From the Department of Cancer Chemotherapy, Institute
for Cancer Research, Faculty of Medicine, Kagoshima University,
Sakuragaoka 8-35-1, Kagoshima 890-8520 and the ¶ Graduate School
of Pharmaceutical Sciences, Osaka University, Yamada-oka 1-6, Suita,
Osaka 565-0871, Japan
Received for publication, February 20, 2001, and in revised form, April 4, 2001
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ABSTRACT |
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MRP1 is a 190-kDa membrane glycoprotein
that confers multidrug resistance (MDR) to tumor cells. MRP1 is
characterized by an N-terminal transmembrane domain
(TMD0), which is connected to a P-glycoprotein-like
core region ( Following exposure to a natural product chemotherapeutic agent,
tumor cells often acquire resistance to several structurally and
functionally unrelated drugs, so-called multidrug resistance (MDR).1 MDR is the major
obstacle to successful cancer chemotherapy. Two membrane proteins,
P-glycoprotein (P-gp) and the human multidrug resistance protein (MRP1)
are frequently overexpressed in MDR cells (for reviews see Refs. 1-4).
Although both MRP1 (190 kDa) and P-gp (170 kDa) are members of the
family of ATP-binding cassette transporters (5), they share only 15%
amino acid sequence identity (6). The amino acid sequence suggests that
P-gp consists of two homologous halves and a variable linker region.
Each half of the protein has six transmembrane segments and one
nucleotide binding domain (NBD). MRP1 differs from P-gp by the presence
of an extra N-terminal extension with five transmembrane segments (TMD0), which is connected to the P-gp-like core ( It has been reported that GSH plays a critical role in MRP1-mediated
MDR (15). Reduction of GSH level in MRP1-expressing cells by buthionine
sulfoximine, an inhibitor of GSH synthesis, resulted in a decrease in
the active efflux of some anticancer agents, and an increase in
cellular sensitivity to adriamycin (ADM), vincristine (VCR), and
etoposide (VP-16) (15). In addition to conjugated anions, MRP1 has been
shown to actively transport some unmodified compounds such as VCR, only
in the presence of GSH (16). There is also evidence that VCR and GSH
are co-transported by MRP1 (16). ATP-dependent VCR
transport into membrane vesicles overexpressing MRP1 could be
stimulated by GSH, and ATP-dependent uptake of
[3H]GSH was stimulated by VCR. Although all of the above
data strongly suggest that GSH is essential for MRP1-mediated MDR, the
role of GSH in binding and/or transport of drugs by MRP1 is unclear. In
contrast to the above studies, Vezmar et al. (17, 18)
reported that a quinoline-based photoactive drug,
N-{4-[1-hydroxy-2-(dibutylamino) ethyl]
quinolin-8-yl}-4-azidosalicylamide (IAAQ), photolabeled MRP1 in both
MRP1-overexpressing intact cells and membrane vesicles, and that GSH is
not required for the binding of the drug. More recently, another
quinoline-based photoaffinity drug,
N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide (IACI), which has a structure very similar to that of IAAQ, has also
been shown to photolabel multiple sites of MRP1 in the absence of GSH
(19). These findings raise the question of whether GSH is essential for
MRP1-mediated MDR, and, if it is essential, which step in the drug
transport is stimulated by GSH: the drug binding step or subsequent steps.
Photoaffinity analogs of some xenobiotics are powerful tools for
studying drug binding sites on proteins (20). Photoaffinity analogs of
some anti-cancer and other reversing agents, such as azidopine (21),
N-(p-azido-(3-I)-salicyl)-N'- Recently, we isolated a novel polyhydroxylated sterol acetate, named
agosterol A (AG-A), from a marine sponge (Spongia sp.). We
found that AG-A completely reversed MDR in human KB carcinoma cells
overexpressing MRP1 (27), and that AG-A competitively inhibited the
transport of LTC4 by MRP1 (28). This suggests that AG-A is
a new MDR-reversing agent.
In the present study, we synthesized a photoaffinity analog of
AG-A, [125I]11-azidophenyl agosterol A
([125I]azidoAG-A), and used it to photolabel MRP1. We
found that GSH is required for [125I]azidoAG-A binding to
the C-terminal half of MRP1. In order to clarify the site of GSH
interaction on MRP1, a series of truncated and mutated MRP1
constructs and co-expressed half-molecules of MRP1 were used to see if
they are photolabeled by the photoanalog of AG-A in the presence or
absence of GSH. We found that the C-proximal fragment of MRP1
(C932-1531) is photolabeled by the photoanalog of AG-A
in the presence of GSH and GSH interacts with the L0
region of MRP1.
Materials--
[125I]NaI (3.7 GBq (100 mCi)/ml)
was purchased from PerkinElmer Life Sciences. AG-A was isolated
from a marine sponge (Spongia sp). collected in Mie
Prefecture, Japan. Details of its isolation and determination of its
structure were described in a previous paper (27).
2-Azido-5-(trimethylstannyl) benzoic acid was attached to the
11-hydroxy group of AG-A with a C-4 linker and then iodinated with
[125I]NaI and chloramine T (Fig. 1) (29). The product was
confirmed as [125I]azidoAG-A by comparison with a
standard sample on thin layer chromatography. 7.2 µ Ci/nmol
[125I]azidoAG-A was used for the experiment. PAK-104P,
MK571, and ONO-1078 were obtained as described previously (30). Mouse
IgG2a and Protein G-Sepharose 4B were obtained from Zymed
Laboratories Inc. (San Francisco, CA). Cellfectin and competent
DH10Bac Escherichia coli cells were purchased from Life
Technologies, Inc. Other drugs and chemicals were obtained from Sigma.
Cell Culture and Membrane Vesicle Preparation--
KB/MRP, human
KB cells transfected with MRP1 cDNA, and KB/CV, KB cells
transfected with an empty vector, were cultured in minimum essential
medium (Nissui Seiyaku Co., Tokyo, Japan) containing 10% newborn calf
serum as described previously (31, 32). Sf21 insect cells were
cultured in serum-free Sf-900 II SFM medium (Life Technologies, Inc.).
Membrane vesicles were prepared from KB/MRP, KB/CV cells, and
Sf21 insect cells infected with various recombinant
baculoviruses as described previously (30). Membrane vesicles were
suspended in buffer containing 10 mM Tris-HCl (pH 7.5), and
250 mM sucrose. Protein concentrations were determined by
the method of Bradford (33).
Generation of Constructs and Viral Infection--
pFastBac
MRP1-His containing the MRP1 coding region was constructed
as described previously (30).
To generate a construct expressing
To generate constructs for expression of the N1-932 and
C932-1531 of MRP1, nucleotides 2505-2995 and 2993-4399 of MRP1 were amplified by PCR from an MRP1 cDNA template. The primers used for the 2505-2995 fragment were
5'-CTGGGGGCCAGAAGCAGCGCGTGAG-3' (forward) and
5'-TTATGCGGTGCTGTTGTGGTGCC-3' (reverse). A stop codon
(underlined) was introduced into the reverse primer. The primers used
for the 2993-4399 fragment were:
5'-ATGGCAGAACTGCAGAAAGCTGAG-3' (forward) and
5'-CCAGACTTCTTCATCCGAGT-3' (reverse). In the forward primer, a
start codon was introduced (underlined). The two PCR fragments were
first cloned into a pBluescript T-vector to make pBluescript
MRPN and pBluescript MRPC. The orientation and
fidelity of the PCR fragments were verified by sequence analysis and
were then excised from the plasmid as XhoI/NotI
and SalI/NcoI fragments, respectively. The
XhoI/NotI fragment was then ligated to pFastBac MRP1-His predigested with XhoI and
NotI to construct pFastBac1 MRPN encoding
N1-932. The SalI/NcoI fragment,
together with the MRP1 NcoI/KpnI
fragment, was cloned between the XhoI and KpnI sites in the multiple cloning site II of pFastBac DUAL donor plasmid (Life Technologies, Inc.) to construct pFastBac DUAL MRPC
for C932-1531. The construct pFastBac DUAL MRP
full resulted from ligation of the two large
BssHII/PvuI fragments of the N and C donor plasmids.
To express TMD0L0 and
To facilitate detection of the expressed CoreN of MRP1, a sequence
encoding a myc tag was inserted by PCR using pBluescript MRPN as a template. For this purpose, two primers:
5'-TCTGAAGAGGATCTGTAAACTAGTTCTAGAGCGGCCGC-3' (forward) and
5'-GATGAGTTTCTGTTCTGCGGTGCTGTTGTGGTGCC-3' (reverse), encoding the myc epitope EQKLISEEDL (underlined) were used. The PCR
products were digested with DpnI to remove template plasmid DNA and then self-ligated in the presence of T4 kinase (34). Following
verification by DNA sequencing, the insertion was removed as an
XhoI/NotI fragment and exchanged with the
corresponding fragment in the
Constructs encoding L0
Baculoviruses encoding the above wild type and mutant MRP1s were
generated using the Bac to Bac expression system (Life Technologies, Inc.) as described previously (30).
Photoaffinity Labeling of MRP1 with
[125I]AzidoAG-A--
Membrane vesicles (100 µg of
protein) were incubated for 20 min at room temperature with 5 µM photoanalog of AG-A labeled with 125I
(1.2 × 106 cpm) in the absence or presence of various
agents and different concentrations of GSH as indicated. Following
continuous irradiation of the samples with a short wavelength
(366 nm) UV lamp for 20 min at 25 °C, the samples were solubilized
in SDS sample buffer as described by Debenham et al. (35)
and subjected to SDS-PAGE. Autoradiograms were developed after 6-24 h
of exposure. Gel slices corresponding to specific bands on the
autoradiogram were cut, and the radioactivity (cpm) was measured with
an Auto Well Gamma System (ARC-380, Aloka Co., Ltd., Tokyo, Japan).
Background radioactivity in equal areas of the gel in each lane was
measured and subtracted from the radioactivity of the band.
Immunoprecipitation of MRP1 and Inhibition of Photoaffinity
Labeling--
Membrane proteins (300 µg) were photolabeled by
[125I]azidoAG-A as described above. Membrane vesicles
were then solubilized in 2 ml of Buffer A (50 mM Tris-HCl
(pH 8.0), 150 mM NH4Cl, 2 mM MgCl2) containing 1% CHAPS and incubated for 30 min at
4 °C. The solubilized membranes were centrifuged at 12,000 × g, and the supernatant was incubated at 4 °C for 2 h
with 4 µg of anti-MRP1 monoclonal antibody, QCRL-3, or 8 µg of
control mouse IgG2a. The mixture was then incubated with 200 µg of
20% Protein G-Sepharose in buffer A for 30 min at 4 °C with
continuous mixing, and the precipitates were washed four times with
Buffer A containing 1% CHAPS. The precipitates were then analyzed by
SDS-PAGE and autoradiography.
Treatment of Vesicles with Trypsin and
Immunoblotting--
Membrane vesicles were treated with
diphenylcarbamyl chloride-treated trypsin (ICN Biomedicals, St.
Laurent, Quebec, Canada) according to Hipfner et al. (7)
with some modifications. Briefly, membrane vesicles (50 µg) were
diluted in buffer (10 mM Tris-HCl (pH 7.5), 250 mM sucrose) to a final concentration of 5 mg/ml and then
treated with 2-50 µg/ml trypsin for 30 min at 37 °C. The reaction
was stopped by the addition of 2 mM
aminophenylmethylsulfonyl fluoride (Wako Chemical, Osaka, Japan) and
0.5 TIU (trypsin inhibitory unit)/ml aprotinin, and the samples were
subjected to SDS-PAGE and immunoblotting as described previously (30).
The N- and C-terminal fragments of trypsinized MRP1 were detected with
specific monoclonal antibodies MRPr1 (epitope amino acids MRPr1
(epitope aa 238-247) (36, 41) and MRPm6 (epitope aa 1511-1520) (36, 41), respectively. Treatment of membrane vesicles with trypsin was
carried out either before or after the photolabeling as indicated.
GSH-dependent Binding of
[125I]AzidoAG-A to a Drug Binding Site on MRP1--
We
recently demonstrated that AG-A reverses MDR in human KB carcinoma
cells overexpressing MRP1. AG-A competitively interacts with the
binding site for LTC4 on MRP1 and inhibited the transport of LTC4 by MRP1 (28).
To examine whether AG-A directly binds to the drug binding site of
MRP1, we synthesized the photoaffinity analog of AG-A, [125I]azidoAG-A (Fig. 1).
[125I]AzidoAG-A photolabeled the 190-kDa cellular
component only in the presence of GSH. The labeled band was not
detected even in the presence of GSH in membrane vesicles from KB/CV
cells that did not express detectable levels of MRP1 (Fig.
2A). The photolabeling increased with increasing concentrations of GSH up to 2 mM
(Fig. 2B).
To determine if the 190-kDa component was MRP1,
photolabeled membrane vesicles from KB/MRP cells were solubilized with
CHAPS and immunoprecipitated with a monoclonal anti-MRP1 antibody,
QCRL-3 (37). The labeled protein was immunoprecipitated by the
monoclonal antibody, but not by nonimmune mouse IgG2a confirming that
the [125I]azidoAG-A-binding protein is MRP1 (Fig.
2C).
We then characterized the effect of GSH on
[125I]azidoAG-A binding to MRP1. Loe et al.
(16) reported that, in addition to GSH, some short
S-alkyl-GSH derivatives stimulated [3H]VCR
uptake by MRP1. We therefore investigated whether GSH derivatives could
also stimulate [125I]azidoAG-A binding to MRP1. As shown
in Fig. 3A,
S-methyl-GSH stimulated photolabeling of MRP1 to an extent
similar to that for GSH. Stimulation of labeling was less efficient for
S-ethyl-GSH, and S-octyl-GSH was ineffective. The
role of the reducing activity of GSH in stimulation of the AG-A-MRP1
binding was tested by determining the ability of the sulfhydryl
reducing agents, dithiothreitol, 2-mercaptoethanol, and thioredoxin,
and a glucuronic acid, to substitute for GSH in binding assay (Fig.
3B). It seems that the reducing activity of GSH is not
essential for the photolabeling.
In order to determine if [125I]azidoAG-A binds to
the biologically relevant site(s) of MRP1, quantitative inhibition
studies were performed with a molar excess of AG-A, LTC4,
or several anti-cancer and reversing agents and conjugated organic
anions. The effect of AG-A, ADM, VCR, and LTC4 on
photolabeling of MRP1 in membrane vesicles from KB/MRP cells is shown
in Fig. 4A. The data for all agents used were quantified by removing slices of the gel corresponding to the bands on the autoradiograms (including those not shown) and
measuring their radioactivity (Fig. 4B). AG-A inhibited the photolabeling in a dose-dependent manner and completely
inhibited it at 200 µM. Inhibition by LTC4
was similar to that of AG-A. Among the anticancer agents, VCR and ADM
inhibited photolabeling most strongly, with half-maximal inhibition at
33.9 and 30.4 µM, respectively, and almost complete
inhibition at 200 µM. VP-16 and cisplatin inhibited the
labeling by only 30% and 7% at 200 µM, respectively.
Among the reversing agents,
4-oxo-8-[p-(4-phenylbutyl-oxy)benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate (ONO-1078) (38) had the highest inhibitory activity, and
completely inhibited the labeling at 200 µM. ONO-1078,
3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethylamino-3-oxopropyl)-thio}-methyl]thio)propanoic acid (MK571) (39), and
2-[4-(diphenylmethyl)-1-piperazinyl]-5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P) (40) inhibited the labeling by 50% at 10.6, 18, and
24.9 µM, respectively. Among the conjugated organic
anions, LTC4 inhibited the photolabeling most strongly,
with half-maximal inhibition at 1.3 µM.
17- GSH Stimulates AG-A Binding to the C-terminal Half of
MRP1--
In order to determine the drug binding site of AG-A on MRP1,
the MRP1 molecule was cleaved into defined fragments by trypsin digestion and the fragments tested for binding to
[125I]azidoAG-A. Membrane vesicles from KB/MRP cells were
digested with trypsin and immunoblotted with antibodies specific for
the N- and C-terminal half of MRP1, i.e. MRPr1 (epitope aa
238-247) and MRPm6 (epitope aa 1511-1520) (36, 41). Fig.
5 shows that both antibodies detect the
intact 190-kDa MRP1 prior to trypsin digestion. In addition, a 120-kDa
band (N1) was detected with MRPr1 and a minor band at 78 kDa was
detected with MRPm6. Following treatment with 10 µg/ml trypsin, the
amounts of the 120-kDa (N1) and 78-kDa (C1) peptides increased. When
the concentration of trypsin was increased to 20 µg/ml, other diffuse
bands (N2 (50-60 kDa) and C2 (40 kDa)) appeared, which were detected
with MRPr1 and MRPm6, respectively. Fig. 5B shows the time
courses of the appearance of N1 and C1 during treatment with trypsin at
10 µg/ml. N1 and C1 increased simultaneously during 5-30 min of
incubation, indicating that both of them came from MRP1. These results
are in accordance with previous studies (7) and suggest that the trypsin-accessible sites of MRP1 are located on the cytoplasmic NBD1 and L0 of MRP1. Thus, N1 and C1 bands
represent the N-terminal half (i.e. TMD0
+TMD1+NBD1), and the C-terminal half
(i.e. TMD2+NBD2) of MRP1,
respectively.
Since considerable amounts of N- and C-terminal halves of MRP1 were
produced by digestion of KB/MRP membrane vesicles with 10 µg/ml
trypsin, it was possible to use these fragments to characterize the
drug binding sites on MRP1. Membrane vesicles were photolabeled with
[125I]azidoAG-A and then trypsinized under the same
conditions used for immunoblotting (Fig.
6A). We found that the
photoaffinity analog of AG-A labeled MRP1 and a 78-kDa fragment (C1).
Together with previous reports (7, 42), this indicates that AG-A
photolabeled the C-terminal half of MRP1. To determine whether the
N-terminal half is photolabeled with increasing concentrations of the
photoanalog, the concentration of the [125I]azidoAG-A was
varied from 1 to 10 µM (Fig. 6B). As shown in Fig. 6B, the labeling of the native form of MRP1 and the C1
fragment were saturable. When the concentration of
[125I]azidoAG-A was greater than 5 µM, a
weakly photolabeled 120-kDa having the same size as the N-terminal
fragment appeared. We think this band is nonspecific and not the
N-terminal half of MRP1 since a large excess of AG-A almost completely
inhibited the labeling of intact MRP1 but not of this 120-kDa band
(Fig. 6C).
A baculovirus expression system was used as an alternative approach to
study the binding site of AG-A on MRP1. Truncated, co-expressed, and
mutated MRP1s were summarized in Fig. 7.
It was reported that, although it was underglycosylated, MRP1 expressed in Sf21 insect cells transported LTC4 with kinetic
parameters similar to those of MRP1 expressed in transfected mammalian
cells, and that a functional transporter could be reconstituted when both halves of MRP1 were co-expressed (43). Gao et al.
reported that reconstitution of a functional transporter in cells
infected with a dual expression vector was extremely efficient compared with co-infection with two recombinant baculoviruses (44). cDNAs encoding N1-932 (N-proximal molecule of MRP1) and/or
C932-1531 (the C-proximal molecule of MRP1) were cloned in
a baculovirus dual expression vector. Spodoptera
frugiperda Sf21 insect cells were infected with a
recombinant baculovirus encoding wild type MRP1, N1-932,
or C932-1531 alone, or with both N1-932 and
C932-1531 from a dual expression vector with a
multiplicity of infection of 5-10. MRPr1 and MRPm6 antibodies were
used to determine the expression levels of the proteins in membrane
vesicles prepared from the cells. As shown in Fig.
8A, both N1-932 and C932-1531 were efficiently expressed as 104- and
67-kDa peptides, respectively. Under the conditions used for the
infection, the levels of the half-molecules were comparable both when
they were expressed alone or co-expressed from a dual vector. The
ATP-dependent LTC4 transport by membrane
vesicles prepared from cells infected with a dual expression vector
encoding both N1-932 and C932-1531 was almost
the same as that with vesicles prepared from cells infected with a wild
type MRP1 virus (data not shown). In membrane vesicles expressing
N1-932 or C932-1531 alone,
C932-1531 was only weakly photolabeled by the photoanalog
of AG-A, and the labeling was not stimulated by GSH. However, when
C932-1531 was co-expressed with N1-932, the
binding of AG-A to C932-1531 was greatly enhanced in the
presence of the indicated concentrations of GSH (Fig. 8, B
and C). These results indicate that GSH stimulates AG-A
binding to C932-1531, and that N1-932 is
required for this binding.
GSH Is Not Required for Photolabeling of the P-gp-like Core Region
of MRP1 with [125I]AzidoAG-A--
GSH is required for
the binding of AG-A to C932-1531. To investigate the site
of interaction of GSH on MRP1, TMD0L0 region of
MRP1 (aa 1-281) (8) was co-expressed with
In order to investigate the GSH-independent binding site of AG-A on
L0 Is the Site of GSH Interaction on MRP1--
Since
GSH is hydrophilic, the GSH binding site on MRP1 may be located in a
hydrophilic domain. To determine if L0 is the site of
GSH-MRP1 interaction, we constructed a plasmid encoding
L0
Glutathione S-transferases (EC 2.5.1.18) (GSTs) are a family
of isoenzymes that conjugate GSH to a variety of organic compounds that
contain an electrophilic center (46). They are dimeric enzymes composed
of two identical subunits, each of which contains one binding site for
GSH (G-site) and a second binding site for hydrophobic substrates
(H-site). Despite the low amino acid sequence homologies among GSTs
belonging to different classes, all GST isoenzymes have a conserved
G-site (47). The G-site of the human placental GST ( There is considerable experimental evidence showing that P-gp
mediates MDR by directly binding and then transporting natural chemotherapeutic agents in an ATP-dependent manner (12,
13). It has been shown that MRP1 is an organic anion transporter, which can also transport non-anionic agents, such as VCR, only in the presence of GSH (16, 50). However, the role of GSH in facilitating drug
transport is unclear. As an organic anion transporter, it was
speculated that MRP1 confers resistance by eliminating drugs conjugated
with GSH or other small molecules (24). However, there is some evidence
against this idea. First, conjugated forms of some
anti-cancer drugs, such as ADM and VCR, were not found in the culture
medium of MRP1-transfected drug-resistant cells exposed to these agents
(15). Second, phase I and II biotransformation reactions that form the
GSH conjugates are known to occur primarily in liver, but it seems
unlikely that these reactions occur at the same rate in all of the
MRP1-overexpressing cells from other organs. Alternative speculations
concerning the role of GSH are that GSH is either required as an
activator, facilitating substrate binding and/or transport, or as a
cotransported substrate (4). In order to clarify these issues, we
synthesized a photoaffinity analog of AG-A that reversed MRP1-mediated
MDR, and demonstrated that GSH is required for the binding of AG-A to
MRP1.
Loe et al. (16) found that, in addition to GSH,
S-methyl-GSH, but not dipeptides such as cysteinylglycine
and MRP1 and P-gp belong to the same ATP-binding cassette transporter
superfamily. However, MRP1 is characterized by an extra ~200 amino
acid residues, TMD0, which is connected to the P-gp-like core region by a linker region L0. The precise functions of
these two MRP1 specific domains are not known. In the present study, when the MRP1-specific TMD0L0 was truncated,
AG-A bound to both halves of the core region, and GSH was not required
for this binding. However, when L0 was introduced into the
P-gp-like core region, GSH-dependent binding was recovered.
Mutations of two amino acids in L0 considerably abrogated
the GSH-dependent binding of AG-A to L0 Concerning the function of TMD0, a previous study suggested
that TMD0 is not required for LTC4 transport by
MRP1 (8). When TMD0 alone was introduced into the core
region of MRP1, AG-A did not bind to the resultant polypeptide, even in
the presence of GSH (data not shown). The photolabeling study suggested
that TMD0 is not required for and inhibits drug binding to
MRP1.
The drug binding site(s) on MRP1 is unknown. Photoaffinity labeling
studies using photoaffinity analogs revealed that P-gp contains at
least two drug-binding regions situated within the transmembrane
segments 5 and 6 in its N-terminal half, and 11and 12 in its C-terminal
half. These sites may form one large drug interaction site, or may
represent two or more distinct drug binding sites (23, 51). Our data
suggest that the labeling site of [125I]azidoAG-A on MRP1
is within C932-1531. However N1-932 is
required for the labeling, since C932-1531 expressed alone
was not efficiently photolabeled with the photoanalog of AG-A. It is
still not clear why AG-A photolabeled It has been demonstrated that murine mrp1 confers MDR when expressed in
human cells (52). However, the mouse protein did not confer resistance
to anthracycline. Recent studies with murine/human hybrid proteins
demonstrated that amino acids 959-1187, which are in the C-terminal
half of MRP1, are critical for anthracycline resistance (53). Our
finding that the AG-A labeling site on MRP1 is in
C932-1531 is consistent with this previous report.
Our findings also suggest that the site of AG-A binding on MRP1 is
similar to or partially overlaps the binding sites of LTC4, E217 KB/MRP cells were 30.4-, 24.5-, and 39-fold more resistant to VCR, ADM,
and VP16, respectively, than KB/CV cells, but were not resistant to
cisplatin (28, 11). The present study showed that VCR and ADM
considerably inhibited the photolabeling of MRP1 by AG-A, while
cisplatin did not inhibit, even at 200 µM. We recently demonstrated that AG-A completely reversed the resistance to VCR, ADM,
and VP16 of KB/MRP cells and found that AG-A, a compound with a
chemical structure different from known MDR reversing agents, reversed
MRP1-mediated MDR by two means. One was by directly interacting with
MRP1 and the other by reducing the cellular concentration of GSH (28).
In the present photolabeling study, the inhibitory activity of VP-16
was considerably lower than that of VCR and ADM, although resistance to
VP-16 in KB/MRP was high (28). AG-A may reverse resistance to VP-16
mainly by lowering the cellular concentration of GSH. Alternatively,
metabolite(s) of VP-16, but not VP-16 itself, may be a substrate for
MRP1, or more than one drug binding site may exist on MRP1, and the
binding site of VP-16 on MRP1 may partially overlap but be different
from that of AG-A.
Previous studies showed that MRP1 is photolabeled by LTC4
(25, 26). However, it is impossible to investigate the role of GSH in
drug binding using LTC4 since LTC4 itself is a
GSH conjugate. Recently, some photoactive quinoline-based drugs (IAAQ,
IACI) have been shown to bind directly to MRP1 in the absence of GSH (17-19). These authors demonstrated that MRP1-overexpressing cells are
less sensitive to these quinoline-based drugs than the parental cells,
and that these drugs are substrates of MRP1 on the basis of the results
of MTT assays and drug accumulation studies. Daoud et al.
(19) reported that IACI photolabeled three small peptides located in
the N- and C-halves of MRP1 in the absence of GSH. We found that AG-A
bound to the C-terminal half of MRP1 only in the presence of GSH, and
that AG-A bound to both halves of To investigate whether AG-A is a substrate for MRP1, we examined the
accumulation of the photoanalog of AG-A in KB/MRP and KB/CV cells in
the dark. The accumulation of the photoanalog in KB/MRP cells was
similar to that in KB/CV cells, suggesting that [125I]azidoAG-A bound to MRP1 but was not transported by
it. The affinity of [125I]azidoAG-A to the drug binding
site of MRP1 may be too high for it to be dissociated.
Borst et al. (54) recently proposed a model for MRP1/2 with
two drug binding sites. He proposed that one site may have a relatively
high affinity for GSH and a low affinity for drug (G-site), and the
other site may have a relatively high affinity for drug and a low
affinity for GSH (D-site). Our findings suggest that the G- and the
D-sites of this model are located in the L0 and C-terminal
half of MRP1, respectively. GSH may bind to L0 and cause a
conformational change (55), which facilitates the specific binding of
AG-A to MRP1. LTC4 and compounds conjugated with
glutathione, glucuronide, or sulfate do not need free GSH to facilitate
their binding and (or) transport by MRP1. Borst et al. (54)
suggested that such compounds have a relatively high affinity for both
the G- and D-sites, and therefore are transported efficiently in the absence of GSH. Both this report and the previous reports indicate that
mutated MRP1 lacking the entire TMD0 but containing
L0 can transport LTC4, whereas a mutant MRP1
lacking both TMD0 and L0 cannot. The role of
L0 in LTC4 transport has been unclear. We now
know that the lack of LTC4 transporting activity in
membrane vesicles overexpressing the P-gp-like core region of MRP1 is
probably due to the loss of the GSH binding site. Elucidation of the
topology and interaction of GSH- and drug-binding sites should
contribute to understanding the molecular basis for the transport of
unmodified drugs by MRP1. The agents that compete with GSH for binding
to L0 may reverse MRP1-mediated drug resistance.
In conclusion, our results indicate that GSH is required
for the binding of an unconjugated agent to MRP1 and suggested that GSH
interacts with L0 of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role
of GSH in transporting substrates by MRP1.
MRP) by a cytoplasmic linker domain zero
(L0). It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates
MDR and the precise roles of TMD0 and L0 are
not known. We synthesized [125I]11-azidophenyl agosterol
A ([125I]azidoAG-A), a photoaffinity analog of the
MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found
that the analog photolabeled the C-proximal molecule of MRP1
(C932-1531) in a manner that was
GSH-dependent. The photolabeling was inhibited by
anticancer agents, reversing agents and leukotriene C4.
Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and
mutated MRP1s, we found that L0 is the site on MRP1 that
interacts with GSH. This study demonstrated that GSH is required for
the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L0 of MRP1. The photoanalog of AG-A will be
useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MRP)
by a cytoplasmic linker domain zero (L0) (7, 8). The
precise roles of TMD0 and L0 are unknown.
Although MRP1 and P-gp both confer multidrug resistance by actively
effluxing drugs from the cells (9, 10), there is compelling evidence
that they function very differently in drug transport (6, 11). P-gp
confers multidrug resistance by directly binding and transporting
unmodified drugs (12, 13). MRP1, however, is an active transporter of
amphiphilic conjugated organic anions, including a number of compounds
conjugated with GSH, glucuronide, and sulfate. To date, leukotriene
C4 (LTC4) is the best substrate for MRP1
(14).
-aminoethylvindesine (12, 22),
N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine (13), and iodoarylazidoprazosin (23), have been successfully used to
photolabel P-gp and determine its drug binding site(s). Although the
resistance phenotypes conferred by expression of MRP1 are similar to
those of P-gp expression, a photoaffinity labeling study using known
photoaffinity analogs for P-gp failed to photolabel MRP1 under the same
conditions used for P-gp (24). Leier et al. (25, 26)
reported that LTC4 photolabeled MRP1; however, the labeling
site of LTC4 on MRP1 has not been determined.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MRP, an adaptor was constructed
using two oligonucleotides
(5'-TCGACGCCGCCATGGATGCATCG-3', 5'-GATCCGATGCATCCATGGCGGCG-3')
encoding a consensus Kozak sequence (bold and underlined), a
NsiI site (underlined), and cohesive ends of SalI
(italic) and BamHI (bold). The adaptor was then ligated to
pFastBac MRP1-His in which the
SalI/BamHI fragment encoding MRP1 amino acids
1-280 was removed. The correct insertion of the adaptor was identified
by the presence of the NsiI site and sequence analysis.
MRP from a dual
vector, pFastBac MRP1-His was digested with BamHI
and SphI. After being blunted by T4 polymerase, the two
sites were then ligated to each other. The fidelity of the blunting was
confirmed by reconstitution of a BamHI site. The fragment
encoding TMD0L0 of MRP1 was removed from the
plasmid as a SalI/KpnI fragment and then cloned
between the XhoI and KpnI sites in multiple
cloning site II of the pFastBac DUAL donor plasmid. Translation of the
inserted fragment terminated at a stop codon in the DUAL vector
resulting in the addition of eight amino acids, PVPGDGGG. The
BssHII/PvuI fragment was then ligated with the
large BssHII/PvuI fragment of the
MRP
expression construct.
MRP expression construct to construct
pFastBac MRP CoreN-myc. In order to co-express the CoreN of
MRP1 with C932-1531, the BssHII/PvuI
vector part of the above plasmid was exchanged with the large
BssHII/PvuI fragment of the pFastBac DUAL
MRPC.
MRP, ML0
MRP, and
L0full were prepared as described above. The PCR primer
used for L0
MRP was
5'-GCCGCCATGGACCCTAATCCCTGCCCAGAG-3' encoding a consensus
Kozak sequence (underlined). The primers used to introduce two
mutations, K267M and W261A, into MRP1 were 5'-GAGTGCGCCATGACTAGGAAG-3' (forward) and
5'-CTTCTTCGCGTTCTTTACCAAAAC-3' (reverse) encoding
mismatched bases (bold). The underlined mismatched base introduces a
silent mutation, which destroys an original BsmI site in
MRP1 and acts as a marker for the mutant cDNA. The primer used for
L0full was a reverse primer:
5'-GGATCCTGGATGGTTTCCGAGAAC-3'.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The structural formulas of AG-A and
[125I]azidoAG-A.
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Fig. 2.
The effect of GSH on
[125I]azidoAG-A binding to MRP1-enriched membrane
vesicles. Membrane vesicles were photolabeled with
[125I]azidoAG-A as indicated, detergent-solubilized, and
analyzed by SDS-PAGE and autoradiography. A, KB/MRP and
KB/CV membrane vesicles were photolabeled with 5 µM
[125I]azidoAG-A in the presence or absence of 2 mM GSH. An arrow indicates the 190-kDa labeled
protein. B, KB/MRP membrane vesicles were photolabeled with
[125I]azidoAG-A in the presence of increasing
concentrations of GSH (0.1-5 mM) (inset). The
data were quantified by removing gel slices corresponding to the
190-kDa bands on the autoradiogram and determining their radioactivity.
The background activity was measured and subtracted from the
radioactivity of the band. C, SDS-PAGE of immunoprecipitates
of [125I]azidoAG-A-labeled, detergent-solubilized, KB/MRP
membrane vesicles. Photoaffinity labeling of KB/MRP membrane vesicles
with the photoanalog of AG-A was carried out as described under
"Experimental Procedures." Photolabeled membrane vesicles (300 µg) were solubilized with a buffer containing CHAPS, and
immunoprecipitated with QCRL-3 (left lane) or
nonimmune mouse IgG2a (right lane). The immune
complex precipitated with protein G-Sepharose was collected and
subjected to 7.5% SDS-PAGE and autoradiography. Molecular size markers
at the left are in kDa. Autoradiograms were developed after
3 days (left lane) and 6 days (right
lane) of exposure, respectively.
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Fig. 3.
The effect of various GSH derivatives,
sulfhydryl reducing agents, and glucuronic acid on
[125I]azidoAG-A binding to MRP-enriched membrane
vesicles. Photolabeling was carried out as described in the legend
to Fig. 2 in the presence of 2 mM GSH or its derivatives
(A), or 2 mM glucuronic acid, dithiothreitol,
2-mercaptoethanol, or thioredoxin (25 mg/ml) (B).
Autoradiograms were developed after 8 h of exposure.
-Estradiol-17 (
-D-glucuronate)
(E217
G) inhibited the photolabeling by 50% at 129 µM and by 40% at 200 µM. These results
indicate that [125I]azidoAG-A bound to the biologically
relevant site on MRP1.
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Fig. 4.
Inhibition of [125I]azidoAG-A
photolabeling of MRP1 in KB/MRP membrane vesicles by various
agents. A, KB/MRP membrane vesicles were photolabeled
with 5 µM [125I]azidoAG-A and 2 mM GSH in the absence or presence of the indicated
concentrations of AG-A, ADM, VCR, and LTC4.
Autoradiograms were developed after 12 h of exposure.
B, inhibition of [125I]azidoAG-A labeling of
MRP1 by anti-cancer agents, MDR-reversing agents, E217 G,
and LTC4. Quantification was achieved as described in the
legend to Fig. 2. Data are expressed as the percentage of photolabeling
of MRP1 in the absence of agents.
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Fig. 5.
Immunoblots of the proteolytic products of
MRP1 generated by trypsin digestion. A, membrane
vesicles (50 µg) from KB/MRP cells were treated at 37 °C for 30 min with the indicated concentrations of trypsin and then subjected to
7.5% SDS-PAGE followed by immunoblotting with MRPr1 and MRPm6.
B, membrane vesicles were treated at 37 °C for the
indicated periods with 10 µg/ml trypsin and then subjected to 7.5%
SDS-PAGE and immunoblotting with MRPr1 and MRPm6. N1,
N2, C1, and C2 are 120-, 50-60-, 78-, and 40-kDa tryptic fragments, respectively.
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Fig. 6.
Photoaffinity labeling of trypsinized MRP1
with [125I]azidoAG-A. Photolabeling of MRP1 with
[125I]azidoAG-A was performed in the presence of 2 mM GSH as described under "Experimental Procedures."
Membrane vesicles from KB/MRP cells (100 µg of protein/lane) were
treated as follows. A, photolabeling of MRP1 was carried out
with 5 µM [125I]azidoAG-A followed by MRP1
cleavage with the indicated concentrations of trypsin. B,
photolabeling of MRP1 was carried out with 1-10 µM
[125I]azidoAG-A followed by treatment for 30 min at
37 °C with 10 µg/ml trypsin. C, vesicles were
pretreated for 30 min at 37 °C with 10 µg/ml trypsin before
photolabeling MRP1 with 7 µM
[125I]azidoAG-A in the presence or absence of 100 µM AG-A. Trypsin reactions were stopped by adding 2 mM phenylmethylsulfonyl fluoride and 0.5 TIU (trypsin
inhibitory unit)/ml aprotinin, and the samples were subjected to 7.5%
SDS-PAGE and autoradiography.
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Fig. 7.
Membrane topology model of MRP1 and the
various constructs expressed in Sf21 insect cells. The
topology of MRP1 is characterized by the presence of an extra
N-terminal domain (TMD0) with five transmembrane segments,
which is connected to the P-gp-like core by a cytoplasmic linker
(L0). Positions of nucleotide binding domains
(NBD1 and NBD2) are also shown. A schematic
diagram depicting Sf21-expressed MRP1 constructs is aligned and
compared with the full-length MRP1.
N1-932+C932-1531,
TMD0L0+ MRP, and CoreN+C932-1531
represent two fragments of MRP1 co-expressed by the dual expression
vector.
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Fig. 8.
GSH stimulates AG-A binding to
C932-1531 in membrane vesicles from Sf21 insect
cells co-expressing half-molecules of MRP1. A, membrane
vesicles were prepared from Sf21 insect cells infected with
recombinant baculovirus encoding full-length MRP1 or
N1-932 and/or C932-1531 half-molecules.
Membrane proteins (20 µg) of each sample were subjected to 7.5%
SDS-PAGE and immunoblotted with MRPr1 and MRPm6. Left,
detection of N1-932 by MRPr1; right, detection
of C932-1531 by MRPm6. B, membrane vesicles
containing full-length MRP1 or N1-932 and/or
C932-1531 of MRP1 (50 µg of protein/lane) were
photolabeled with 5 µM [125I]azidoAG-A in
the absence ( ) or presence of the indicated concentrations of GSH.
Samples were subjected to 7.5% SDS-PAGE, and autoradiograms were
developed after 10 h of exposure. Membrane vesicles from
Sf21 insect cells without infection were used as a control.
C, the data in B were quantified as described in
the legend to Fig. 2. Open triangles,
N1-932 alone; filled circles,
N1-932 in membrane vesicles co-expressing both
half-molecules; open squares,
C932-1531 alone; filled squares,
C932-1531 in membrane vesicles co-expressing both
half-molecules.
MRP (aa 281-1531), the
P-gp-like core region of MRP1, from a dual vector in Sf21 insect
cells. The expression levels of TMD0L0 and
MRP were determined by immunoblotting with the MRP1 N- and
C-terminal specific mAbs, MRPr1 and MRPm6, respectively.
MRP was
detected as a 139-kDa band by MRPm6, but not by MRPr1.
TMD0L0 was detected as a band of about 32 kDa
by MRPr1 but not by MRPm6 (Fig.
9A). ATP-dependent
LTC4 transport was examined using membrane vesicles expressing
MRP alone or both
MRP and
TMD0L0. The LTC4 transport activity
in the membrane vesicles expressing both
MRP and
TMD0L0 was similar to that in membrane vesicles
expressing wild type MRP1, while it was almost abrogated in membrane
vesicles expressing
MRP alone (data not shown). These data are in
accordance with previous reports (8, 45). Membrane vesicles expressing
MRP alone or co-expressing both
MRP and
TMD0L0 were photolabeled with
[125I]azidoAG-A in the absence or presence of the
indicated concentrations of GSH. In membrane vesicles co-expressing
both
MRP and TMD0L0, the
MRP was weakly
photolabeled by AG-A in the absence of GSH, and the labeling was
enhanced 13-fold when the concentration of GSH was increased to 10 mM. In the absence of GSH,
MRP in membrane vesicles expressing
MRP alone was photolabeled by
[125I]azidoAG-A more efficiently than that in membrane
vesicles co-expressing both
MRP and TMD0L0.
However, this labeling was not enhanced by GSH (Fig. 9, B
and C).
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Fig. 9.
Comparison of GSH-dependent
photolabeling of membrane vesicles expressing P-gp-like core region of
MRP1 ( MRP) alone or both
MRP and TMD0L0.
A, immunoblots of membrane proteins prepared from
Sf21 insect cells expressing P-gp-like core region of MRP1
(
MRP) alone or together with TMD0L0.
Membrane proteins (20 µg) of each sample were subjected to 7.5% and
12% SDS-PAGE and immunoblotted with MRPm6 and MRPr1, respectively.
Membrane vesicles (20 µg) expressing full-length MRP1 were applied to
7.5% gel as a control. Left, detection of
TMD0L0 by MRPr1; right, detection of
core MRP by MRPm6. B, membrane vesicles expressing either
MRP alone or both
MRP and TMD0L0 (50 µg
of protein/lane) were incubated with 5 µM
[125I]azidoAG-A in the absence or presence of GSH
(0.2-10 mM), the samples were then separated by 7.5%
SDS-PAGE. Autoradiograms were developed after 12 h of exposure at
room temperature. C, radioactive bands corresponding to the
core region of MRP1 were excised and bound
[125I]azidoAG-A was quantified as described in the legend
to Fig. 2. Filled circles,
MRP;
filled squares,
MRP and
TMD0L0.
MRP, CoreN (amino acids 281-932), which contains the TMD1 and NBD1 domains of MRP1, was co-expressed
with C932-1531 using a dual expression vector. A myc tag
was introduced to the C terminus of core N for the detection of the
expressed protein. As shown in Fig.
10A, CoreN was detected as a
band of 72 kDa by the anti-Myc antibody whereas C932-1531
was detected as a 67-kDa band by MRPm6. Fig. 10B shows the
GSH-independent binding of [125I]azidoAG-A to both halves
of the core region of MRP1. Inhibition studies using ADM and
LTC4 as inhibitors showed that the GSH-independent binding
of AG-A to the core region of MRP1 was not inhibited by either ADM or
LTC4 (Fig. 10C). Based on the finding that
truncation of the MRP-specific TMD0L0 abolished
the specific GSH-dependent binding of AG-A to MRP1, it
seems probable that the GSH interaction site on MRP1 is located on the
truncated region.
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Fig. 10.
Photolabeling of both halves of the
P-gp-like core region of MRP1 without GSH. A, membrane
proteins from Sf21 cells expressing both the N-proximal molecule
(core N) and the C932-1531 of MRP1 from a dual vector
(CoreN+C932-1531) and both halves of MRP1
(N1-932+C932-1531) as a control were
separated by 7.5% SDS-PAGE and transferred to a polyvinylidene
difluoride membrane. Left and right, detection of
CoreN by myc-specific antibody but not MRP1-specific mAb MRPr1;
middle, detection of C932-1531 by MRP1-specific
MRPm6 antibody. B, membrane proteins (50 µg/lane) isolated
from Sf21 insect cells infected with a recombinant virus
encoding both CoreN and C932-1531 were photolabeled with 5 µM [125I]azidoAG-A in the absence or
presence of the indicated concentrations of GSH. Autoradiograms were
developed after 20 h of exposure. C, membrane vesicles
co-expressing both halves of the core region of MRP1were incubated with
5 µM [125I]azidoAG-A in the absence or
presence of the indicated concentrations of ADM or LTC4.
Photolabeling was conducted in the absence of GSH. Autoradiograms were
developed after 20 h of exposure.
MRP (aa 203-1531), a polypeptide lacking the first
transmembrane domain TMD0. L0
MRP expressed
in membrane vesicles reacted with both MRPr1 and MRPm6 antibodies and
the expression levels were similar to that of full-length MRP1.
LTC4 transporting activity was retained in this truncated
MRP1 (data not shown), which is in accordance with a previous report
(8). Photolabeling was performed in the absence or presence of
increasing concentrations of GSH. GSH stimulated the binding of AG-A to
L0
MRP in a dose-dependent manner. Together
with the data showing that TMD0L0 is required
for GSH-dependent binding of AG-A to MRP1, it seems likely
that L0 is the site of GSH interaction on MRP1.
GST), which has
been studied in three dimensions, is formed by an irregular
-helix
(helix 2) which, through Trp38 and Lys44,
provides important electrostatic interaction with the carboxylate group
of the glycine moiety of GSH (48, 49). We examined the L0
region of MRP1 for the presence of conserved residues that might
interact with GSH. There are four tryptophan residues in the
L0 region of MRP1, but only two of them are followed by
lysine. Furthermore, hydropathy analysis of the region indicated that only Trp261 and Lys267 exist in a predicted
-helix. These residues were thus likely candidates for GSH binding.
This was tested by substituting Trp261 and
Lys267 with Ala and Met, respectively, and the subsequent
mutated L0 region tested in binding studies. The mutated
L0 (ML0) region was linked to the N terminus of
MRP to construct ML0
MRP. As shown in Fig.
11A, ML0
MRP
protein, which has a level of expression comparable to that of wild
type L0
MRP, reacted with both MRPr1 and MRPm6
antibodies. Photolabeling was performed using ML0
MRP membrane vesicles in the absence or presence of increasing
concentrations of GSH. Fig. 11 (B and C) shows
that the binding of AG-A to ML0
MRP was increased only
2.7-fold in the presence of 10 mM GSH compared with a
7.5-fold increase in the binding of AG-A to L0
MRP. This observation suggests that the L0 region of MRP1 may indeed
form part of the GSH binding site.
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Fig. 11.
Comparison of GSH-dependent
photolabeling of L0 MRP and
ML0
MRP by
[125I]azidoAG-A. A, membrane proteins (20 µg/lane) from Sf21 cells expressing L0
MRP or
ML0
MRP were separated by 7.5% SDS-PAGE, transferred to
a polyvinylidene difluoride membrane, and immunoblotted with
MRP1-specific mAb MRPr1. B, the L0
MRP or
mutant L0
MRP (ML0
MRP) proteins were
photolabeled as described under "Experimental Procedures." The two
proteins were photolabeled at the same time to compare labeling
efficiency. Autoradiograms were developed after 25 h of exposure
at room temperature. C, the labeled bands were excised,
radioactivities were determined. Filled circles,
L0
MRP; filled squares,
ML0
MRP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glutamylcysteine, could also stimulate VCR transport by MRP1,
suggesting that the stimulation of drug transport requires the complete
GSH tripeptide but is not dependent on a free thiol. The free thiol of
GSH was also not needed for the binding of AG-A to MRP1, since
S-methyl-GSH stimulated AG-A binding to MRP1 with the same
efficiency as GSH and a number of sulfhydryl reducing agents could not
substitute for GSH in binding assays. It has been reported that
S-decyl-GSH inhibited LTC4 uptake with a
Ki of 116 nM, indicating that alkylated
GSH is a competitive inhibitor of LTC4 uptake by MRP1-enriched membrane vesicles (14). We found that
S-octyl-GSH did not stimulate AG-A binding to MRP1. On the
contrary, it inhibited the GSH-dependent AG-A binding to
MRP1 in a dose-dependent manner (data not shown). This long
S-alkyl-GSH derivative may have bound to the GSH binding
site on MRP1 and competitively inhibited the interaction of GSH with
the site. Our findings suggest that GSH may be required for the binding
of VCR to the drug binding site of MRP1.
MRP.
These findings strongly suggest that L0 is the site of GSH
interaction on MRP1.
MRP more efficiently than
intact MRP1 in the absence of GSH, and why this labeling was not
inhibited by ADM, VCR, and LTC4.
MRP may form a tertiary structure different from that of MRP1. As a result, nonphysiological drug binding site(s) may be formed on
MRP.
G, ADM, VCR, VP-16, PAK-104P, MK-571, and ONO-1078.
It has been shown that MRP1 is an organic anion transporter, with
LTC4 being one of the highest affinity MRP1 substrates
identified to date (14). In accordance with this, LTC4 had
the highest inhibitory activity among the agents we used in our
inhibition study.
MRP in the absence of GSH. The
GSH-dependent binding of AG-A to the C-terminal half of
intact MRP1, but not the GSH-independent binding of AG-A to
MRP, was
inhibited by LTC4, ADM, and VCR. Therefore, we consider
that the GSH-independent binding of AG-A to
MRP, the P-gp-like core
region of MRP1, differs from the physiological drug binding site on
MRP1. The reason why the photoactive quinoline-based drugs can
photolabel MRP1 in the absence of GSH is unknown. The photoanalog of
AG-A is different from the photoactive quinoline-based drugs in its
requirement for GSH for binding to the specific drug binding site(s) of
MRP1. Quinoline-based drugs may bind to other sites of MRP1 besides the
drug binding site in the C-terminal half, and that may enable them to
be transported by MRP1.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Piet Borst (Netherlands Cancer Institute) for the MRP1 cDNA, Dr. Susan P. C. Cole (Queen's University) for the QCRL-3 antibody, Dr. Kazumitsu Ueda (Kyoto University) for KB/MRP cells, Dr. Noriyuki Suzuki (AGENE Research Institute) for Sf21 cells and technical advice, Drs. Tatsuya Maeda (University of Tokyo), Kazutake Tsujikawa (Osaka University) and Yutaka Hoshikawa (Tokyo Metropolitan Institute of Medical Science) for technical advice and useful discussion, and Dr. Pauline O'Grady for her critical reading the manuscript. We also thank Hiromi Kakura for excellent secretarial assistance, and Etsuko Sudo, Hui Gao, Kana Kamimura, and Minae Tada for excellent technical assistance.
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FOOTNOTES |
---|
* 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.
§ Supported by a research fellowship from the Japan Society for the Promotion of Science.
To whom correspondence should be addressed. Tel.:
81-99-275-5490; Fax: 81-99-265-9687; E-mail:
akiyamas@m3.kufm.kagoshima-u.ac.jp.
Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M101554200
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ABBREVIATIONS |
---|
The abbreviations used are:
MDR, multidrug
resistance;
P-gp, P-glycoprotein;
MRP1, human multidrug resistance
protein;
TMD, transmembrane domain;
NBD, nucleotide-binding domain;
GSH, glutathione;
VCR, vincristine;
ADM, adriamycin;
VP-16, etoposide;
LTC4, leukotriene C4;
E217G, 17-
-estradiol-17 (
-D-glucuronate);
AG-A, agosterol
A;
MK571, 3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethylamino-3-oxopropyl)-thio}
-methyl]thio)propanoic acid;
ONO-1078, 4-oxo-8-[p-(4-phenylbutyl-oxy)benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran
hemihydrate;
PAK-104P, 2-[4-(diphenylmethyl)-1-piperazinyl]-5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide;
IAAQ, N-{4-[1-hydroxy-2-(dibutylamino)ethyl]
quinolin-8'-yl}-4-azidosalicylamide;
IACI, N-(hydrocinchonidin-8-yl)-4-azido-2-hydroxybenzamide;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel
electrophoresis;
GST, glutathione S-transferase;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
L, linker domain.
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REFERENCES |
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