From the Departments of Pathology and ** Pharmacology
and Toxicology and the § Cancer Research Laboratories,
Queen's University, Kingston, Ontario K7L 3N6, Canada
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ABSTRACT |
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Multidrug resistance in tumor cells
is often accompanied by overexpression of multidrug resistance protein
(MRP), a 190-kDa transmembrane protein that belongs to the ATP-binding
cassette superfamily of transport proteins. MRP mediates
ATP-dependent transport of a variety of conjugated organic
anions and can also transport several unmodified xenobiotics in a
glutathione-dependent manner. To facilitate
structure-function studies of MRP, we have generated a panel of
MRP-specific monoclonal antibodies (mAbs). Four of these mAbs, QCRL-2,
-3, -4, and -6, bind intracellular conformation-dependent
epitopes, and we have shown that they can inhibit the transport of
several MRP substrates. Binding competition and immunoprecipitation
assays indicated that mAbs QCRL-4 and -6 probably recognize the same
detergent-sensitive epitope in MRP, whereas mAbs QCRL-2, -3, and -4 each bind distinct, non-overlapping epitopes. Fab fragments inhibit
transport as effectively as the intact mAbs, suggesting that inhibition
results from direct interactions of the mAbs with MRP. Immunodot blot
and immunoprecipitation analyses revealed that the minimal regions of
MRP sufficient for full reactivity of mAbs QCRL-2 and -3 are amino
acids 617-858 and 617-932, respectively, which encompass the
NH2-proximal nucleotide-binding domain (NBD). In
contrast, the epitope bound by mAb QCRL-4 localized to amino acids
1294-1531, a region that contains the COOH-proximal NBD. However, none
of the mAbs inhibited photolabeling of intact MRP with
8-azido-[ The development of resistance to multiple natural product and
semi-synthetic drugs commonly used in cancer chemotherapy, termed multidrug resistance, is a serious obstacle to successful treatment. This phenomenon has been extensively studied in vitro using
tumor cell lines selected for resistance by chronic or intermittent exposure to drugs. The multidrug resistance observed in these cell
lines is most commonly accompanied by overexpression of one or both of
the integral membrane transporters,
MRP1 and P-glycoprotein
(1-3). The 190-kDa MRP and 170-kDa P-glycoprotein are members of the
ATP-binding cassette superfamily of transport proteins, which in
eukaryotes are typically composed of one or two highly conserved
nucleotide-binding domains (NBDs) and one to three membrane-spanning
domains (MSDs) (4). The NBDs of ATP-binding cassette transporters are
characterized by the presence of three short sequences, the so-called
Walker A and B motifs and the transport family signature motif (3-6).
Outside of their two NBDs, however, MRP and P-glycoprotein show
relatively little sequence homology, and it is now clear that there are
significant structural and functional differences between these two
proteins (7-11).
Transfection studies have confirmed that overexpression of either MRP
or P-glycoprotein is sufficient to confer resistance to a similar, but
not identical, spectrum of drugs (1, 12-15). A substantial body of
experimental evidence indicates that P-glycoprotein confers multidrug
resistance by binding and transporting unmodified chemotherapeutic
drugs and other compounds out of cells (1, 2). In contrast, direct
binding of drugs to which it confers resistance has not been
demonstrated for MRP (3, 16-19). However, MRP has been shown to be a
primary active transporter of amphiphilic conjugated organic anions.
Established substrates of MRP include a number of GSH-, glucuronide-,
and sulfate-conjugated compounds, with the cysteinyl leukotriene
LTC4 and the GSH conjugate of the potent carcinogen
aflatoxin B1 being the highest affinity substrates identified to date (17, 18, 20, 21). In addition to conjugated anions,
MRP has been shown to mediate ATP-dependent transport of
some unmodified compounds, such as aflatoxin B1 and the
anticancer drugs vincristine and daunorubicin, but only in a
GSH-stimulatable manner (16, 19, 21-23). Recently, we have shown that
the GSH-stimulated transport of unmodified vincristine by MRP-enriched
membrane vesicles involves cotransport of GSH (19). It may be that MRP
confers resistance to at least some classes of xenobiotics in intact
cells by such a cotransport mechanism.
We previously reported the isolation and initial characterization of a
panel of five MRP-specific mAbs (QCRL-1, -2, -3, -4, and -6) (24).
These mAbs were raised against crude membranes prepared from
multidrug-resistant H69AR small cell lung cancer cells, which express
very high levels of MRP. All five mAbs reacted only with permeabilized
cells in immunocytochemical analyses, indicating that they recognize
intracellular epitopes (24). The linear epitope bound by one of the
mAbs, QCRL-1, was subsequently mapped to amino acids 918-924 in the
cytoplasmic region connecting the two halves of MRP (25). This
knowledge has facilitated the use of mAb QCRL-1 for a number of
purposes, including analysis of MRP expression in clinical tumor
specimens (26-31), structure-function studies (10, 25, 32, 33), and
immunoaffinity purification of
MRP.2
In contrast to mAb QCRL-1, the remaining four mAbs recognize
conformation-dependent epitopes in the MRP molecule (24).
mAbs QCRL-2, -3, and -4 also have the ability to inhibit the transport of several conjugated substrates (including LTC4,
17 Cell Lines and Antibodies--
The MRP-overexpressing
multidrug-resistant H69AR small cell lung cancer cell line has been
described previously (7, 35). T5 cells are HeLa cells that have been
transfected with a full-length MRP cDNA expression vector,
pRc/CMV-MRP1, and C1 cells are HeLa cells transfected with empty vector
(13, 14). mAbs QCRL-1, -2, -3, -4, and -6 are murine mAbs raised
against cell membranes from MRP-overexpressing H69AR cells (24). The
mAbs were used in the form of crude or DEAE Blue-purified ascites fluid
(26), were obtained as purified preparations from Centocor (Malvern, PA), or were purified using GammaBind Plus Sepharose resin (Amersham Pharmacia Biotech). Fab fragments of mAbs QCRL-1, -2, -3, and -4 were
prepared using the ImmunoPure Fab kit (Pierce). MRP-1 and -2 are
polyclonal antisera raised against homologous 15-amino acid peptides
from the first and second NBDs of MRP (amino acids 765-779 and
1427-1441, respectively) (24, 25, 36). The rat mAb MRPr1 and the mouse
mAbs MRPm5 and MRPm6 were kindly provided by Dr. R. J. Scheper
(Free University Hospital, Amsterdam) (37, 38). mAbs MRPr1 and MRPm6
recognize linear epitopes between MRP amino acids 238-247 and
1511-1520, respectively (39), whereas the epitope for mAb MRPm5 is
located between MRP amino acids
986-1096.3
Immunoprecipitations--
For immunoprecipitation of MRP,
membrane-enriched fractions were prepared from H69AR cells that had
been metabolically labeled with [35S]methionine (24).
Membrane proteins were solubilized in PBS containing 1% CHAPS at
detergent/protein ratios ranging from 1:1 to 10:1 (w/w) for 3 h at
4 °C. After centrifugation at 100,000 × g, 1 µl
of crude ascites fluid was mixed with 22.5 µg of solubilized membrane
protein and incubated overnight at 4 °C. For immunoprecipitation of
polypeptides MRP-(617-932) and MRP-(1294-1531) (prepared as described
below), ~4 µg of total cytosolic protein from infected Sf21
cells was diluted to 100 µl with PBS, and 1 µl of ascites (mAb
QCRL-1, -2, -3, or -4) or 5 µl of MRP-2 antiserum was added. After
incubation for 2 h at 4 °C, immune complexes were recovered by
the addition of 30 µl of 25% GammaBind Plus Protein G-Sepharose (Amersham Pharmacia Biotech) in PBS. After incubation for 1 h at
room temperature, the beads were washed four times with PBS, and
precipitated proteins were eluted with Laemmli buffer and analyzed by
SDS-PAGE and fluorography.
125I Labeling of mAbs--
Sixty µg each of DEAE
Blue-purified mAbs QCRL-1, -2, -3, -4, and -6 were passed over an
Econo-Pac 10DG column (Bio-Rad) and eluted in PBS. Protein-containing
fractions were pooled, and the volume was brought up to 850 µl with
PBS. IODO-BEADS (Pierce) were washed with PBS and dried on filter
paper. For each mAb, one IODO-BEAD was added to 150 µl of PBS
containing 400 µCi of 125I (17 Ci/mg of iodine; ICN,
Montreal, Quebec, Canada) and incubated for 5 min. The purified mAb was
then added, and labeling was allowed to proceed for 15 min. To separate
125I-labeled mAb from free 125I, the reaction
mixture was loaded onto an Econo-Pac 10DG column, the mAbs were eluted
with PBS, and radioactive fractions were pooled.
Dot Blot Assay of Direct Binding Competition between Unlabeled
and 125I-Labeled mAbs--
To quantitate the relative
amount of mAb in DEAE blue-purified mAb samples and crude ascites
fluid, the purified samples were separated by SDS-PAGE together with
serial dilutions of the corresponding ascites fluid. The amount of IgG
heavy chain protein in each was assessed by densitometric analysis of
the Coomassie-stained gels on a Molecular Dynamics computing
densitometer using ImageQuant software. Dot blots were prepared on
Immobilon P membrane in a 96-well manifold by blotting 4 µg of H69AR
crude membranes in 50 µl of TBS/well and draining slowly by gravity
(see below). After washing with TBS containing Tween 20 (TBS-T), 100 µl of blocking solution (10% calf serum and 1% bovine serum albumin in TBS-T) per well was added and allowed to drain slowly by gravity. Approximately 30 ng of each 125I-labeled mAb was mixed with
QCRL-1, -2, -3, -4, and -6 crude ascites or with mouse IgG (Pierce) in
200 µl of blocking solution/well at ratios of unlabeled competing
antibody to 125I-labeled antibody ranging from 1:125 to
125:1. The labeled/unlabeled antibody mixtures were added to the blot
and drained slowly by gravity. Finally, the wells were rinsed four
times with 200 µl of TBS-T, the blot was removed from the manifold
and dried, and bound antibody was detected by exposure to Kodak X-Omat
AR film at Generation of Expression Constructs, Production of Recombinant
Baculovirus, and Viral Infection--
The preparation of constructs
encoding wild-type and truncated (MRP-(1-858), MRP-(1-932),
MRP-(229-1531), MRP-(281-1531), and MRP-(932-1531)) MRP molecules
has been described previously (32, 33). Additional constructs
(MRPins708, MRP-(281-932), MRP-(1-616), MRP-(281-1295),
MRP-(932-1295), MRP-(617-932), and MRP-(1294-1531)) were also
prepared using similar
methods.4 All constructs were
transferred into the recombinant donor plasmid pFASTBAC1 (Life
Technologies, Inc., Burlington, Ontario, Canada), and recombinant
bacmids and baculovirus were produced and used to infect
Spodoptera frugiperda Sf21 cells as described
previously (32).
Cytosolic and Membrane Protein Isolation--
H69AR, empty
vector-transfected HeLa C1, MRP-transfected HeLa T5, or Sf21
cells were harvested, and crude membrane fractions were prepared (24,
32). For some experiments, plasma membrane vesicles and cytosolic
fractions were isolated. Briefly, cells were homogenized in buffer
containing 50 mM Tris-HCl, 250 mM sucrose, 0.25 mM CaCl2, pH 7.5, and protease inhibitors
(CompleteTM, Roche Molecular Biochemicals). Cell pellets
were frozen at MRP-mediated LTC4 Transport in Inside-out Membrane
Vesicles--
ATP-dependent
[3H]LTC4 uptake into membrane vesicles was
measured by a rapid filtration technique (16, 40). Plasma membrane vesicles from MRP-transfected T5 cells (2 µg of protein in a 60-µl reaction volume) were preincubated alone or with mAb QCRL-1, -2, -3, or
-4 (10 µg/ml intact mAb or Fab fragment) for 1 h at room temperature. Uptake of [3H]LTC4 (50 nM, 40 nCi/reaction, 146 Ci/mmol; Life Science Products, Markham, Ontario) was then determined in the presence of 10 mM MgCl2 and 4 mM ATP for 1 min at
23 °C. Uptake was stopped by filtration through glass-fiber filters.
Filters were washed with transport buffer, and associated
radioactivity was quantitated.
Preparation of Dot Blots and Immunoblotting--
Membrane
proteins in TBS were blotted onto Immobilon P membrane by gravity using
a 96-well vacuum manifold. After washing with TBS-T, the blots were
removed from the manifold, cut into strips, and transferred to a
24-slot incubation tray. Blots were blocked for at least 1 h in
4% skim milk powder in TBS-T, incubated with primary antibody diluted
in blocking solution for 1-18 h, and then processed as described
previously (24). Primary antibody binding was visualized by enhanced
chemiluminescence detection (Amersham Pharmacia Biotech) using
horseradish peroxidase-conjugated F(ab')2 fragments of goat
anti-rat IgG (H+L; for mAb MRPr1), goat anti-mouse IgG + IgM (H+L; for
mAbs MRPm6, MRPm5, and QCRL-1, -2, -3, -4, and -6), or goat anti-rabbit
IgG (H+L; for antisera MRP-1 and MRP-2).
Labeling of MRP with
8-Azido-[32P]ATP--
Labeling of MRP-enriched T5 or
control C1 membrane proteins was carried out as described previously
(36, 41). Briefly, 30 µg of T5 or C1 membrane proteins was incubated
with intact mAbs or their Fab fragments (40 µg/ml) for 15 min at room
temperature. 8-Azido-[ Immunoprecipitation of Full-length MRP--
We demonstrated
previously that mAbs QCRL-1, -2, and -3 could immunoprecipitate MRP
from 1% CHAPS-solubilized membranes, confirming that they are
MRP-specific (24). In contrast, mAbs QCRL-4 and -6, which bound
strongly and specifically to crude membranes from MRP-overexpressing
cell lines in immunodot blots, did not immunoprecipitate MRP
solubilized under similar conditions or with different detergents, including 0.1% SDS, 1% Nonidet P-40, or 0.5% deoxycholate (data not
shown). Consequently, it could not be concluded with absolute certainty
that they reacted exclusively with MRP. To test the possibility that
mAbs QCRL-4 and -6 recognized conformation-dependent epitopes that were particularly sensitive to denaturation, milder conditions were used for extracting MRP from cell membranes prior to
immunoprecipitation. At a detergent/protein ratio of 1:1, mAb QCRL-3
immunoprecipitated the 190-kDa MRP, as expected (Fig.
1). mAbs QCRL-4 and -6 also
immunoprecipitated a protein of the same size under these conditions,
indicating that they are indeed MRP-specific. Higher detergent/protein
ratios (e.g. 10:1) had little effect on the interaction of
mAb QCRL-3 with MRP. However, binding and precipitation of MRP by both
mAbs QCRL-4 and -6 were substantially reduced, confirming the detergent
sensitivity of the epitopes recognized by these mAbs.
Assay of Binding Competition between MRP-specific
mAbs--
Binding competition assays were carried out to determine
whether mAbs QCRL-1, -2, -3, -4, and -6 recognize overlapping epitopes using H69AR membrane dot blots. A constant amount of
125I-labeled mAb QCRL-1, -2, -3, -4, or -6 either alone or
premixed with increasing amounts of unlabeled competing antibody (mouse IgG or mAb QCRL-1, -2, -3, -4, or -6) was added to individual wells. As
expected, binding of each 125I-labeled mAb was inhibited by
the unlabeled form of the same mAb at an ~1:1 ratio or by an excess
of unlabeled competitor (Fig. 2). This
competition was specific because total mouse IgG did not compete for
binding with any of the 125I-labeled mAbs even when present
at 125-fold excess. Of all the mAbs tested, only mAbs QCRL-4 and -6 competed reciprocally for binding to MRP. This finding, together with
observations that they behave similarly in immunoprecipitations (Fig.
1) and other immunoassays (data not shown), suggested that mAbs QCRL-4
and -6 probably bind to the same epitope. Consequently, mAb QCRL-6 was
not characterized further. No significant overlap between the epitopes
of the other mAbs was apparent.
Inhibition of MRP-mediated LTC4 Transport by Fab
Fragments of mAbs QCRL-2, -3, and -4--
mAb QCRL-3 and, in some
cases, mAbs QCRL-2 and -4 have been shown to inhibit MRP-mediated
transport of several conjugated substrates as well as GSH-stimulatable
transport of several unmodified xenobiotics (16, 19, 21, 23, 34).
However, intact (bivalent) mAbs were used in these studies, raising the
possibility that inhibition resulted from steric effects caused by the
Fc regions of these mAbs or by cross-linking of adjacent MRP molecules.
To address this possibility, Fab fragments of mAbs QCRL-2, -3, and -4 were prepared and tested to determine whether these monovalent fragments are also capable of inhibiting MRP-mediated transport activity. As expected, the Fab fragment of the non-inhibitory mAb
QCRL-1 had little effect on MRP-mediated LTC4 transport
relative to the control sample with no mAb (Fig.
3). In contrast, Fab fragments of mAbs
QCRL-2, -3, and -4 inhibited transport by >85% at 10 µg/ml. Similar
results were obtained with the intact mAbs, in agreement with previous
observations. These results indicate that inhibition of MRP-mediated
transport by mAbs QCRL-2, -3, and -4 is caused by their direct
interaction with the protein.
Binding of mAbs to Sf21-expressed Modified MRP
Molecules--
To localize the conformation-dependent
epitopes of mAbs QCRL-2, -3, and -4, the reactivity of these mAbs with
a panel of truncated or otherwise altered MRP molecules expressed in
Sf21 insect cells was examined in a series of immunodot blot
analyses. In the first set of experiments, the mAbs were tested against
MRP polypeptides with NH2-proximal truncations
(MRP-(229-1531) and MRP-(932-1531)) or COOH-proximal truncations
(MRP-(1-858) and MRP-(1-932)). In addition, a mutated protein
(MRPins708) that contains 13 additional amino acids
(DIRTINVRFLREI) inserted at amino acid 708 between the Walker A and B
motifs of NBD1 was examined. These amino acids are present in the
comparable location of P-glycoprotein, but are absent from most
MRP-related ATP-binding cassette transporters (3, 7, 11). A diagram of
MRP showing the approximate locations of its three MSDs and two NBDs as
well as the portions of MRP contained in the various polypeptides used
in this analysis is shown in Fig.
4A.
As positive controls, the dot blots were probed in parallel with mAbs
MRPr1, MRPm6, and QCRL-1, the epitopes of which have been defined
previously (Fig. 4A) (25, 39). These blots provided standards against which the immunoreactivity of mAbs QCRL-2, -3, and -4 with each polypeptide could be compared (25, 39). All three of the
positive control mAbs showed patterns of immunoreactivity with the MRP
fragments consistent with the locations of their epitopes (Fig.
4B). However, mAb MRPr1 reacted more strongly with the
MRP-(229-1531) polypeptide than the other two mAbs, and mAb QCRL-1
reacted more strongly with the MRP-(1-932) polypeptide than mAb MRPr1.
In each case, the truncation in the MRP molecule is very near the site
of the epitope of the more strongly reacting mAb (amino acids 238-247
for mAb MRPr1 and amino acids 918-924 for mAb QCRL-1) (25, 39). This
may increase accessibility of the mAbs to these epitopes and hence
account for the stronger signal observed.
mAbs QCRL-2 and -3 showed similar patterns of immunoreactivity with the
MRP fragments (Fig. 4B). Both mAbs bound to the
NH2-proximal half-molecule MRP-(1-932) and to the
polypeptide lacking MSD1 (MRP-(229-1531)), and neither bound to the
COOH-proximal half-molecule MRP-(932-1531). In addition, the binding
of both mAbs was markedly diminished by the insertion of 13 amino acids
in the NH2-proximal NBD (MRPins708). The only
major difference between the two mAbs was that mAb QCRL-2 did not bind
to MRP-(1-858), whereas mAb QCRL-3 reacted strongly with this
polypeptide. These results suggest that the sequences bound by mAbs
QCRL-2 and -3 are located somewhere in MSD2 and/or NBD1. In addition,
at least part of the mAb QCRL-2 epitope is between amino acids 859 and
932 in the region connecting NBD1 to MSD3. In contrast, mAb QCRL-4
reacted only with those polypeptides containing the COOH-proximal half
of MRP (MRP-(932-1531) and MRP-(229-1531)) and bound as well to
MRPins708 as the control mAbs. These observations suggest
that the QCRL-4 epitope is in MSD3 and/or NBD2.
To further localize the QCRL-2 and -3 epitopes, a second series of
immunodot blotting was carried out with these mAbs using MRP
polypeptides containing MSD2 or MSD2 + NBD1 (Fig.
5, A and B). The
polypeptides used were MRP-(281-932), MRP-(281-1531), and
MRP-(1-616). As positive controls, blots were probed with mAb MRPr1
and the MRP-1 polyclonal antiserum. MRP-(229-1531) was included in
these analyses to allow normalization of signal intensity since this
polypeptide contains the epitopes bound by all four antibodies used.
mAb MRPr1 reacted with MRP-(1-616), and the MRP-1 antiserum reacted
with MRP-(281-932) and MRP-(281-1531), consistent with the known
locations of their epitopes (Fig. 5B). mAbs QCRL-2 and -3 showed a pattern of reactivity similar to that of the MRP-1 antiserum,
reacting only with polypeptides containing MRP amino acids 617-932
(Fig. 5B). This suggests that the
conformation-dependent epitopes recognized by these mAbs
are wholly or partially contained within this region corresponding to
NBD1.
To further localize the QCRL-4 epitope, immunodot blotting was
performed using MRP fragments containing MSD3 or MSD3 + NBD2 (Fig. 5,
A and C). In these experiments, the polypeptides
used were MRP-(932-1531), MRP-(281-1295), and MRP-(932-1295). As a positive control, the blot was also probed with mAb MRPm5, whose linear
epitope lies between amino acids 986 and 1096.3 mAb MRPm5
bound to all three polypeptides as expected, whereas mAb QCRL-4 reacted
only with MRP-(932-1531) (Fig. 5C). These results suggest
that the QCRL-4 epitope is contained wholly or in part within the
region corresponding to NBD2.
Immunoprecipitation of Soluble Polypeptides Corresponding to NBD1
(MRP-(617-932)) and NBD2 (MRP-(1295-1531))--
To determine whether
the epitopes recognized by the mAbs were entirely contained between
amino acids 617 and 932 (QCRL-2 and -3) or amino acids 1296 and 1531 (QCRL-4), the ability of the mAbs to immunoprecipitate the
MRP-(617-932) or MRP-(1294-1531) polypeptides was examined.
MRP-(617-932) migrated as a polypeptide of ~36 kDa in SDS-PAGE and
could be immunoprecipitated from the cytosolic fraction of Sf21
cells with mAbs QCRL-2 and -3 as well as with mAb QCRL-1, as expected
(Fig. 6A). Immunoprecipitation of MRP-(617-932) with mAbs QCRL-2 and -3 was specific because this
polypeptide was not immunoprecipitated with mAb QCRL-4.
MRP-(1294-1531) was also present in the cytosolic fraction of
Sf21 cells and migrated as a doublet between 25 and 30 kDa (Fig.
6B). The exact nature of these doublet MRP NBD-derived bands
is not known. However, we speculate that there is a protruding
"tail" at the NH2 or COOH terminus of the folded NBD
polypeptide that is susceptible to proteolysis. Cleavage of the tail
would result in lower levels of the larger, but less stable full-length
polypeptide and higher levels of the slightly smaller, but more stable
protein. MRP-(1294-1531) could be immunoprecipitated with mAb QCRL-4
as well as with the MRP-2 antiserum, as expected (Fig. 6B).
Immunoprecipitation with these antibodies was specific because the only
proteins detectable in the mAb QCRL-2 and -3 immunoprecipitates were
the immunoglobulin light chains of the precipitating antibodies, which
migrated at ~25 kDa.
Fab Fragments of mAbs QCRL-2, -3, and -4 Do Not Inhibit Azido-ATP
Labeling of MRP--
To determine whether mAbs QCRL-2, -3, and -4 interfere with the ability of MRP to bind nucleotide, membrane vesicle
proteins from MRP-transfected T5 cells and control C1 cells were
incubated with Fab fragments of the mAbs individually or together and
then photolabeled with 8-azido-[32P]ATP. As shown in Fig.
7, labeling of MRP in T5 membranes was unaffected by the Fab fragments, suggesting that the mAbs do not prevent nucleotide binding by the protein.
To provide molecular probes for studying both the structure and
transport mechanism of MRP, we isolated a panel of mAbs that reacted
preferentially with cell membranes from MRP-overexpressing cell lines
(24). Four of these mAbs (QCRL-2, -3, -4, and -6) recognize
intracellular conformation-dependent epitopes and are able
to inhibit the transport activity of MRP. At the time of their initial
characterization, immunodot blot analyses strongly suggested that mAbs
QCRL-4 and -6 recognize MRP, but we were unable to confirm this
directly by immunoprecipitation (24). By using very low
detergent/protein ratios, we have now shown that mAbs QCRL-4 and -6 immunoprecipitate a single 190-kDa protein from solubilized membranes
(Fig. 1). These observations establish the MRP specificity of mAbs
QCRL-4 and -6 and indicate that both mAbs recognize detergent-sensitive
epitopes. Binding competition assays revealed that mAbs QCRL-4 and -6 recognize identical or overlapping epitopes (Fig. 2). None of the
remaining mAbs competed reciprocally for binding, demonstrating that
mAbs QCRL-2, -3, and -4 recognize distinct, non-overlapping epitopes of
MRP.
mAbs QCRL-2, -3, and -4 have been shown to be effective inhibitors of
MRP-mediated ATP-dependent transport of several conjugated organic anion substrates, and mAb QCRL-3 has been shown to inhibit GSH-stimulatable transport of unmodified vincristine, daunorubicin, and
aflatoxin B1 (16, 19, 21, 23, 34). This inhibitory property
is not shared by mAb QCRL-1, which may be explained by the fact that
its linear heptapeptide epitope is within a region of MRP that is not
required for LTC4 transport activity in Sf21 cell
membranes (25, 32). The Fab fragments of mAbs QCRL-2, -3, and -4 inhibited MRP-mediated transport as effectively as the intact mAbs
(Fig. 3). This finding indicates that inhibition is caused by direct
interaction of the antigen-binding sites of these mAbs with MRP, rather
than being the result of a steric effect caused by the Fc regions of
the immunoglobulins or by cross-linking of adjacent MRP molecules.
As an initial step toward understanding the mechanism by which mAbs
QCRL-2, -3, and -4 inhibit the transport activity of MRP, we have
localized the conformation-dependent epitopes bound by these mAbs. With respect to mAbs QCRL-2 and -3, we have determined that
the minimal regions of MRP sufficient for full reactivity are amino
acids 617-932 and 617-858, respectively. These regions include NBD1
and different amounts of the segment that connects NBD1 to MSD3. Thus,
a portion of the epitope recognized by mAb QCRL-2 is located between
amino acids 859 and 932 in the connector region of the protein.
Insertion of 13 amino acids at position 708 in NBD1 of MRP resulted in
a markedly decreased reactivity of mAbs QCRL-2 and -3 and also reduced
the transport activity of the protein.4 Taken together,
these observations indicate that the determinants of the epitopes bound
by both of these mAbs are contained within NBD1 and that the relative
positioning of these determinants is perturbed when the distance
between the Walker A and B motifs of this domain is altered. In
contrast to mAbs QCRL-2 and -3, the epitope bound by mAb QCRL-4 appears
to reside wholly within the second NBD of MRP, between amino acids 1294 and 1531. Since mAbs QCRL-2, -3, and -4 do not react with MRP in its
denatured form, the ability of these mAbs to immunoprecipitate
MRP-(617-932) (NBD1) and MRP-(1296-1531) (NBD2) indicates that both
of these soluble truncated polypeptides have folded correctly and
assumed a configuration similar or identical to the NBDs when they are present in the context of the full-length protein in its native state.
Consistent with this conclusion is our observation that the individual
NBDs have the ability to bind and hydrolyze ATP.4 This
knowledge is important with respect to ongoing structural and
functional studies of these MRP domains and their interactions with
other regions of the protein as well as in comparisons with the NBDs of
other ATP-binding cassette transporters such as P-glycoprotein and the
cystic fibrosis transmembrane conductance regulator
(42-46).4
The location of the QCRL-2, -3, and -4 epitopes in the NBDs of MRP
raised the possibility that one or more of the mAbs might inhibit
transport by interfering directly with ATP binding. However, the
inability of both of the intact mAbs (data not shown) and their Fab
fragments to inhibit labeling of MRP by 8-azido-ATP suggests that this
is not the case (Fig. 7). We have shown previously that intact mAb
QCRL-3 can inhibit photolabeling of MRP by
[3H]LTC4 (16), indicating that this mAb may
inhibit transport by directly limiting access of LTC4 to
its binding site. Further experiments examining the effects of the Fab
fragments of mAbs QCRL-2, -3, and -4 on
[3H]LTC4 photolabeling of MRP and MRP-derived
polypeptides will be required to determine conclusively whether these
mAbs inhibit transport of this substrate by preventing its binding to
the protein. In turn, these studies may provide important information
about the location of the LTC4 and other substrate binding
site(s) on MRP.
Structure-function studies of P-glycoprotein have been aided by several
P-glycoprotein-specific mAbs that have been reported to interfere with
substrate interactions and/or the transport activity of this protein.
The first P-glycoprotein-reactive mAb to be characterized was mAb C219,
which recognizes a linear cytoplasmic epitope located six amino acids
downstream from the Walker B motif in both the NH2- and
COOH-proximal NBDs of this protein (47, 48). mAb C219 inhibited
photolabeling of P-glycoprotein with the calcium channel blocker
photoaffinity analog [3H]azidopine (49), but unlike mAbs
QCRL-2, -3, and -4 in the present study, also inhibited photolabeling
with 8-azido-ATP (49, 50). The inhibition of ATP binding by mAb C219
was associated with a decrease in ATPase activity present in membranes
from P-glycoprotein-overexpressing cells (50), whereas we have noted no
such effect of the MRP-specific mAbs on the ATPase activity of
MRP.2 Despite these effects on drug binding and ATPase
activity, inhibition of P-glycoprotein transport activity by mAb C219
has not been reported. On the other hand, several other mAbs that bind
extracellular epitopes of P-glycoprotein specifically reverse or reduce
the drug resistance and/or drug accumulation deficit of viable,
P-glycoprotein-overexpressing tumor cells (51-54). The mechanism by
which one of these mAbs, UIC2, inhibits P-glycoprotein function appears
to involve trapping of the protein in an inactive conformation (55).
Similarly, mAbs QCRL-2, -3, and -4 may stabilize or trap MRP in a given
conformational state and, in doing so, prevent the conformational
changes that are presumed to occur during the transport cycle of the
protein. To explore this possibility, it will be necessary to determine if the mAbs are able to modulate MRP conformational transitions. mAbs
QCRL-2, -3, and -4 cannot be used to detect MRP in intact, non-permeabilized cells because their epitopes are cytoplasmic. Nevertheless, it should be possible to test their reactivity with functional MRP in cell membranes, in the presence and absence of ATP
and transport substrates, using filter binding assays. Such studies
will provide important insight into the mechanism by which MRP
transports its conjugated organic anion and drug substrates.
-32P]ATP. This suggests that rather than
preventing nucleotide binding, the mAbs inhibit transport by
interfering with substrate binding or by trapping MRP in a conformation
that does not allow transport to occur. Our results also demonstrate
for the first time that the NBDs of MRP can be expressed as soluble
polypeptides that retain a native conformation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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-estradiol 17-(
-D-glucuronide), and aflatoxin
B1-glutathionyl aflatoxin B1) into inside-out
MRP-enriched plasma membrane vesicles (16, 21, 23, 34). Similarly,
GSH-stimulatable transport of unmodified vincristine and aflatoxin
B1 is inhibited by these mAbs (19, 21, 23). These results
suggest that the conformation-dependent epitopes recognized
by mAbs QCRL-2, -3, and -4 are localized in or near functionally
important regions of MRP. In this study, we show that mAbs QCRL-2, -3, and -4 recognize distinct, non-overlapping epitopes of MRP and that
their ability to inhibit MRP-dependent transport results
from direct interactions with the NBDs of the protein that do not
prevent the binding of ATP.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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70 °C.
70 °C for at least 1 h, thawed, and then
disrupted by N2 cavitation. EDTA was added to 1 mM, and after centrifugation at 500 × g
for 15 min, the supernatant was layered over 35% (w/w) sucrose in 50 mM Tris-HCl and 1 mM EDTA and centrifuged at
100,000 × g for 1 h. The interface was collected
and washed twice by centrifugation. The membrane pellet was resuspended
in transport buffer (50 mM Tris-HCl and 250 mM
sucrose, pH 7.5) and passed 20 times through a 27-gauge needle for
vesicle formation. For Sf21 cells expressing polypeptides
MRP-(617-932) and MRP-(1294-1531), the layer above the 35% sucrose
interface containing soluble cytosolic proteins was retained.
-32P]ATP (4 µCi, 20 Ci/mmol;
ICN) was added, and incubation was continued on ice for 5 min. The
samples were irradiated at 302 nm for 8 min, and proteins were then
resolved by SDS-PAGE followed by autoradiography.
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MATERIALS AND METHODS
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Fig. 1.
Immunoprecipitation of MRP with mAbs QCRL-4
and -6. Crude membranes prepared from H69AR cells that had been
metabolically labeled with [35S]methionine were
solubilized in PBS containing 1% CHAPS at a detergent/protein ratio of
1:1 or 10:1 (w/w) as indicated. MRP was immunoprecipitated from 22.5 µg of solubilized membrane protein using mAb QCRL-3, -4, or -6 crude
ascites (1 µl). Immune complexes were recovered using Protein
G-Sepharose, and precipitated proteins were analyzed by SDS-PAGE and
fluorography.
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Fig. 2.
Radioimmunoassay of binding competition
between unlabeled and 125I-labeled mAbs. Dot blots
were prepared by blotting H69AR membrane protein (4 µg/spot) onto
Immobilon P membrane using a 96-well vacuum manifold. To each well was
added a constant amount of 125I-labeled mAb (QCRL-1, -2, -3, -4, or -6; indicated to the left of each blot) that had been
preincubated with unlabeled competing antibodies (mAb QCRL-1, -2, -3, -4, or -6 crude ascites or mouse IgG; indicated to the right of each
blot). The unlabeled/125I-labeled mAb ratios are indicated
at the top of the blots. After allowing the antibody mixtures to drain
slowly by gravity, the wells were rinsed. The blots were dried, and
125I-labeled mAb binding was detected by
autoradiography.
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Fig. 3.
Inhibition of MRP-mediated LTC4
transport by mAb Fab fragments. Plasma membrane vesicles prepared
from MRP-transfected T5 cells were preincubated alone
(Control), with intact mAbs (QCRL-1, -2, -3, or -4), or with
the Fab fragments of these mAbs (10 µg/ml). Uptake of
[3H]LTC4 into the vesicles during 60 s
was then determined as described under "Materials and Methods."
Data points represent the mean ± S.D. of triplicate
determinations.
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Fig. 4.
Immunodot blot analysis of
Sf21-expressed truncated MRP constructs. A, a
schematic diagram depicting Sf21-expressed wild-type
(WT) MRP and mutant MRP molecules lacking portions of the
NH2 terminus (MRP-(229-1531) and MRP-(932-1531)) or the
COOH terminus (MRP-(1-858) and MRP-(1-932)) or with 13 amino acids
inserted at amino acid 708 in NBD1 (MRPins708) is shown
below the box model of MRP. The locations of the three MSDs
and two NBDs in MRP are indicated, as are the positions of the epitopes
recognized by the three mAbs used as positive controls (MRPr1, QCRL-1,
and MRPm6) (dotted lines). B, identical dot blots
of crude membrane protein isolated from Sf21 cells expressing
MRP constructs (5 µg/spot for MRP-(1-858) and 2 µg/spot for all
other polypeptides) were prepared and probed with mAb QCRL-2, -3, or -4 or with control mAbs MRPr1, QCRL-1, or MRPm6.
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Fig. 5.
Immunodot blot analysis of
Sf21-expressed MRP domains. A, shown is a
schematic diagram depicting the domain structure of MRP and approximate
locations of amino acids that mark the five domains of the protein.
Locations of the epitopes recognized by mAbs MRPr1 and MRPm5 and
polyclonal antiserum MRP-1 are indicated below the box model
of MRP. B, identical dot blots of crude membrane protein
prepared from Sf21 cells expressing constructs encoding linear
combinations of MRP domains (1 µg/spot) were probed with mAb QCRL-2
or -3 or with mAb MRPr1 or the MRP-1 antiserum as a control. Membrane
protein from cells expressing the NH2-terminally truncated
MRP-(229-1531) construct (3 µg/spot) was included on the dot blots
for normalization of signal intensities. C, identical dot
blots of crude membrane protein prepared from Sf21 cells
expressing constructs encoding linear combinations of MRP domains (3 µg/well for MRP-(932-1531) and 1 µg/well for others) were probed
with mAb QCRL-4 or with control mAb MRPm5.
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Fig. 6.
Immunoprecipitation and immunoblotting of
Sf21-expressed MRP fragments encoding the NH2- and
COOH-proximal NBDs of MRP. Soluble polypeptides containing NBD1
(MRP-(617-932)) or NBD2 (MRP-(1294-1531)) of MRP were expressed in
Sf21 cells, and cytosolic protein fractions were isolated by
differential centrifugation as described under "Materials and
Methods." MRP-(617-932) and MRP-(1294-1531) were immunoprecipitated
from 4 µg of total cytosolic protein, and one-fourth of each
precipitate was separated by SDS-PAGE and transferred to Immobilon P,
and the polypeptides were detected by immunoblotting. A,
MRP-(617-932) was immunoprecipitated with mAb QCRL-1, -2, -3, or -4 and detected by immunoblotting with the MRP-1 antiserum. Cytosolic
protein (1 µg) from Sf21 cells expressing MRP-(617-932) was
loaded onto the gel in an adjacent lane. B, MRP-(1294-1531)
was immunoprecipitated with mAb QCRL-2, -3, or -4 or with the MRP-2
polyclonal antiserum and detected by immunoblotting with mAb MRPm6.
Cytosolic protein (1 µg) from Sf21 cells expressing
MRP-(1294-1531) was loaded onto the gel in an adjacent lane. Molecular
mass markers (in kilodaltons) are indicated to the right of each blot.
Ippt'n, immunoprecipitation.
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Fig. 7.
Photolabeling of MRP with
8-azido-[ -32P]ATP in the
presence of Fab fragments of MRP-specific mAbs. MRP-enriched T5 or
control C1 membrane vesicle proteins (30 µg) were photolabeled with
8-azido-[
-32P]ATP (4 µCi) in the presence of Fab
fragments of mAb QCRL-1, -2, -3, or -4 (40 µg/ml) individually or
with a mixture of QCRL-2, -3, and -4 Fab fragments together, as
indicated. Membrane vesicles were irradiated at 302 nm for 8 min and
then analyzed by SDS-PAGE and autoradiography.
DISCUSSION
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Dr. D. W. Loe for technical assistance and advice and Dr. R. Scheper for providing mAbs MRPr1, MRPm6, and MRPm5.
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FOOTNOTES |
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* This work was supported in part by grant MT-10519 from the Medical Research Council of Canada.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 Medical Research Council of Canada Studentship Award.
Present address: EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Supported by an Ontario Graduate Student Award.
§§ Supported by a Queen's University graduate award.
¶¶ Stauffer Research Professor of Queen's University.
|| Senior Scientist of Cancer Care Ontario. To whom correspondence should be addressed: Cancer Research Laboratories, Botterell Hall, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-6507; Fax: 613-533-6830; E-mail: coles{at}post.queensu.ca.
2 Q. Mao, E. M. Leslie, R. G. Deeley, and S. P. C. Cole, submitted for publication.
3 D. R. Hipfner, R. G. Deeley, and S. P. C. Cole, unpublished results.
4 M. Gao, K. C. Almquist, D. W. Loe, C. E. Grant, S. P. C. Cole, and R. G. Deeley, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: MRP, multidrug resistance protein; NBD, nucleotide-binding domain; MSD, membrane-spanning domain; LTC4, leukotriene C4; mAb, monoclonal antibody; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline.
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