From the Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada
Received for publication, December 13, 2000, and in revised form, February 19, 2001
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ABSTRACT |
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Multidrug resistance protein 1 (MRP1/ABCC1) belongs to the ATP-binding cassette transporter
superfamily and is capable of conferring resistance to a broad range of
chemotherapeutic agents and transporting structurally diverse
conjugated organic anions. In this study, we found that substitution of
a highly conserved tryptophan at position 1246 with cysteine
(W1246C-MRP1) in the putative last transmembrane segment (TM17)
of MRP1 eliminated 17 The 190-kDa multidrug resistance protein 1 (MRP1)1 is a member of a
branch of the ATP-binding cassette (ABC) superfamily of transport
proteins designated ABCC (1-3). When overexpressed in tumor
cells, MRP1 (gene symbol ABCC1) confers resistance to anticancer drugs and other xenobiotics of remarkably diverse structures and charge. Thus, the resistance spectrum associated with MRP1 expression extends from cationic and neutral natural product drugs (e.g. vincristine, doxorubicin, and VP-16) to the
antimetabolite methotrexate to arsenical and antimonial oxyanions (4).
MRP1 is also a primary active transporter of conjugated organic anions that include GSH-, glucuronide-, and sulfate-conjugated derivatives of
both endo- and xenobiotics, suggesting a role for MRP1 in the disposition and elimination of these compounds (2).
The ability of MRP1 to confer drug resistance and transport conjugated
organic anions is shared by at least two other proteins belonging to
the ABCC subfamily, MRP2 (ABCC2) and MRP3 (ABCC3) (5, 6). These three
proteins share a common five-domain structure that distinguishes them
from most other ABC transporters, which more typically have four
structural domains. Thus, MRP1, -2, and -3 have a third
membrane-spanning domain (MSD) with an extracytosolic N terminus that
precedes a core structure consisting of two tandemly arranged units
containing an MSD and a nucleotide-binding domain (see Fig.
1A) (7, 8). The drug resistance profiles and the specificity
and relative affinities of the three proteins for organic anions are
similar, but not identical (5, 8-10). For example, cisplatin
resistance is associated with elevated expression of MRP2, but not MRP1
or MRP3 (8, 11). Moreover, MRP3 is a relatively poor transporter of
GSH-conjugated organic anions compared with MRP1 and MRP2 (9). Such
differences in substrate specificity are not unexpected given that the
amino acid sequences of MRP2 and MRP3 are only 48 and 58% identical, respectively, to that of MRP1.
Two of the best characterized substrates of MRP1 are the GSH-conjugated
arachidonic acid derivative leukotriene C4
(LTC4) and the glucuronidated estrogen 17 The specific amino acids involved in the recognition, binding, and
transport of MRP1 substrates are largely unknown. In previous studies,
the differing abilities of human MRP1 and its murine ortholog to
confer resistance to anthracycline drugs have allowed us to identify a
nonconserved glutamic acid residue at position 1089 in putative
transmembrane segment 14 (TM14) in the third C-proximal MSD (MSD3) of
human MRP1 that is essential for its ability to confer resistance to
this class of drugs (19). Anthracyclines such as doxorubicin and
daunorubicin exist predominantly as cations at physiological pH, and
consequently, it seems reasonable for a negatively charged
membrane-embedded amino acid to be involved in conferring resistance to
these drugs (20). In this study, we have identified a highly conserved
tryptophan residue in predicted TM17, the last TM segment in MSD3, that
is critical not only for enabling the transport of the conjugated
organic anion E217 Materials--
[6,7-3H]E217 Vector Construction and Site-directed Mutagenesis--
The MRP1
expression vector pcDNA3.1(
Mutations of Trp1246 in MRP1 were generated
using the TransformerTM site-directed mutagenesis kit
(CLONTECH, Palo Alto, CA). The template for
mutagenesis was prepared by cloning a 2-kb XmaI fragment from pcDNA3.1( Construction of MRP1-GFP Fusion Proteins--
The
238-amino acid coding sequence of jellyfish GFP with S65T was cloned
in-frame to the 3'-end of MRP1 to generate a wild-type MRP1-GFP
construct as follows. First, the TGA stop codon of MRP1 was eliminated
using polymerase chain reaction to generate a 0.55-kb fragment encoding
MRP1 amino acids 1353-1531 (nucleotides 4058-4593) with
ClaI and SacII restriction sites engineered at
the 5'- and 3'-ends, respectively, with
pcDNA3.1( Transient and Stable Transfections of MRP1 Expression
Vectors--
For transient transfections, wild-type and mutant
pcDNA3.1( Measurement of Protein Levels in Transfected Cells--
The
levels of wild-type and mutant MRP1 proteins were determined by
immunoblot analysis of membrane protein fractions from transfected
cells essentially as described (22). Proteins were resolved on a 6-7%
polyacrylamide gel and electrotransferred to a nylon membrane. Blots
were blocked with 4% (w/v) skim milk powder for 1 h, followed by
incubation with primary antibody. Wild-type and mutant MRP1 proteins
were detected with the human MRP1-specific murine mAb QCRL-1 (diluted
1:10,000), which recognizes a linear epitope consisting of amino acids
918-924 (23). After washing, blots were incubated with horseradish
peroxidase-conjugated goat anti-mouse antibody (Pierce, Edmonton,
Alberta, Canada), followed by application of Renaissance®
chemiluminescence blotting substrate (PerkinElmer Life Sciences). Relative levels of protein expression were estimated by densitometric analysis using a ChemiImagerTM 4000 (Alpha Innotech, San
Leandro, CA).
Confocal Microscopy--
HEK293T cells were seeded at 3.5 × 105 cells/well in a 6-well plate on coverslips coated
with 0.1% gelatin in 2 ml of Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum and transiently transfected with the
MRP1-GFP cDNA constructs (1 µg of DNA/well) using
FuGENETM-6. Forty-eight hours later, the coverslips were
washed once with PBS and fixed with 4% paraformaldehyde for 10 min at
room temperature. After washing twice with PBS, the cells were
permeabilized by adding 0.2% Triton X-100 in PBS. The coverslips were
then incubated in RNase A (10 µg/ml in 0.1% Triton X-100 and 1%
bovine serum albumin in PBS) for 60 min at room temperature. The
coverslips were washed again, and cell nuclei were stained with 1 ml of
propidium iodide (2 µg/ml in PBS) for 45 min in the dark. Finally,
the coverslips were placed on slides containing 1 drop of Antifade
solution (Molecular Probes, Inc., Eugene, OR), and cells were examined
using a Meridian InSight Plus confocal microscope equipped with an
air-cooled argon laser. Images obtained at 488 nm excitation were
pseudo-colored and overlaid using Maxim DL software.
Flow Cytometry and Cloning by Limiting Dilution of Stably
Transfected HeLa Cells--
To determine the proportion of stably
transfected cells expressing MRP1, populations of HeLa cells selected
in G418 for >3 weeks were analyzed by flow cytometry with the
MRP1-specific mAb QCRL-3, which recognizes a
conformation-dependent epitope within the first
nucleotide-binding domain of MRP1 (22). Briefly, cells were washed
twice with cold PBS and fixed with 1% paraformaldehyde in PBS for 30 min at 4 °C. After washing with blocking solution (1% bovine serum
albumin and 0.1% Triton X-100 in PBS), cells were incubated with mAb
QCRL-3 (diluted 1:100) for 1 h. The cells were washed twice with
blocking solution and incubated for 30 min with fluorescein-conjugated
goat anti-mouse IgG (H + L) F(ab')2 fragment (Pierce)
diluted 1:100 in blocking solution. After two washes with blocking
solution, cells were resuspended in 1% paraformaldehyde in PBS and
analyzed on a Coulter Epic flow cytometer. Several populations
containing the highest level of MRP1 with the largest proportion of
MRP1-positive cells were cloned by limiting dilution and expanded to
obtain stable cell lines with >90% of the cells expressing wild-type
or mutant MRP1.
MRP1-mediated Transport by Inside-out Membrane
Vesicles--
Inside-out membrane vesicles were prepared from
transiently transfected HEK293T cells or stably transfected HeLa cells,
and ATP-dependent transport of 3H-labeled
substrates by the membrane vesicles was measured using a rapid
filtration technique as described previously (13). Briefly, LTC4 transport assays were performed at 23 °C in a
50-µl reaction containing 50 nM LTC4 (40 nCi), 4 mM AMP or ATP, 10 mM MgCl2, 10 mM creatine phosphate, 100 µg/ml creatine kinase, and
2-4 µg of vesicle protein in transport buffer (50 mM
Tris-HCl, 250 mM sucrose, pH 7.4). Uptake was
stopped at selected times by rapid dilution in ice-cold buffer, and
then the reaction was filtered through glass-fiber filters (type A/E)
that had been presoaked in transport buffer. Radioactivity was
quantitated by liquid scintillation counting. All data were corrected
for the amount of [3H]LTC4 that remained
bound to the filter, which was usually <10% of the total
radioactivity. Transport in the presence of AMP was subtracted from
transport in the presence of ATP to determine ATP-dependent
LTC4 uptake. All transport assays were carried out in
triplicate, and results are expressed as means ± S.D.
Uptake of [3H]E217 Chemosensitivity Testing and 3H-Labeled Drug
Accumulation in Stably Transfected HeLa Cell Lines--
The relative
drug resistance of the HeLa cell lines stably transfected with
wild-type and mutant MRP1 cDNAs was determined using a
tetrazolium-based microtiter plate assay (24). Cells were seeded in
96-well plates (1 × 104 cells/well), incubated at
37 °C for 24 h before the addition of drug, and then incubated
for a further 72 h before the addition of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (2 mg/ml
of PBS). After 3 h at 37 °C, isopropyl alcohol and 1 N HCl (24:1) were added to solubilize the formazan
crystals, and the absorbance at 570 nm was determined using an
ELX800 microplate reader (Bio-Tek Instruments, Inc.).
IC50 values were obtained from the dose-response curves
using GraphPAD Prism® software. All assays were carried
out in quadruplicate and reported as means ± S.D. Relative
resistance was calculated as the IC50 of the wild-type or
mutant MRP1-transfected HeLa cell line divided by the IC50
of the HeLa cell line transfected with empty pcDNA3.1(
[3H]Vincristine accumulation in intact cells was measured
as described previously (25). Briefly, HeLa cells (1.25 × 106/ml) were incubated at 37 °C in the presence of 1 µM [3H]vincristine (1 µCi/ml) in RPMI
1640 medium supplemented with 5 mM HEPES (pH 7.0), 10 mM glucose, and 5% fetal bovine serum. Aliquots of
suspended cells were removed at 0 and 60 min, and accumulation of
[3H]vincristine was stopped by rapid dilution into
ice-cold PBS. Cells were washed twice with PBS, and the cell pellets
were solubilized in 1% SDS. Cell-associated radioactivity was
determined by liquid scintillation counting.
Identification of Trp1246 as a Functionally Important
Amino Acid in MRP1--
During the course of a mutational analysis of
proline residues in MRP1, we generated an MRP1 mutant in which proline
at position 196 was replaced with alanine (P196A). When stably
expressed in HeLa cells, the ability of this mutant to transport
organic anions was reduced, and it no longer conferred resistance to
natural product drugs. In contrast, a second independently derived
P196A mutant HeLa cell line displayed a phenotype similar to that of cells expressing wild-type
MRP1.2 Using three sets of
overlapping primers, the integrated MRP1-transfected cDNAs of the
wild-type MRP1 and the first P196A mutant cell lines were amplified by
polymerase chain reaction. The DNA fragments obtained were of the size
expected, and when sequenced, it was discovered that, in addition to
the P196A mutation, the transfected copies of MRP1 cDNA in the
first cell mutant line with an altered phenotype contained a G A W1246C Substitution Causes Complete Loss of
E217 Conservative and Nonconservative Substitutions of MRP1
Trp1246 Cause Loss of E217 Verapamil-stimulated Transport of GSH Remains Intact after
Substitution of Trp1246--
To determine whether the
Trp1246 substitution might affect the transport of other
MRP1 substrates, GSH uptake was measured in membrane vesicles prepared
from HEK293T cells transfected with wild-type and Trp1246
mutant MRP1 cDNAs. We have shown previously that MRP1 mediates low
level ATP-dependent GSH transport that is stimulated by the calcium channel blocker verapamil and certain of its dithiane analogs (17, 27). Consequently, GSH transport by wild-type MRP1 and the
Trp1246 mutant proteins was measured in the presence of 100 µM verapamil. As shown in Fig.
4, all four Trp1246 mutant
MRP1 proteins retained the ability to mediate verapamil-stimulated GSH
uptake at levels proportional to the expressed protein.
Trp1246 Mutant MRP1 Molecules Are Correctly Routed to
the Plasma Membrane--
To ensure that the loss of transport activity
in the Trp1246 MRP1 mutants was not caused by impaired
trafficking of the mutant molecules to the plasma membrane, GFP-tagged
constructs of wild-type MRP1 and mutants W1246C and W1246A were
generated and transiently transfected into HEK293T cells. When viewed
under the confocal microscope, both the wild-type and mutant MRP1
proteins were observed to localize strongly to the plasma membrane,
indicating that in the transiently transfected cells, the mutants were
correctly routed to the cell surface (Fig.
5). Studies with HEK293T and HeLa cells
transfected with wild-type MRP1-GFP cDNA showed that fusion of GFP
to the C terminus of wild-type MRP1 did not affect the LTC4
and E217 Drug Resistance Is Lost and Accumulation Is Restored in Stably
Transfected Trp1246 Mutant MRP1 HeLa Cells--
In
addition to its ability to transport organic anions such as GSH,
E217
The drug resistance phenotype of the W1246C-MRP1-transfected cells was
further characterized by determining the sensitivity of these cells to
the cationic anthracyclines doxorubicin and daunorubicin as well as the
electroneutral epipodophyllotoxin VP-16 (etoposide). As shown in Fig.
7, the IC50 values of the W1246C-MRP1 mutant cells for all three drugs were similar to those of
the vector control-transfected cells. Thus, in addition to eliminating
the ability to bind and transport the conjugated estrogen E217 Resistance to Potassium Antimony Tartrate Is Retained, but
Resistance to Sodium Arsenite Is Reduced, in W1246C-MRP1-expressing
Cells--
We have previously shown that in addition to conferring
resistance to anticancer drugs, both human and murine MRP1 can confer low level resistance to arsenical and antimonial oxyanions (4, 28).
When tested for sensitivity to potassium antimony tartrate, the
IC50 of the W1246C mutant HeLa cells was comparable to that of cells expressing wild-type MRP1 (40 versus 30 µg/ml)
(Fig. 8A). Thus, both cell
lines showed a similar level of resistance (6-8-fold) to this heavy
metal oxyanion. In contrast, W1246C mutant cells displayed reduced
resistance to sodium arsenite (Fig. 8B). Thus, the
IC50 of W1246C mutant HeLa cells for sodium arsenite was 1 µg/ml compared with 3.5 µg/ml for cells expressing wild-type MRP1
and 0.6 µg/ml for vector control-transfected cells.
MRP1 was first cloned and identified as a protein capable of
effluxing cationic and electroneutral chemotherapeutic agents and
subsequently demonstrated to be a transporter of GSH-, glucuronide-, and sulfate-conjugated organic anions (1, 2, 28-31). Earlier studies
from our group and others demonstrating competitive inhibition between
structurally diverse substrates indicated that MRP1 contains mutually
exclusive substrate-binding sites and TM structures that allow the
recognition and passage of cationic, anionic, and electroneutral molecules. Thus, among ABC transporters, MRP1 and its related proteins
have a remarkably broad capacity to transport substrates of striking
physical and chemical diversity. However, the molecular basis for the
breadth of this transport capacity is largely unknown.
Previous studies have shown that portions of the first 280 amino acids
of MRP1 encompassing the N-terminal MSD (MSD1) and the cytoplasmic loop
connecting it to the second MSD (MSD2) are important for its expression
in mammalian cell membranes as well as its ability to transport at
least some of its substrates (32, 33). In the course of a mutational
analysis of this region of MRP1, we derived a mutant in which
Pro196 was replaced with Ala by site-directed mutagenesis
(34). When stably expressed in HeLa cells, the ability of this MRP1
mutant to transport organic anions was diminished, and in particular, E217 When transiently transfected into HEK293T cells, W1246C-MRP1 could be
expressed at levels comparable to those of wild-type MRP1 and also
transported LTC4 with a similar efficiency. Moreover, verapamil-stimulated GSH transport was still intact. However, as
observed for the double P196A/W1246C mutant, transport of
E217 To examine the drug resistance phenotype of the W1246C mutation, stably
transfected HeLa cells were generated. In addition to the selective
loss of E217 Current topological models of MRP1 are based on predictions from
computer-based algorithms for which there are limited supporting biochemical data. Although similar in many respects, some models differ
in their placement of several TM segments in the MSDs (7, 35). However,
with the exception of the PredictProtein model, which predicts only
four TM helices in MSD3, all of the algorithms place
Trp1246 in the last TM segment close to the cytoplasmic
face of the membrane (Fig. 1A). This tryptophan residue is
extremely well conserved among MRP (ABCC) subfamily members, including
MRP2, -3, -4, and -6 and murine MRP1. It is also found in the cystic
fibrosis transmembrane conductance regulator, the sulfonylurea
receptor SUR1, the Saccharomyces cerevisiae cadmium
resistance protein Ycf1, Leishmania tarentolae PGPA, and the Arabidopsis thaliana conjugate
transporter AtMRP3 (Fig. 1B). However, it is not conserved in human
P-glycoprotein (MDR1) or other members of the ABCB subfamily to which
this multidrug resistance protein belongs. The last TM helix in MSD3 of
MRP1 has a highly amphipathic character, with Trp1246 and
other amino acids with hydrogen-bonding side chains densely clustered
on one side of the Our data clearly demonstrate that Trp1246 is critical for
the recognition and transport of E217 It has been proposed (and there is considerable evidence to support the
notion) that substrates for P-glycoprotein are taken up from the inner
leaflet of the plasma membrane (39). Whether or not this is also true
for every MRP1 substrate is not known, but may depend on the physical
characteristics of the substrate, such as its hydrophobicity,
aromaticity, and charge, as well as certain aspects of its chemical
structure. The fact that a hydrogen-bonding aromatic amino acid close
to the cytosolic face of the protein is of such critical importance for
the binding and transport of E217 As mentioned previously, we have recently shown that the ability of
MRP1 to confer resistance to cationic anthracyclines is lost when
Glu1089 is substituted with a glutamine residue (19). This
finding, together with our present data, indicates that recognition and transport of these drugs depend not only on the presence of a membrane-embedded negatively charged amino acid in putative TM14, but
also on the presence of the bulky indole Trp1246 in TM17.
Thus, these data raise the possibility that interactions between TM14
and TM17 are required for MRP1 to confer drug resistance. The
comparable interaction in P-glycoprotein would be between TM9 and TM12.
However, such an interaction has not been reported, providing further
evidence of significant differences between these two drug resistance
proteins with respect to the mechanisms by which they recognize and
transport xenobiotics.
As has been proposed for other multidrug transporters (20), MRP1
substrate molecules may penetrate the hydrophobic core of the protein,
where they form a number of van der Waals and stacking interactions
with the surrounding hydrophobic and aromatic residues. Our data
suggest that for a broad range of MRP1 substrates, aromatic stacking
interactions, hydrogen-bonding interactions, and possibly -estradiol
17-(
-D-glucuronide) (E217
G)
transport by membrane vesicles prepared from transiently transfected
human embryonic kidney cells while leaving the capacity for leukotriene
C4- and verapamil-stimulated glutathione transport intact.
In addition, in contrast to wild-type MRP1, leukotriene C4
transport by the W1246C-MRP1 protein was no longer inhibitable by
E217
G, indicating that the mutant protein had lost the
ability to bind the glucuronide. A similar phenotype was observed when
Trp1246 was replaced with Ala, Phe, and Tyr. Confocal
microscopy of cells expressing Trp1246 mutant MRP1
molecules fused at the C terminus with green fluorescent protein showed
that they were correctly routed to the plasma membrane. In addition to
the loss of E217
G transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to the
Vinca alkaloid vincristine and accumulated levels of
[3H]vincristine comparable to those in vector
control-transfected cells. Cells expressing W1246C-MRP1 were also not
resistant to cationic anthracyclines (doxorubicin, daunorubicin) or the
electroneutral epipodophyllotoxin VP-16. In contrast, resistance to
sodium arsenite was only partially diminished, and resistance to
potassium antimony tartrate remained comparable to that of cells
expressing wild-type MRP1. This suggests that the structural
determinants required for transport of heavy metal oxyanions differ
from those for chemotherapeutic agents. Our results provide the first
example of a tryptophan residue being so critically important for
substrate specificity in a eukaryotic ATP-binding cassette transporter.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-estradiol
17-(
-D-glucuronide) (E217
G) (5, 12). MRP1
has also been shown in vitro to transport certain
unconjugated xenobiotics, but only in the presence of GSH (13-16).
Conversely, transport of GSH itself by MRP1 is markedly stimulated by a
variety of structurally diverse heterocyclic molecules that may or may
not be cotransported with this tripeptide (16, 17). The mechanism by
which MRP1-mediated transport and/or cotransport of its conjugated and
unconjugated substrates occurs is presently unclear. Nevertheless, it
has been proposed that MRP1 contains a bipartite binding site(s) that
can accommodate the structural and physical diversity of its substrates
(3, 13, 18).
G, but also for the ability of MRP1 to
confer resistance to both cationic and electroneutral natural product drugs.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G
(55 Ci/mmol) and [glycine-2-3H]GSH (40-44.8
Ci/mmol) were purchased from PerkinElmer Life Sciences.
[3H]Vincristine (44.8 Ci/mmol) and
[14,15-3H]LTC4 (115.3 Ci/mmol) were from
Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom).
LTC4 was purchased from Calbiochem, and nucleotides, GSH,
verapamil, 2-mercaptoethanol, acivicin, E217
G, and
dithiothreitol were purchased from Sigma. Drugs and heavy metal
oxyanions used in chemosensitivity assays were obtained as described
previously (4).
)-MRP1K was constructed by
cloning the EagI/KpnI fragment from pMRP-fc-AT
engineered with a perfect Kozak sequence into the
NotI/KpnI site of the eukaryotic expression
vector pcDNA3.1(
) (Invitrogen, Carlsbad, CA) (21).
)-MRP1K (containing nucleotides
2532-4518 of the MRP1 sequence encoding amino acids 843-1506) into
pGEM-3Z® (Promega, Madison, WI). Mutagenesis was then
performed according to the manufacturer's instructions with the
following sense mutagenic primers (substituted nucleotides are
underlined): W1246C, 5'-CCACGTACTTGAACTGCCTGGTTCGGATGTC-3'; W1246A, 5'-CCACGTACTTGAACGCGCTGGTTCGGATGTC-3'; W1246F,
5'-CCACGTACTTGAACTTCCTGGTTCGGATGTC-3'; and W1246Y,
5'-CCACGTACTTGAACTATCTGGTTCGGATGTC-3'. After confirming all
mutations by sequencing or diagnostic restriction enzyme digests, a
1.3-kb BsmBI/EcoRI fragment was subcloned back
into pcDNA3.1(
)-MRP1K, and the fragments in the
full-length constructs were sequenced once again.
)-MRP1K as template, primers 5'-GCCCatcgatGGCATCAACATCGCCAAGATC-3' (forward) and
5'-CATAATAATCCccgcggTTTCACCAAGCCGGCGTCTTTG-3' (reverse) (substituted nucleotides are underlined; letters in italics
indicate restriction enzyme recognition sequences), and cloned
Pfu DNA polymerase (Stratagene). Next, a 0.7-kb polymerase chain reaction product encoding GFP with SacII and
AflII restriction sites engineered at the 5'- and 3'-ends,
respectively, was prepared using the pECE-GFP(S65T) plasmid as template
(gift from Dr. P. Greer, Queen's University) and primers
5'-CATCCccgcggATGAGTAAAGGAGAAGAAC-3' (forward) and
5'-CGGCGGcttaagGTTATTTGTATAGTTCATCCATG-3' (reverse). Finally, the two fragments were digested with
ClaI/SacII and SacII/AflII, respectively, and ligated together with T4 DNA ligase, and then the
single 1.26-kb fragment was cloned into the
ClaI/AflIII sites of
pcDNA3.1(
)-MRP1K to generate a vector designated
pcDNA3.1(
)-MRP1K-GFP. All cloning boundaries of the
final product were confirmed by sequencing. Mutant MRP1-GFP fusion
proteins were generated by replacing the 1.3-kb
BsmBI/EcoRI fragment in the
pcDNA3.1(
)-MRP1K-GFP construct with the comparable
fragment containing either the W1246C or W1246A mutation generated
above and designated pcDNA-3.1(
)-W1246C-MRP1-GFP and
pcDNA3.1(
)-W1246A-MRP1-GFP, respectively.
)-MRP1 expression vectors were transfected into
SV40-transformed human embryonic kidney cells (HEK293T). Briefly,
~3.9 × 106 cells were seeded in 175-cm2
flasks, and 24 h later, DNA (16 µg) was added using
FuGENETM-6 (Roche Molecular Biochemicals, Laval, Quebec,
Canada) according to the manufacturer's instructions. After 48-60 h,
the HEK293T cells were harvested, and inside-out membrane vesicles were
prepared as described previously (13). Empty vector and vector
containing the wild-type cDNAs were included as controls in all
experiments. Levels of wild-type and mutant MRP1 proteins were
determined by immunoblotting as described below. For stable
transfections, ~1.5 × 105 HeLa cells were seeded in
each well of a 6-well plate, and 24 h later, DNA (1 µg) was
added with FuGENETM-6. After 48 h, the cells were
subcultured 1:12, and the medium was replaced with fresh medium
containing G418 (1000 µg/ml; Geneticin, Life Technologies, Inc.).
After ~14 days, cell colonies were removed individually using cloning
cylinders and subcultured in 600 µg/ml G418. Levels of MRP1 protein
in G418-resistant cell populations were then determined by
immunodot blotting, and the proportion of cells expressing MRP1 was
determined by flow cytometry, followed by cloning by limiting dilution
as described below.
G was measured in a
similar fashion, except that membrane vesicles (2-4 µg of protein)
were incubated at 37 °C in a total reaction volume of 50 µl
containing E217
G (400 nM, 40 nCi) and the
components as described for LTC4 transport. [3H]GSH uptake was also measured by rapid filtration with
membrane vesicles (20 µg of protein) incubated at 37 °C in a
60-µl reaction volume with 100 µM [3H]GSH
(300 nCi/reaction) (17). To minimize GSH catabolism by
-glutamyltranspeptidase during transport, membranes were
preincubated in 0.5 mM acivicin for 10 min at 37 °C
prior to measuring [3H]GSH uptake in the presence of
verapamil (100 µM) (16).
) vector alone.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C nucleotide mutation, resulting in substitution of cysteine for
tryptophan at position 1246. This finding, together with the highly
conserved nature of Trp1246 among ABC transporters
belonging to the ABCC subfamily (Fig. 1B), prompted us to initiate a
more extensive analysis of the structural and functional importance of
this amino acid.
View larger version (28K):
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Fig. 1.
A, schematic diagram of a
predicted secondary structure of MRP1. The position of
Trp1246 in MSD3 of MRP1 is highlighted. The amino acids in
the transmembrane helices are those predicted by the MEMSAT algorithm.
B, alignment of putative transmembrane segment TM17 of MRP1
and related ABCC proteins showing conservation of Trp1246
in this subfamily of the ABC superfamily of transporter proteins. The
numbers to the right indicate the number of amino acids in
the sequences that are identical to the 23 amino acids (positions
1228-1250) of TM17 of MRP1. C, helical wheel projection of
the amino acid sequence of putative TM17 of MRP1. Shaded
circles indicate amino acids that can participate in
hydrogen-bonding interactions. NBD, nucleotide-binding
domain; Hum, human; mus, Mus musculus;
Ysc, yeast S. cerevisiae; Lt, L. tarentolae; At, A. thaliana;
CFTR, cystic fibrosis transmembrane conductance
regulator.
G Transport Activity in Membrane Vesicles from
Transiently Transfected HEK293T Cells--
As a first step to
confirming the importance of Trp1246, this amino acid was
substituted with cysteine in pcDNA3.1(
)-MRP1K by site-directed mutagenesis, and the resulting construct was then transiently transfected into HEK293T cells. Control transfections with
the wild-type construct and the empty pcDNA3.1(
) vector were
carried out at the same time. Membrane vesicles were prepared, and
protein expression levels were determined by immunoblotting. For the
experiments shown in Fig. 2, the levels
of W1246C-MRP1 in the transient transfectants were ~70% those of
wild-type MRP1 (Fig. 2A). A time course of LTC4
uptake was performed, and the levels of uptake by the W1246C-MRP1
mutant relative to wild-type MRP1 were observed to be essentially
proportional to their relative levels of expression (Fig.
2B). Subsequent kinetic analyses of LTC4
transport by the W1246C-MRP1 mutant indicated that its affinity for
this substrate was reduced compared with wild-type MRP1
(Km = 189 nM versus 79 nM), but the Vmax values for the two
proteins were similar (40.6 versus 35.1 pmol/mg/min).3 These results
indicate that substitution of Trp1246 with Cys reduces the
affinity of MRP1 for this substrate, but that the transport capacities
of the mutant and wild-type proteins are comparable. In contrast,
uptake of E217
G by the W1246C-MRP1 mutant was
dramatically reduced and was comparable to that of the
vector-transfected controls (Fig. 2C). We have previously shown that E217
G is a competitive inhibitor of
MRP1-mediated LTC4 transport, with Ki ~ 25 µM (26). Thus, the ability of E217
G
to inhibit LTC4 transport by the W1246C-MRP1 mutant was
examined, and the results are shown in Fig. 2D.
E217
G (25 µM) inhibited LTC4
transport by wild-type MRP1 by 54%, as expected. In contrast,
E217
G had no effect on LTC4 transport by
W1246C-MRP1, indicating that the loss of E217
G transport
by the mutant protein is associated with a loss of binding of this
substrate.
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Fig. 2.
Transport activity of wild-type MRP1 and
mutant W1246C-MRP1 in transiently transfected HEK293T cells.
A, relative levels of wild-type (WT-MRP1) and
mutant MRP1 proteins in the membrane vesicles were determined by
immunoblotting with the MRP1-specific murine mAb QCRL-1 as described
under "Experimental Procedures." The numbers below the
blot refer to the relative levels of MRP1 proteins. B, shown
is the time course of LTC4 uptake in membrane vesicles
prepared from HEK293T cells transiently transfected with wild-type MRP1
( ), mutant W1246C-MRP1 (
), and empty control (
)
cDNA expression vectors. Membrane vesicles were incubated at
23 °C with 50 nM [3H]LTC4 in
transport buffer for the times indicated. Results shown are means ± S.D. of triplicate determinations in a single experiment. Similar
results were found in three additional independent experiments.
C, shown is the time course of E217
G uptake
in membrane vesicles prepared from HEK293T cells transiently
transfected with wild-type MRP1 (
), mutant W1246C-MRP1 (
), and
control empty (
) cDNA expression vectors. Membrane vesicles were
incubated at 37 °C with 400 nM
[3H]E217
G in transport buffer for the
times indicated. Results shown are means ± S.D. of triplicate
determinations in a single experiment. Similar results were found in
three additional independent experiments. D, membrane
vesicles prepared from wild-type MRP1- and W1246C-MRP1-transfected
cells (A) were incubated for 2 min with
[3H]LTC4 at 23 °C in the absence
(white bars) and presence (black bars) of 25 µM E217
G. The results shown are means ± S.D. of triplicate determinations in a single experiment. Similar
results were obtained in a second experiment.
G Transport
Activity--
To determine whether the loss of E217
G
transport activity was related specifically to a Cys substitution at
position 1246, several different amino acids were introduced in place
of Trp1246. These included substitution with a nonpolar
non-aromatic amino acid (Ala; W1246A-MRP1) as well as conservative
substitutions with polar (Tyr; W1246Y-MRP1) and nonpolar (Phe;
W1246F-MRP1) aromatic amino acids. Membrane vesicles were prepared from
HEK293T cells transiently transfected with these constructs, and the
ATP-dependent transport of LTC4 and
E217
G was determined (Fig.
3). As shown in Fig. 3A, the
levels of expression of the mutant proteins were comparable or slightly
higher than those of wild-type MRP1. The LTC4 transport
levels of the W1246A-MRP1 mutant (Fig. 3B) and the
W1246Y-MRP1 and W1246F-MRP1 mutants (Fig. 3C) were similar to those of wild-type MRP1 and the W1246C-MRP1 mutant. In contrast, like the W1246C mutant, the W1246A mutant did not transport
E217
G (Fig. 3D). E217
G
transport by the W1246Y and W1246F mutants was also extremely low
(~10% of wild-type MRP1) (Fig. 3E).
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Fig. 3.
Expression and transport activity of
wild-type and Trp1246 mutant MRP1 cDNAs in transfected
HEK293T cells. A, immunoblots of membrane vesicles
prepared from HEK293T cells transiently transfected with
pcDNA3.1( )-MRP1K (wild-type MRP1
(WT-MRP1)), pcDNA3.1(
)-W1236A-MRP1,
pcDNA3.1(
)-W1246C-MRP1, pcDNA3.1(
)-W1246F-MRP1,
pcDNA3.1(
)-W1246Y-MRP1, and pcDNA3.1(
) alone as a control.
MRP1 proteins were detected with the MRP1-specific mAb QCRL-1 as
described under "Experimental Procedures." B and
C, time course of [3H]LTC4 uptake
by inside-out membrane vesicles prepared from HEK293T cells expressing
MRP1 mutants W1246A (
) and W1246C (
) (B) and
W1246F (
) and W1246Y (
) (C). Membrane vesicles from
HEK293T cells transfected with wild-type MRP1 (
) and vector control
(
) were included in each assay. Membrane vesicles were incubated at
23 °C with 50 nM [3H]LTC4 in
transport buffer for the times indicated. Results shown are means ± S.D. of triplicate determinations in a typical experiment.
D and E, time course of
[3H]E217
G uptake by inside-out membrane
vesicles prepared from MRP1 mutants W1246A (
) and W1246C (
)
(D) and W1246F (
) and W1246Y (
) (E).
Membrane vesicles from cells transfected with wild-type MRP1 (
) and
vector control (
) were included in each assay. Membrane vesicles
were incubated at 37 °C with 400 nM
[3H]E217
G in transport buffer for the
times indicated. Results shown are means ± S.D. of triplicate
determinations in a typical experiment.
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Fig. 4.
Verapamil-stimulated
ATP-dependent [3H]GSH uptake in transfected
cells expressing Trp1246 MRP1 mutants. A,
shown are immunoblots of membrane vesicles prepared from transiently
transfected HEK293T cells as described in the legend to Fig.
3A. B, membrane vesicles were preincubated with
acivicin and then incubated with 300 nM
[3H]GSH in the presence of 100 µM verapamil
in transport buffer for 30 min at 37 °C as described under
"Experimental Procedures." Results shown are means ± S.D. of
triplicate determinants in a single experiment. Similar results were
obtained in two additional experiments. WT-MRP1, wild-type
MRP1.
G transport activities or the drug
resistance-conferring properties of the
protein.4
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Fig. 5.
Confocal laser scanning fluorescence
micrographs of HEK293T cells expressing GFP-tagged wild-type and mutant
MRP1 cDNA constructs. HEK293T cells were transfected with
pcDNA3.1( )-MRP1K-GFP (A, D, and
G), pcDNA3.1(
)-W1246C-MRP1-GFP (B,
E, and H), and pcDNA3.1(
)-W1246A-MRP1-GFP
(C, F, and I); and 48 h later,
cells were processed for confocal fluorescence microscopy as described
under "Experimental Procedures." A-C, shown are merged
images of GFP and nuclear propidium iodide signals shown in
D-F and G-I, respectively. D-F, GFP
signals were collected with a 530/30-nm band-pass filter.
G-I, nuclear propidium iodide signals were collected with a
620/40-nm band-pass filter. Scale bars = 20 µm.
G, and LTC4, MRP1 confers resistance to
natural product anticancer drugs by reducing drug accumulation in cells
in which it is overexpressed. To examine whether Trp1246
plays a role in conferring drug resistance, stably transfected cell
lines were established by transfection of HeLa cells with pcDNA3.1(
)-MRP1K and pcDNA3.1(
)-W1246C-MRP1.
After G418 selection and cloning by limiting dilution, stable cell
lines were obtained, and uniformity of protein expression levels was
established by flow cytometric and immunoblot analyses. The transfected
HeLa cell lines were then tested for vincristine resistance using a tetrazolium salt-based chemosensitivity assay. As expected, HeLa cells
expressing wild-type MRP1 displayed ~8-fold resistance to this drug
(Fig. 6A). In contrast, the
IC50 of the W1246C-MRP1 mutant for vincristine was similar
to that of the vector control-transfected cell line. When accumulation
of [3H]vincristine was measured, steady-state
concentrations of the drug in the W1246C-MRP1-transfected cells were
comparable to those in vector control-transfected cells (33.83 ± 1.94 versus 34.25 ± 2.24 pmol/106 cells/h)
(Fig. 6B). However, in wild-type MRP1-transfected cells, vincristine accumulation was reduced to 19.44 ± 1.11 pmol/106 cells/h (~57% of vector control-transfected
cells), consistent with results obtained in previous studies (4).
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Fig. 6.
Vincristine resistance and
accumulation in stably transfected HeLa cells. A,
vincristine sensitivity of HeLa cell lines stably transfected with the
pcDNA3.1( ) vector (
),
pcDNA3.1(
)-MRP1K (
), and
pcDNA3.1(
)-W1246C-MRP1 (
) was determined using a tetrazolium
salt-based chemosensitivity assay as described under "Experimental
Procedures." Each point represents the mean ± S.D.
of quadruplicate determinations in a single experiment. Similar results
were obtained in two additional experiments. B,
[3H]vincristine (VCR) accumulation in HeLa
cell lines stably transfected with the pcDNA3.1(
) vector
(white bar), pcDNA3.1(
)-MRP1K (wild-type
MRP1 (WT-MRP1); black bar), and
pcDNA3.1(
)-W1246C-MRP1 (gray bar). Cells
(1 × 106) were incubated with
[3H]vincristine for 1 h at 37 °C, and
accumulation was stopped by the addition of ice-cold PBS.
Cell-associated radioactivity was quantitated by liquid scintillation
counting. Each bar represents the mean ± S.D. of
triplicate determinations in a single experiment. Similar results were
obtained in one additional experiment.
G, the W1246C substitution in MRP1 results in loss
of resistance to all classes of natural product drugs tested.
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Fig. 7.
Resistance of MRP1-transfected cells to
chemotherapeutic drugs. HeLa cell lines stably transfected with
the pcDNA3.1( ) vector (
),
pcDNA3.1(
)-MRP1K (
), and
pcDNA3.1(
)-W1246C-MRP1 (
) were exposed to doxorubicin
(A), daunorubicin (B), and VP-16 (C)
for 72 h at 37 °C, and then cell viability was measured as
described under "Experimental Procedures." The results shown are
those of a typical experiment in which each data point
represents the mean ± S.D. of quadruplicate determinations in a
single experiment. Similar results were obtained in a second
experiment.
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Fig. 8.
Resistance of MRP1-transfected HeLa cells to
heavy metal oxyanions. HeLa cell lines stably transfected with the
pcDNA- 3.1( ) vector (
),
pcDNA3.1(
)-MRP1K (
), and
pcDNA3.1(
)- W1246C-MRP1 (
) were exposed to potassium
antimony tartrate (A) or sodium arsenite (B) for
72 h at 37 °C, and then cell viability was measured as
described under "Experimental Procedures." The results shown are
those of a typical experiment in which each data point
represents the mean ± S.D. of quadruplicate determinations in a
single experiment. Similar results were obtained in two additional
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G transport was not detectable. Moreover, this
mutant protein failed to confer drug resistance. In contrast, a second
independently derived transfected cell line also expressing an MRP1
protein with the same P196A mutation displayed a phenotype similar to that of cells expressing wild-type MRP1.2 It was
subsequently discovered that the transfected MRP1 in the first P196A
mutant cell line had acquired a second non-engineered mutation causing
the substitution of Cys for Trp at position 1246. Consequently, we
generated a single W1246C mutant of MRP1 so that its phenotype could be
compared with that of the double P196A/W1246C mutant.
G by W1246C-MRP1 membrane vesicles was not
detectably different from that obtained with vesicles from vector
control-transfected cells, and although LTC4 transport
appeared unchanged in the W1246C mutants, it could no longer be
inhibited by E217
G. Taken together, these results
allowed us to conclude that the altered phenotype of the
P196A/W1246C-transfected cells was attributable to the single
tryptophan substitution at position 1246 in MSD3 and did not require
the additional proline substitution at position 196 in the cytoplasmic
loop between MSD1 and MSD2. Additional mutants in which
Trp1246 was substituted with Ala, Phe, and Tyr displayed
transport characteristics similar to those of W1246C-MRP1, indicating
that replacement of the tryptophan residue rather than the introduced
cysteine was responsible for the phenotype.
G transport, we found that
W1246C-MRP1-expressing cells were no longer resistant to natural
product chemotherapeutic agents, including the electroneutral VP-16 and
the cationic vincristine and anthracyclines. Consistent with this loss
of drug resistance, vincristine accumulation in intact cells expressing
W1246C-MRP1 was comparable to that in vector control-transfected cells,
which was ~2-fold higher than vincristine accumulation in HeLa cells expressing wild-type MRP1. On the other hand, the
W1246C-MRP1-expressing cells were still resistant to antimony tartrate
and partially resistant to sodium arsenite. Taken together, these
observations suggest that the structural determinants in MRP1 necessary
for recognition and transport of these heavy metal oxyanions differ from those for the natural product chemotherapeutic agents.
-helix (Fig. 1C) and predominantly in
the region predicted to be in the inner leaflet of the plasma membrane
(Fig. 1A). The last predicted TM helix of other ABCC subfamily members in which Trp1246 is conserved such as
MRP2 and MRP3 has a similar amphipathic character, lending support to
the idea that the extensive hydrogen-bonding capacity of putative TM17
may play an important role in the substrate-binding and transport
properties of these proteins. Interestingly, the topologically
comparable TM segment in P-glycoprotein (TM12) has also been
demonstrated to contain key determinants of substrate specificity, but
TM12 of this ABC transporter is significantly less amphipathic than
TM17 of MRP1, -2, and -3 (36-38). This difference may be relevant to
the apparent differences in the substrate specificity and transport
mechanisms of MRP1 and P-glycoprotein (2, 3). MRP1 Trp1246
may function to either maintain or form part of a substrate-binding pocket, and/or it may interact directly with the natural product drugs
and E217
G. The facts that the Trp1246
mutants were expressed at comparable levels and correctly routed to the
plasma membrane in the transiently transfected cells and that their
LTC4- and verapamil-stimulated GSH transport activities remained intact indicate that the mutations did not perturb the global
structural integrity of the protein. The inability of the variously
substituted Trp1246 MRP1 mutants to transport
E217
G suggests that tryptophan possesses specific
physical and/or chemical properties that are essential for recognition
and transport of some MRP1 substrates. Thus, the absence of
E217
G transport activity and drug resistance in the W1246C-MRP1 mutant can be explained by the fact that although cysteine
can participate in hydrogen bonding, it is considerably smaller than
tryptophan and lacks aromaticity. However, the introduction of a
phenylalanine or tyrosine residue at position 1246 also resulted in the
loss of E217
G transport, indicating that aromatic
interactions and general hydrogen-bonding capability are not sufficient
to maintain the architecture of the binding site in MRP1 for this substrate and that specific tertiary interactions involving the indole
residue are required. To our knowledge, our data represent the first
example of a tryptophan residue being critically important for
substrate specificity in a eukaryotic ABC transporter. It will be of
interest to determine whether the analogous tryptophan residue in other
members of the ABCC subfamily is also important for their transport functions.
G by MRP1. However,
although the human and murine orthologs of MRP1 differ markedly in
their ability to transport this conjugated estrogen, this amino acid is
conserved in the two proteins, suggesting that other, nonconserved
residues are important as well (28). Thus, it seems probable that
several conserved and nonconserved determinants within the MSDs and
possibly cytosolic regions of MRP1 come together to form a multipartite binding pocket for this organic anion and, furthermore, that
substitution of just one amino acid can be sufficient to abrogate
E217
G recognition and transport activity as well as the
ability to confer drug resistance. Our recent finding that wild-type
MRP1, but not W1246A-MRP1, transports the O-glucuronide of
the tobacco-specific carcinogen
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol indicates that
Trp1246 is important for the transport of other glucuronide
conjugates as well.5
G suggests that this
organic anion may be taken up by MRP1 from the cytoplasm. Why
LTC4 transport is not affected by the Trp1246
substitutions is unclear. It is possible that steric complementarity is
more important for interaction of MRP1 with E217
G than
with LTC4. In support of this idea are our earlier findings
that a change in the site of glucuronidation on the D-ring of
16
,17
-estriol from the 17
-position to the 16
-position
increased the Ki for E217
G transport
~30-fold (26). The LTC4 molecule is significantly less
rigid and more lipophilic than E217
G. These physical
properties of LTC4 may make its recognition and binding
less affected by the changes in the architecture of a multipartite
substrate-binding pocket caused by replacement of the
Trp1246 residue as well as possibly favoring its uptake
from the membrane leaflet.
-bonding
interactions with Trp1246 (and possibly other amino acids
in TM17) are also important, as are electrostatic interactions with
negatively charged amino acids such as Glu1089 in the case
of cationic drugs (19). Whether or not anionic substrates have
electrostatic interactions with positively charged residues in MRP1 is
not yet known. However, the predicted MSDs of MRP1 and related proteins
contain a significant number of membrane-embedded arginine and lysine
residues. Indeed, it has recently been reported that mutants of the
related protein MRP2 in which several basic residues in MSD2 and MSD3
have been replaced by other amino acids show decreased transport
activity (40, 41). Thus, additional molecular and pharmacological
studies are anticipated to continue to provide important information on
the structural features that determine how MRP1 binds and transports
both its charged and electroneutral substrates.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Elaine Leslie, Kevin Weigl, and Shelagh Mirski for helpful advice and discussions; Kathy Sparks, Jennifer Bryant, Hisaki Nakamura, and Taro Nanko for technical assistance; and Derek Schulze for advice on confocal microscopy. We thank Maureen Rogers for expert word processing and assistance with the preparation of the figures.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant MT-10519 from the Medical Research Council of Canada (Canadian Institutes for Health Research).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.
Recipient of a fellowship from the International Agency for Cancer Research.
§ Senior Scientist of Cancer Care Ontario. To whom correspondence should be addressed: Cancer Research Laboratories, Botterell Hall, Rm. 328, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-6507; Fax: 613-533-6830; E-mail: coles@post.queensu.ca.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M011246200
2 K. Ito, R. G. Deeley, and S. P. C. Cole, unpublished observations.
3 K. Ito, R. G. Deeley, and S. P. C. Cole, manuscript in preparation.
4 W. Qiu, A. Haimeur, R. G. Deeley, and S. P. C. Cole, unpublished observations.
5 E. M. Leslie, K. Ito, P. Upadhyaya, S. S. Hecht, R. G. Deeley, and S. P. C. Cole, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MRP, multidrug
resistance protein;
ABC, ATP-binding cassette;
MSD, membrane-spanning
domain;
LTC4, leukotriene C4;
E217G, 17
-estradiol
17-(
-D-glucuronide);
TM, transmembrane;
kb, kilobase;
GFP, green fluorescent protein;
mAb, monoclonal antibody;
PBS, phosphate-buffered saline.
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REFERENCES |
---|
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