MTX
inhibits transport of E217G by MRP2 with an approximate
IC50 of ± 1 mM, whereas we found that transport
of 1 µM E217G by MRP2 is slightly stimulated by
MTX up to 4.4 mM. We have no explanation for this discrepancy, but
our results are readily explained by our model. The affinity of MTX for the
substrate site is low, making it a poor competitive inhibitor of transport.
Moreover, MTX weakly stimulates transport of E217G
(Fig. 2F), indicating
that it binds to the M site as well. Hence, MTX will inhibit
E217G transport only under conditions where it can no longer
stimulate it, i.e. close to E217G substrate
saturation (Fig. 7). Using a
similar reasoning, we also expected to find conditions where GSH, a low
affinity substrate of MRP2
(34,
41), would inhibit
E217G or MTX transport, but no effect was found up to 10
mM GSH. In contrast, 3 mM GSH has been shown to inhibit
transport of NNAL-glucuronide by MRP2 nearly completely
(27). A speculative
explanation for these large differences is that GSH binding within the
drug-transporting site of MRP2 disrupts the binding of some cosubstrates
(e.g. NNAL-glucuronide) but not of other ones (e.g.
E217G), as proposed previously by Leslie et al.
(27).
Complex interactions between multiple drug binding sites in ABC transporters have been described already for P-glycoprotein (17–20, 42, 43) and MRP1 (27, 44–49). The two-site model proposed here is patterned on results obtained for the cytochrome P-450 monooxygenases, which contain a single complex binding site able to bind two ligands (50, 51). There are other explanations for apparent cooperativity, however (52). Structural studies on ligand-bound MRP2 will be required to determine how the protein works.
Given the complex heterotropic positive drug interactions found for MRP1
and MRP2, it is necessary to reconsider the results interpreted as cotransport
of drug and GSH reported previously
(26,
45,
46). If GSH can bind both to
the M site and the S site of MRP2, as shown unambiguously for MRP1
(47,
48), apparent cotransport
could be the result of cross-stimulation, in which GSH in the M site
stimulates transport of drug in the S site and vice versa. At present, we see
no compelling evidence for the alternative that the M site can function as a
transport site, but given the postulated presence of at least two transport
sites in P-glycoprotein, this alternative remains open for the MRPs. It
follows that we cannot exclude cotransport either, as proposed previously by
Evers et al. (26). It
should be noted that the transport of E217G by human
(23) and rat MRP2
(23,
53) has been analyzed before.
In both cases saturation kinetics and Km values
of 4–7 µM were reported
(23,
53). More experiments are
required to resolve the discrepancy with our results.
Some of the drugs studied here are used in patients (e.g. glibenclamide for the treatment of diabetes), and the allosteric properties of MRP2 could therefore result in adverse or beneficial drug-drug interactions, as pointed out before (25, 28). On the one hand, stimulation of intestinal and hepatic MRP2 could lead to a decrease in bioavailability of drugs and thus to a lower treatment efficacy. On the other hand, stimulation of the secretion of toxic metabolites could be beneficial (see also Ref. 20). Many, but not all drugs able to stimulate MRP2 are aromatic compounds containing a sulfoxide (S = O) or tosyl (O = S = O) group. In fact, two of the stimulators in the present study, sulfanitran and glibenclamide, were chosen by us solely based on this structural property. The exact requirements for MRP2 stimulation remain to be determined.
Allosteric interactions seem to be a general feature of MRPs, and such interactions have been described for MRPs originating from plants (54), rodents (53, 55), as well as for human MRP1 and MRP3 (25, 38). In recent years, several MRP2 point mutants and chimeric constructs have been generated and characterized (40, 53, 56). These offer the opportunity to study the regions involved in the drug interactions reported here.
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FOOTNOTES |
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* This work was supported by the Dutch Cancer Society Grants NKI 2001-2473, 2001-2474, and 1998-1794 (to P. Bo.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|| To whom correspondence should be addressed: Dept. of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands. Tel.: 31-20-512-2880; Fax: 31-20-512-2886; E-mail: p.borst{at}nki.nl.
1 The abbreviations used are: ABC, ATP binding cassette;
E217G, estradiol-17--D-glucuronide; GS-DNP,
glutathione-S-dinitrophenol; MDCK, Madin-Darby canine kidney; MRP,
multidrug resistance protein; MTX, methotrexate; NNAL-glucuronide,
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol glucuronide;
S1/2, apparent half-maximal rate; Sf9, Spodoptera
frugiperda.
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ACKNOWLEDGMENTS |
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We thank Henk de Vries, Nobuhito Ono, Hein te Riele (Division of Molecular Biology, The Netherlands Cancer Institute), Johan W. Jonker (Division of Experimental Therapeutics, The Netherlands Cancer Institute), and Ronald Oude Elferink (Department of Experimental Hepatology, University of Amsterdam) for critically reading this manuscript. We thank Balazs Sarkadi (National Institute of Hematology, Budapest, Hungary) for providing the recombinant baculoviruses encoding MRP1 and MRP2. We appreciate that Bodó and colleagues (57) allowed us to read their paper and critically reviewed our manuscript.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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