(Received for publication, November 30, 1995; and in revised form, January 17, 1996)
From the
The 190-kDa multidrug resistance protein (MRP) has recently been
associated with the transport of cysteinyl leukotrienes and several
glutathione (GSH) S-conjugates. In the present study, we have
examined the transport of leukotriene C (LTC
)
in membrane vesicles from MRP-transfected HeLa cells (T14), as well as
drug-selected H69AR lung cancer cells which express high levels of MRP. V
and K
values for
LTC
transport by membrane vesicles from T14 cells were 529
± 176 pmol mg
min
and 105
± 31 nM, respectively. At 50 nM LTC
, the K
(ATP) was 70
µM. Transport in T14 vesicles was osmotically-sensitive
and was supported by various nucleoside triphosphates but not by non-
or slowly-hydrolyzable ATP analogs. LTC
transport rates in
membrane vesicles derived from H69AR cells and their parental and
revertant variants were consistent with their relative levels of MRP
expression. A 190-kDa protein in T14 membrane vesicles was photolabeled
by [
H]LTC
and immunoprecipitation
with MRP-specific monoclonal antibodies (mAbs) confirmed that this
protein was MRP. LTC
transport was inhibited by an
MRP-specific mAb (QCRL-3) directed against an intracellular
conformational epitope of MRP, but not by a mAb (QCRL-1) which
recognizes a linear epitope. Photolabeling with
[
H]LTC
was also inhibitable by mAb
QCRL-3 but not mAb QCRL-1. GSH did not inhibit LTC
transport. However, the ability of alkylated GSH derivatives to
inhibit transport increased markedly with the length of the alkyl
group. S-Decylglutathione was a potent competitive inhibitor
of [
H]LTC
transport (K
116 nM),
suggesting that the two compounds bind to the same, or closely related,
site(s) on MRP. Chemotherapeutic agents including colchicine,
doxorubicin, and daunorubicin were poor inhibitors of
[
H]LTC
transport. Taxol, VP-16,
vincristine, and vinblastine were also poor inhibitors of LTC
transport but inhibition by these compounds was enhanced by GSH.
Uptake of [
H]vincristine into T14 membrane
vesicles in the absence of GSH was low and not dependent on ATP.
However, in the presence of GSH, ATP-dependent vincristine transport
was observed. Levels of transport increased with concentrations of GSH
up to 5 mM. The identification of an MRP-specific mAb that
inhibits LTC
transport and prevents photolabeling of MRP by
LTC
, provides conclusive evidence of the ability of MRP to
transport cysteinyl leukotrienes. Our studies also demonstrate that MRP
is capable of mediating ATP-dependent transport of vincristine and that
transport is GSH-dependent.
Multidrug resistance can be conferred by overexpression of
either the multidrug resistance protein, MRP()(1) ,
or by P-glycoprotein(2) . Both MRP and P-glycoprotein belong to
the ATP-binding cassette transporter superfamily but share only
approximately 15% amino acid identity(3) . Considerable
evidence suggests that P-glycoprotein reduces cellular drug
accumulation by acting as an ATP-dependent drug efflux
pump(2, 4) , but the mechanism of action of MRP is
much less certain. The ability to transport drugs directly into plasma
membrane vesicles has been firmly established for
P-glycoprotein(5, 6) , but not for MRP. There is also
no evidence that xenobiotics bind directly to
MRP(7, 8) , as has been demonstrated for
P-glycoprotein by cross-linking studies with photoaffinity analogs of
chemotherapeutic agents(9) . These observations suggest that
these two ATP-binding cassette proteins may confer multidrug resistance
by different mechanisms.
Some insight into the normal physiological
role of MRP has been obtained by the demonstration that membrane
vesicles from MRP-overexpressing drug-selected and transfected cells
support ATP-dependent transport of cysteinyl leukotrienes (e.g. LTC) and certain other GSH S-conjugates(10, 11, 12) . Further
evidence of a role for MRP in LTC
transport was suggested
by the observation that photolabeling of a 190-kDa protein with
LTC
in MRP-expressing cells is inhibited by MK571 (an
inhibitor of LTC
transport)(10) . The cysteinyl
leukotrienes are potent mediators of inflammation that increase
vascular permeability and smooth muscle contraction(13) .
LTC
is synthesized from GSH and LTA
by
LTC
synthase(14) . It is then exported from the
cell by an ATP-dependent transport mechanism(15) . LTC
synthase and the LTC
transporter protein are
expressed in eosinophils and mast cells(16) , as well as
endothelial cells(13) . LTC
is also transported in
the liver by at least two routes: uptake from the blood circulation
into hepatocytes, and excretion from hepatocytes across canalicular
membranes into the bile(13) .
Although current evidence strongly suggests that MRP mediates an active transport process, the mechanistic relationship between the involvement of MRP in GSH S-conjugate transport and its role in multidrug resistance is unclear. GSH conjugation is not known to be an important pathway for the biotransformation of chemotherapeutic agents to which MRP confers resistance and there is no evidence that this reaction occurs to any significant extent in tumor cells or drug-resistant tumor cell lines(17) .
In the present study, the mechanism by which MRP
confers resistance to multiple drugs was investigated in a plasma
membrane vesicle model system. For these studies, we used membrane
vesicles from a population of HeLa cells, termed T14, which had been
transfected with a MRP expression vector(8) . By using
transfected cells as the source of membrane vesicles, those aspects of
LTC and drug transport kinetics that are solely
attributable to MRP overexpression can be
identified(8, 18) . Our initial objective was to
characterize MRP-mediated LTC
transport in membrane
vesicles from this new population of transfected HeLa cells so that
subsequent studies of drug, or drug conjugate, transport could be
evaluated within this context. We provide evidence that
[
H]LTC
binds specifically to MRP and
demonstrate that a MRP-specific mAb inhibits both
[
H]LTC
transport and binding. We have
also characterized the ability of alkylated GSH derivatives and
hydrophobic chemotherapeutic agents to inhibit
[
H]LTC
transport. Finally, we show
that GSH not only enhances the ability of vincristine to inhibit
LTC
transport, but also results in ATP-dependent transport
of the drug itself.
The sidedness of the membrane vesicles was assessed
by determining the activities of two plasma membrane-associated
ectoenzymes (alkaline phosphatase and 5`-nucleotidase) in the presence
or absence of 0.2% Nonidet P-40 (18, 23) and it was
determined that 30-34% of T14 PM vesicles were inside-out. T14
vesicles were treated with concanavalin A (1:1 (w/w) ratio) overnight
at 4 °C to agglutinate outside-out vesicles(24) . Following
centrifugation to remove agglutinated material, the supernatant
displayed a 2-fold increase in the rate of
[H]LTC
uptake (see below), consistent
with ATP-dependent transport being attributable to inside-out vesicles.
ATP-dependent uptake of
[H]VCR (200 nM; 70 nCi) was measured as
described for [
H]LTC
except that
100-120 µg of vesicle protein was used and the incubations
were carried out at 37 °C. In some experiments, GSH (1-5
mM) was added to the transport buffer. Uptake of
[
H]VCR was stopped by rapid dilution in transport
buffer and immediate filtration through glass fiber (Type A/E) filters
(Gelman Sciences, Dorval, PQ), which had been presoaked overnight at 37
°C in 10% (w/v) bovine serum albumin(5) .
Figure 1:
Time course of
[H]LTC
uptake by membrane vesicles
from MRP and control transfected HeLa cells, parental and drug-selected
H69 cells, and 8226/Dox40 cells. Membrane vesicles were incubated with
50 nM [
H]LTC
in transport
buffer (50 mM Tris-HCl, 250 mM sucrose, pH 7.5) for
the times indicated. Closed symbols represent uptake in the
presence of 4 mM AMP; open symbols represent uptake
in the presence of 4 mM ATP. Vesicles were derived as
described under ``Experimental Procedures'' from the
following cell lines: Panel A, HeLa C6 (
,
) and T14
(
,
); Panel B, H69 (
,
) and H69AR
(
,
); Panel C, H69PR (
,
); Panel
D, 8226/Dox40 (
,
). The dotted curve in Panel A (T14) and Panel B (H69AR) indicate
ATP-dependent uptake, which was calculated by subtracting
ATP-independent transport from transport in the presence of ATP. Data
points are means of triplicate determinations (±S.E.) in a
typical experiment.
Figure 2:
Osmotic sensitivity and nucleotide and
cation specificity of [H]LTC
transport by T14 membrane vesicles. Panel A, T14
membrane vesicles were preincubated for 10 min in transport buffer
containing sucrose (0.25-1 M). Rates of
[
H]LTC
uptake were measured in the
presence of 4 mM ATP (
) or 4 mM AMP (
)
as described under ``Experimental Procedures.'' Panel
B, rates of [
H]LTC
uptake were
measured in the presence of the indicated nucleotides (4 mM).
No regenerating system was included in these experiments and the rate
of transport in 4 mM ATP was not affected by its omission (solid bar). Results obtained with other nucleotides (hatched bars) are plotted as a % of values obtained with 4
mM ATP. The data shown are means of triplicate determinations
(±S.E.) in a single experiment. Panel C, rates of
[
H]LTC
uptake were measured in the
presence of 4 mM ATP and the indicated divalent cations (10
mM) (hatched bars). Results are plotted as a % of
control LTC
uptake values obtained with 10 mM Mg
at 30 s (solid bar). The results
shown are means of triplicate determinations (±S.E.) in a single
experiment and similar results were obtained in one additional
experiment.
Since the activity of many ATPases is
often maintained when other divalent cations are substituted for
Mg, we investigated the level of ATP-dependent
[
H]LTC
transport in T14 vesicles in
the presence of Mn
, Ca
,
Co
, Cd
, Ba
, and
Zn
(10 mM). The relative ability of these
cations to support LTC
transport correlated well with their
abilities to support ATP hydrolysis by known ATPases. Thus, when
Mn
, Ca
, or Co
were substituted for Mg
, the
[
H]LTC
transport rates were
measureable but reduced by 25, 50, and 65%, respectively, whereas
Cd
, Ba
, and Zn
did not support transport (Fig. 2C).
Figure 3:
Effect of substrate and ATP concentration
on [H]LTC
uptake. Panel A,
[
H]LTC
uptake by T14 membrane
vesicles was measured at various LTC
concentrations
(12.5-1000 nM) for 30 s at 23 °C. Data are plotted
as V
versus [S] to confirm that
the appropriate concentration range was selected to observe both
zero-order and first-order rate kinetics. Kinetic parameters were
determined from regression analysis of the Lineweaver-Burk
transformation of the data (inset). Panel B,
ATP-dependent uptake of [
H]LTC
was
measured at various concentrations of ATP (2 µM to 4
mM) in the presence of 50 nM [
H]LTC
. Uptake at ATP
concentrations up to 250 µM are plotted. Kinetic
parameters were determined from regression analysis of the
Lineweaver-Burk data transformation (inset).
The apparent K for ATP (70
µM) was determined by measuring initial rates of
[
H]LTC
uptake at 30 s in the presence
of different concentrations of the nucleotide (2-4000
µM) (Fig. 3B, inset).
Figure 4:
Photolabeling of vesicle proteins and
inhibition of [H]LTC
uptake and
photolabeling by MRP-specific mAb QCRL-3. Panel A, T14
membrane vesicles were photolabeled with
[
H]LTC
and membrane protein (50 or
120 µg) was analyzed by SDS-PAGE and fluorography. Alternatively,
membranes (100 µg of protein) were photolabeled and
immunoprecipitated with a mixture of three MRP-specific mAbs
([
H]LTC
, middle
lane)(19) . For comparison, immunoprecipitations were also
performed on membranes prepared from cells labeled in culture with
[
S]methionine (
S-Ippt, far left lane)(18) . Panel B,
[
H]LTC
uptake was measured in the
presence of MRP-specific mAb QCRL-3. Each point represents the mean of
triplicate determinations (±S.E.) in a single experiment. In the
experiment shown, the rate of uptake in control incubations was 174
pmol mg
min
. Panel C,
membrane vesicle protein (
150 µg) from 8226/Dox40, H69AR, C6,
or T14 cells was incubated with [
H]LTC
(0.5 µCi; 78 nM) in transport buffer, irradiated at
312 nm, and analyzed by SDS-PAGE and fluorography (see
``Experimental Procedures''). T14 membrane vesicles were also
incubated with [
H]LTC
in the presence
of excess unlabeled LTC
, LTB
, MRP-specific mAb
QCRL-3 (10 µg ml
), and murine IgG
(70 µg ml
), prior to irradiation and
SDS-PAGE and fluorography. The autoradiograms shown represent a 14-day
exposure.
To examine
the specificity of the interaction between LTC and MRP, the
ability of unlabeled LTC
or the unconjugated leukotriene,
LTB
, to compete for [
H]LTC
labeling of membrane proteins was determined (Fig. 4C). In the absence of competitor, photolabeling
of MRP was readily detectable in H69AR (Fig. 4C, lane 2) and T14 (Fig. 4C, lane 4),
but not in C6 membranes (Fig. 4C, lane 3), as
expected. The relative degree of labeling was consistent with the
relative level of MRP in these cells. Photolabeling was inhibited by
excess unlabeled LTC
(Fig. 4C, lane
5), but not LTB
(Fig. 4C, lane
6). Labeling was also inhibited by mAb QCRL-3 (Fig. 4C, lane 7) at a concentration which
abolishes [
H]LTC
transport (100 ng
µg protein
). MAb QCRL-1 did not inhibit
photolabeling (not shown), consistent with its inability to inhibit
LTC
transport. Finally, to exclude the possibility that
LTC
labeling of MRP was an artifact resulting from
nonspecific labeling of an abundant integral membrane protein, labeling
experiments were carried out with 8226/Dox40 vesicles known to contain
high levels of P-glycoprotein. As shown in Fig. 4C (lane 1), no labeling with
[
H]LTC
was observed.
Figure 5:
Effect of alkylated GSH derivatives on
[H]LTC
uptake in MRP-enriched
membrane vesicles. Panel A, [
H]LTC
uptake into T14 membrane vesicles was measured in the presence of
the indicated concentrations of GSH derivatives. Results are plotted as
a % of ATP-dependent [
H]LTC
uptake in
the absence of GSH derivative and each bar represent the mean (±
S.E.) of triplicate determinations in a typical experiment. The control
uptake rate in this experiment was 190 pmol mg
min
. Panel B, uptake of
[
H]LTC
was measured in the presence
of various concentrations of S-decyl-GSH (control,
; 50
nM,
; 100 nM,
; 250 nM,
).
Double-reciprocal plots were generated and an apparent K
of 116 nM was calculated from
the apparent K
and V
in the presence of S-decyl-GSH.
Figure 6:
Effect of chemotherapeutic agents and
LTB on [
H]LTC
uptake by
T14 vesicles. The ability of drugs and LTB
to inhibit
[
H]LTC
uptake was measured in the
absence or presence of GSH (1 mM) for 30 s at 23 °C.
Results were calculated as a % of control values obtained in the
absence of both drug and GSH. The bars represent the means
(±S.E.) of triplicate determinations in a single experiment and
similar results were obtained in at least one additional experiment. Panel A, the control uptake rate in this experiment was 185
pmol mg
min
. DOX,
doxorubicin; DNR, daunorubicin; CLC, colchicine; TXL, taxol. Panel B, the control uptake rate in this
experiment was 155 pmol mg
min
. VBL, vinblastine.
Figure 7:
Vincristine uptake in membrane vesicles
from MRP-transfected HeLa cells. Panel A, membrane vesicles
from HeLa C6 (,
) and T14 (
,
) cells were
incubated with 200 nM [
H]VCR in
transport buffer at 37 °C for the times indicated. Closed
symbols represent uptake in the presence of 4 mM AMP; open symbols represent uptake in the presence of 4 mM ATP. Panel B, T14 vesicles were incubated for 10 min in
200 nM [
H]VCR in buffer containing 4
mM AMP, ATP, or ATP and an ATP-regenerating system (ATP/RS) as
described under ``Experimental Procedures.'' Transport was
measured in the absence and presence of the indicated concentrations of
GSH and 2-mercaptoethanol (2-ME). Bars represent the
means (±S.E.) of triplicate determinations in a typical
experiment and similar results were found in three additional
experiments.
Both P-glycoprotein and MRP are capable of causing resistance to a similar spectrum of drugs when overexpressed in mammalian cells(1, 8) . Transfection of MRP or P-glycoprotein into drug-sensitive cells has been shown to result in reduced drug accumulation(8, 30) . In the case of P-glycoprotein, there is considerable experimental evidence the protein causes resistance by binding and transporting drugs out of the cell or plasma membrane in an ATP-dependent fashion(2, 9, 31) . The mechanism by which MRP mediates reduced drug accumulation in resistant cells is less well understood and in contrast to P-glycoprotein, there is no evidence that unmodified chemotherapeutic agents bind directly to, or are transported by, the protein(7, 8) .
In the present study, we
further characterized the transport properties of MRP using membrane
vesicles derived from both drug-selected MRP-overexpressing cells and a
population of transfected HeLa cells(8, 18) . We found
high-affinity rapid transport of LTC in T14 vesicles that
was osmotically sensitive, required hydrolyzable nucleotides, and was
supported only by those divalent cations that support the activity of
other membrane ATPases, thus providing evidence that ATP hydrolysis as
well as binding is required for transport. Transport rates were
somewhat lower in T14 vesicles than in vesicles from the drug-selected
H69AR cells, in keeping with the relative levels of MRP expression in
these two cell types(8) . The low but detectable transport in
revertant H69PR cells was also consistent with the low levels of MRP in
these cells(3, 19) . The absence of significant
LTC
transport in vesicles from
P-glycoprotein-overexpressing 8226/Dox40 cells confirmed the
MRP-specificity of this transport process. These observations confirm
and extend previous studies demonstrating that MRP-enriched vesicles
are capable of LTC
transport(10, 11, 12) . Binding of
LTC
to MRP was shown by immunoprecipitation of a 190-kDa
[
H]LTC
photoaffinity labeled T14
membrane protein with MRP-specific mAbs. MAb QCRL-3 which detects a
conformation-dependent epitope of MRP, also strongly inhibited
LTC
transport and prevented
[
H]LTC
labeling of MRP. Taken
together, these observations provide strong evidence that LTC
binds directly to MRP before being transported and further
suggest that LTC
binds to MRP at a site within or near the
epitope detected by mAb QCRL-3.
LTC is the highest
affinity substrate identified to date for MRP and while some
modifications of the glutathione or arachidonate moieties of the
molecule are tolerated, levels of transport are usually diminished by
these changes(10) . Our present studies demonstrate that
LTC
transport by MRP is effectively inhibited by alkylated
GSH derivatives. The inhibitory potency of alkylated GSH derivatives
with respect to LTC
transport in hepatocanalicular (28, 29) and sarcolemmal (27) membranes has
been shown to increase proportionately with the length of the alkyl
chain. We also found this to be the case in vesicles from MRP
transfectants. Indeed, LTC
transport by MRP was
significantly more sensitive to inhibition by these compounds than
reported for the transporters in rat muscle and
liver(27, 29) . Inhibition of MRP-mediated LTC
transport by the most potent GSH derivative, S-decyl-GSH, was competitive, suggesting that it binds to a
site in MRP that is similar or possibly overlapping the site to which
LTC
binds. The K
(116 nM) for S-decyl-GSH was similar to the apparent K
(105 nM) for LTC
transport in T14 membrane
vesicles, indicating that this GSH derivative is potentially a high
affinity substrate for MRP.
In contrast to cysteinyl leukotrienes
and alkylated GSH derivatives, we and others have found that
chemotherapeutic drugs are poor inhibitors of LTC transport, exerting significant inhibition only at concentrations
200-2000-fold greater than the K
of
LTC
(12, 32) . However, we observed that
inhibition of LTC
transport by certain drugs, most notably
the Vinca alkaloids VCR and vinblastine, could be
significantly enhanced by incubation with physiological concentrations
of GSH. Why this enhancement is more pronounced with these two drugs
than with others is presently unclear. It does not appear to be simply
related to the relative degree of MRP-mediated resistance to a
particular agent since T14 cells are considerably more resistant to
VCR, doxorubicin, and daunorubicin than they are to
vinblastine(8) . Nevertheless, the ability of both Vinca alkaloids to inhibit LTC
transport in T14 membrane
vesicles is similarly and markedly enhanced by GSH while inhibition by
doxorubicin and daunorubicin are unaffected. A similar potentiating
effect of GSH is observed for inhibition of 17
-estradiol
17-(
-D-glucuronide) transport by MRP (44) . This
effect appears specific for GSH since other thiols (2-mercaptoethanol, L-cysteine, and dithiothreitol) or other organic anions such
as glucuronic acid could not substitute for GSH.
We also found that
the presence of GSH resulted in demonstrable ATP-dependent
[H]VCR transport by MRP-enriched vesicles.
ATP-dependent uptake of [
H]VCR was approximately
31 pmol mg
at steady state in the presence of 200
nM VCR and 5 mM GSH. This is substantially lower than
steady state levels of LTC
uptake, GSSG
uptake(33) , and 17
-estradiol
17-(
-D-glucuronide) uptake (44) in MRP-enriched
vesicles but is comparable to that reported for vinblastine uptake in
vesicles from certain cell lines overexpressing
P-glycoprotein(6, 34) .
The mechanism by which GSH
enables ATP-dependent VCR transport by MRP, and possibly other
chemotherapeutic agents as well, is unclear. GSH has also been reported
to potentiate binding of the hydrophobic ligand MK 801 to the integral
membrane NMDA receptor (35) but whether the mechanism involved
is similar to that which potentiates VCR transport is unknown. It is
possible that MRP contains a bipartite binding site for hydrophobic and
anionic moieties that would allow binding of non-covalent drug-GSH
complexes, or the sequential binding of GSH and drug. Occupation of
both elements of the site may be necessary before transport can occur.
At present, there is no convincing evidence that GSH is actively
co-transported with drug. GSH by itself is not a substrate for
transport by MRP(33) , and neither does it inhibit LTC transport, even at 5 mM, the highest intracellular
concentration likely to be encountered in vivo. In contrast,
GSSG caused 50% inhibition of LTC
transport at
approximately 100 µM. Finally, it has been reported that
transport of daunorubicin by MRP does not increase GSH release by
intact cells(36, 37) . Thus it appears unlikely that
MRP transports drugs in association with reduced GSH. Alternatively,
interaction of GSH with MRP may cause a conformational change (37) or an alteration in the exposure of nonpolar residues,
that may favor binding of some hydrophobic compounds prior to
transport. Further studies are required to elucidate precisely how GSH
enhances VCR transport.
In addition to the cysteinyl leukotrienes
and vincristine in the presence of GSH, MRP can transport
17-estradiol 17-(
-D-glucuronide) and possibly
certain other cholestatic steroid glucuronides(44) . We have
determined that LTC
can inhibit 17
-estradiol
17-(
-D-glucuronide) transport and vice versa.
Consequently, the presence of a cysteinyl residue is not absolutely
required for a compound to be a substrate for MRP-mediated transport. A
number of organic anions and cyclic peptides which are neither GSH nor
glucuronide conjugates such as MK 571 (K
0.6
µM) (16, 38) and cyclosporin A (K
5 µM) (10) can behave as
competitive inhibitors of LTC
transport in membrane
vesicles from drug-selected cells known to overexpress MRP. Since
cyclosporin A is not an effective chemosensitizer in MRP-overexpressing
cells(30, 39, 40) , the ability of a compound
to inhibit LTC
transport is clearly not always indicative
of its capacity to reverse MRP-associated resistance nor its ability to
act as a substrate.
We and others have clearly shown that
MRP-overexpressing cells are not resistant to
cisplatin(8, 20, 30, 41, 42) .
The presence of ATP-dependent transport of a glutathione-platinum
complex in a platinum-resistant cell line has been demonstrated and it
was suggested that the transporter or ``GS-X pump'' in these
cells may be MRP(43) . Although this transport activity is
pharmacologically similar to MRP in some respects, in that it is
inhibitable by LTC, GSSG, and S-dinitrophenylglutathione, the overexpression of MRP mRNA and
protein in these cells has not been shown. Furthermore, we found that
cisplatin did not inhibit LTC
transport in T14 vesicles
either by itself or in combination with GSH (results not shown).
Finally, Muller and co-workers (12) were unable to demonstrate
LTC
transport in a platinum-resistant lung cancer cell line
which exhibits reduced drug accumulation. Thus current evidence
suggests that MRP is not involved in conferring resistance to
platinum-containing drugs although it remains possible that a
transporter related to MRP may be responsible, at least in some
platinum-resistant cell lines.
To date, there is little known about
the mechanism of ATP-dependent MRP-mediated transport of cysteinyl
leukotrienes, steroid glucuronides, or drugs in association with GSH.
However, it is possible that substrate binding by MRP (in the presence
of GSH in the case of chemotherapeutic drugs) facilitates the binding
and subsequent hydrolysis of ATP, which in turn induces a
conformational change in MRP that allows for the release of the
transported molecule(s) into the extracellular space. Experiments aimed
at determining whether the ATPase activity of MRP is stimulated by
LTC, 17
-estradiol 17-(
-D-glucuronide),
or chemotherapeutic agents in the absence or presence of GSH are
underway. Mapping of the mAb QCRL-3 epitope is also in progress and
together with proteolytic mapping studies of
[
H]LTC
-labeled MRP, should allow the
LTC
binding site on the MRP molecule to be identified.