From the Cancer Research Laboratories and Department of Pathology, Queen's University, Kingston, Ontario K7L 3N6, Canada
Received for publication, November 2, 2000, and in revised form, December 22, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Murine multidrug resistance protein 1 (mrp1),
unlike human MRP1, does not confer resistance to anthracyclines.
Previously, we have shown that a human/murine hybrid protein containing
amino acids 959-1187 of MRP1 can confer resistance to these drugs. We have now examined the functional characteristics of mutant proteins in
which we have converted individual amino acids in the comparable region
of mrp1 to those present at the respective locations in MRP1. These
mutations had no effect on the drug resistance profile conferred by
mrp1 with the exception of converting glutamine 1086 to glutamate, as
it is in the corresponding position (1089) in MRP1. This mutation
created a protein that conferred resistance to doxorubicin without
affecting vincristine resistance, or the ability of mrp1 to transport
leukotriene C4 (LTC4) and
17 Human multidrug resistance protein 1 (MRP1)1 is a member of the
ATP-binding cassette transporter superfamily that confers resistance to
a wide range of natural product drugs, including anthracyclines, Vinca alkaloids, and epipodophyllotoxins, as well as
methotrexate and certain heavy metal oxyanions (1-4). The predicted
structures of MRP1 and several of its related proteins differ from that
of a typical eukaryotic ATP-binding cassette transporter such as P-glycoprotein (P-gp). MRPs 1, 2, 3, and 6 contain an additional NH2-terminal membrane-spanning domain with an extracellular
NH2 terminus (5-9). Thus, MRP1 is predicted to contain
three membrane-spanning domains with 5+6+6 transmembrane (TM) helices
(6, 7) (Fig. 1). Based on comparisons of amino acid sequence and the
intron/exon organization of their respective genes, the MRPs and the
cystic fibrosis transmembrane conductance regulator appear to have
evolved from a common four-domain ancestor, the progenitor of the C
branch of the ATP-binding cassette superfamily (1, 10).
MRP1 and P-gp confer resistance to many of the same commonly used,
structurally diverse natural product chemotherapeutic agents (2, 4,
11-13). However, unlike P-gp, MRP1 is capable of transporting a wide
range of relatively hydrophilic organic glutathione, glucuronide and
sulfate conjugates (14-17). Considerable evidence indicates that MRP1,
in contrast to P-gp, requires GSH for the transport of some of its
hydrophobic substrates, such as vincristine, daunorubicin and aflatoxin
B1 and that in some cases, GSH may be co-transported with
these compounds (18-22).
Presently, very little is known of the regions of MRP1 that are
important for substrate binding and transport. Studies in P-gp have
demonstrated that drug-binding site(s) involve amino acids in TM
helices 1, 4, 5, 6, 8, 11, and 12, as well as some amino acids in
cytoplasmic regions of the protein (23-30). In an attempt to expedite
identification of amino acid residues important for the transport of
hydrophobic drugs and the more hydrophilic organic anion conjugates
that are established MRP1 substrates, we have taken advantage of well
characterized functional differences between the human and murine
orthologs of the protein (31-33). The murine ortholog, mrp1, and MRP1
are 87% identical, and both proteins confer resistance to
Vinca alkaloids and epipodophyllotoxins with apparently
similar efficiencies. However, mrp1 fails to confer resistance to any
anthracycline that has been tested, including doxorubicin,
daunorubicin, and epirubicin. In addition, mrp1 transports 17 Previously, we have demonstrated using hybrid human/murine proteins
that the COOH-terminal third of MRP1 contains important determinants of
the ability to confer anthracycline resistance and to transport
E217 Materials--
Culture medium and fetal bovine serum were
obtained from Life Technologies, Inc.
[3H]LTC4 (38 Ci/mmol) was purchased from
Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire,
England) and [3H]E217 Site-directed Mutagenesis and Generation of Expression
Vectors--
All mutations were generated using the TransformerTM
site-directed mutagenesis kit (CLONTECH, Palo Alto,
CA). Templates were prepared by cloning ~1.5-1.7-kilobase pair
restriction fragments of human MRP1 or murine mrp1 cDNAs into
pGEM-3Zf (Promega). Mutagenesis was then performed according to the
manufacturer's instructions using a selection primer 5'-GAG AGT GCA
CGA TAT CCG GTG TG-3' that mutates a unique NdeI site in the
vector to an EcoRV restriction site. Oligonucleotides
bearing mismatched bases at the residues to be mutated (underlined)
were synthesized by Cortec DNA Service Laboratories (Kingston, Canada).
They are as follows: mutation mrp1Q955K (5'-GGG CAG GTG AAG
CTG TCA GTG-3'), mutation mrp1N961D (5'-CA GTG TAC TGG GAC
TAC ATG AAG-3'), mutation mrp1L979M (5'-C TTC CTT TTC ATG
TGC AAC CAT G-3'), mutation mrp1L1022S (5'-GCC TTG GGC ATC
TCA CAA GGT GCA GC-3'), mutation mrp1F1040L (5'-C GGG GGC
ATC TTG GCC TCC CGT C-3'), mutation mrp1R1044C (5'-GCC TCC
CGT TGC TTG CAC CTG G-3'), mutation mrp1Y1051H (5'-G GAC
CTG CTA CAC AAT GTT CTT CG-3'), mutation mrp1N1052S (5'-C
CTG CTA TAC AGT GTT CTT CG-3'), mutation
mrp1Y1051H/N1052S (5'-G GAC CTG CTA CAC
AGT GTT CTT CG-3'), mutation mrp1S1097N (5'-GGT TCA CTC TTC
AAC GTC ATT GGA GC-3'), mutation mrp1V1202C (5'-C ATT GGA GCT TGC ATC ATC ATC C-3'), mutation mrp1Q1086E (5'-C TCC
ATG ATC CCG GAG GTC ATC-3'), mutation mrp1Q1086D (5'-C TCC
ATG ATC CCG GAT GTC ATC-3'), mutation
mrp1Q1086N (5'-C TCC ATG ATC CCG GAA GTC
ATC-3'), mutation MRP1E1089Q (5'-C TCC ATG ATC CCG CAG GTC ATC-3'), mutation MRP1E1089D (5'-C TCC ATG ATC CCG GAT GTC
ATC-3'), mutation MRP1E1089A (5'-C TCC ATG ATC CCG GCG GTC
ATC-3'), mutation MRP1E1089L (5'-C TCC ATG ATC CCG CTG GTC
ATC-3'), mutation MRP1E1089K (5'-C TCC ATG ATC CCG AAG GTC
ATC-3'). The double and triple mutants were also generated by this
method. The Q955K/N961D double mutation used the N961D mutagenic primer
with the cDNA containing Q955K mutation as a template. Similarly,
the L979M/F1040L double mutation used the L979M mutagenic primer with
cDNA containing F1040L mutation as a template. The
R1944C/Y1051H/N1052S triple mutation used the R1044C mutagenic primer
with the cDNA containing Y1051H/N1052S mutation as a template.
After confirming all mutations by DNA Thermo Sequenase Cy5.5 and Cy5.0
dye terminator cycle sequencing (Amersham Pharmacia Biotech) according
to the manufacturer's instructions, DNA fragments containing the
desired mutations were transferred into pCEBV7-mrp1 or pCEBV7-MRP1(4,
31-33). After reconstructing the mutations into the respective
full-length clones, the integrity of the entire mutated inserts and
cloning sites was verified by DNA sequencing.
Cell Lines and Tissue Culture--
Stable transfection of HEK293
cells with the pCEBV7 vector containing the wild type MRP1 cDNAs or
wild type mrp1 cDNAs has been described previously (32). All of the
mutated MRP1 or mrp1 constructs were analyzed as stably transfected
HEK293 cells grown in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum and 100 µg/ml hygromycin B (Roche
Molecular Biochemicals, Laval, Quebec). Briefly, HEK293 cells were
transfected with pCEBV7 vectors containing mutant MRP1 or mrp1
cDNAs using Fugene6 (Roche) according to the manufacturer's
instructions. After ~48 h, the transfected cells were supplemented
with fresh medium containing 100 µg/ml hygromycin B. Approximately 3 weeks after transfection, the hygromycin B-resistant cells were cloned
by limiting dilution and the resulting cell lines tested for high level
expression of the mutant proteins.
Determination of Protein Levels in Transfected Cells--
Plasma
membrane vesicles were prepared as described previously (15, 32). After
determination of protein levels by Bradford assay (Bio-Rad), 2 µg of
membrane protein were resolved by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (7.5% gel) and subsequently transferred to
Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford,
MA) by electroblotting. MRP1/mrp1 proteins were identified using the
monoclonal antibody, MRPr1, which cross-reacts with murine and human
mrp1/MRP1 protein (34). Antibody binding was detected with horseradish
peroxidase-conjugated goat anti-rat IgG (Pierce), followed by enhanced
chemiluminescence detection (PerkinElmer Life Sciences).
Confocal Microscopy--
Approximately 5 × 105
stably-transfected HEK293 cells were seeded in each well of a six-well
tissue culture dish on coverslips coated with 0.1% gelatin. When the
cells had grown to confluence, they were washed once in PBS for 10 min
and then fixed with 2% paraformaldehyde in PBS for 10 min. Cells were
permeabilized using digitonin (0.25 mg/ml in PBS) for 10 min, treated
with a blocking solution of 1% BSA in PBS for 10 min, and then
followed by 1-h incubation in blocking solution containing 10 µg/ml
RNase A and an antibody against MRP1. Antibodies used were a 1:5000
dilution of mAb QCRL-1, which recognizes amino acids 918-924 in the
cytoplasmic connector region of MRP1, or a 1:5000 dilution of mAb
MRPm6, which reacts with an epitope close to the COOH terminus of MRP1
(amino acids 1510-1520) (34, 35). After washing with blocking solution for 10 min, cells were incubated with Alexa Fluor 488 anti-mouse IgG
(H+L) (Fab')2 fragment for 1 h, washed with block
solution for 10 min, and then incubated in propidium iodide (2% in
H2O) for 5 min. Coverslips were mounted on the slides with
one drop of Antifade reagent (Molecular Probes, Eugene, OR).
Localization of MRP1 in the transfected cells was determined using a
Meridian Insight confocal microscope (filter, 620/40 nm for propidium
iodide and 530/30 nm for Fluor 488).
Chemosensitivity Testing--
Drug resistance was determined
using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay as described previously (4, 13, 32).
Briefly, cells were seeded at 5 × 103 cells/well in
100 µl of culture medium in 96-well tissue culture plate. The
following day, various concentrations of drug diluted in culture medium
were added to cells (100 µl/well). After incubation for an additional
72 h, 100 µl of medium was removed from each well and the MTT
reagent (25 µl/well, 2 mg/ml) (Sigma) was added. After 3 h, the
formazan was solubilized by mixing with HCl/isopropanol (1:24) (100 µl/well). Color density was determined using the ELX 800 UV
spectrophotometer (Filter4, 570 nm). Mean values of quadruplicate determinations (± S.D.) were plotted using GraphPad software. IC50 values were obtained from the best fit of the data to
a sigmoidal curve. Relative resistance is expressed as the ratio of the
IC50 value of cells transfected with MRP1 and mrp1
expression vectors compared with cells transfected with empty vector.
Resistance was determined in three or more independent experiments.
LTC4, E217 Expression of Mutant mrp1 and MRP1 in Stably Transfected HEK293
Cells--
Previous studies with hybrid human and murine proteins
indicated that sequence differences between mrp1 and MRP1 in the region between amino acids 955 and 1184 of the murine protein contributed to
its inability to confer resistance to anthracyclines (33). This region
includes predicted TM helices 12-15 and cytoplasmic loop (CL) 7 (Fig.
1). Within the predicted
The episomal expression vector, pCEBV7, containing mutated forms of
full-length mrp1 and MRP1 cDNAs was used to stably transfect HEK293
cells. From the initial populations of transfectants, we isolated
subpopulations by limiting cell dilution that, based on immunoblotting,
expressed levels of each mutant protein were approximately equivalent
to the levels of wild type mrp1 and MRP1 in previously characterized
HEK transfectants. The only exceptions were HEK 293 subpopulations
expressing mrp1Q1086N. This mutant protein reached levels that were
slightly higher than those in the other subpopulations used. The levels
of mrp1 and MRP1 mutant proteins used in studies described below,
relative to the respective wild type protein, were determined by
immunoblotting with mAb MRPr1, which reacts with both murine and human
MRP1(34), followed by densitometry (Fig.
2). Endogenous MRP1 in HEK293 cells
transfected with the empty vector was undetectable under the conditions
used (data not shown).
Resistance Profiles of Wild Type Murine mrp1 and Proteins Mutated
in the Region between Amino Acids 955 and 1184--
The relative
resistance of transfectants expressing each of the mrp1 mutant proteins
was determined by MTT assays following exposure to various
concentrations of vincristine and doxorubicin. The results are
summarized as IC50 values and as relative resistance factors (Table I). None of the mutations significantly affected the
level of resistance conferred to vincristine. The only mutation that
affected doxorubicin resistance was the conversion of glutamine 1086 to
glutamate (Q1086E), as it is at the corresponding position, 1089, in
MRP1. The introduction of glutamate resulted in a relative resistance
to doxorubicin of 4-5-fold, as did mutation of this residue to
aspartate (Q1086D). In contrast, a conservative mutation to asparagine
(Q1086N) had no effect (Table I). Typical survival curves for
transfectants expressing wild type mrp1 and the mutant proteins Q1086E,
Q1086D, and Q1086N are shown in Fig. 3.
These results clearly indicate the importance of the residue at this position in influencing substrate specificity and suggest that a
negatively charged side chain may be an important prerequisite for
conferring resistance to doxorubicin.
Mutational Analysis of Glutamate 1089 (Glu-1089) in Predicted TM14
of Human MRP1--
Based on the findings with mrp1 Q1086E and Q1086D
mutations, we investigated how critical glutamate 1089 was for the
ability of the human MRP1 to confer resistance to anthracyclines, and whether or not mutations of this residue also affected resistance to
Vinca alkaloids and epipodophyllotoxins. Thus, glutamate
1089 was mutated to aspartate, glutamine, alanine, leucine, and lysine and chemosensitivity assays were carried out.
Cells expressing MRP1E1089D had a resistance profile indistinguishable
from transfectants expressing the wild type protein (Table
II). In contrast, conversion of glutamate
1089 to glutamine, as it is in the murine protein, essentially
eliminated the ability of MRP1 to confer resistance to doxorubicin,
daunorubicin, and epirubicin, as did mutations MRP1E1089A, MRP1E1089L,
and MRP1E1089K (Table II). In addition, mutation of glutamate 1089 to
glutamine, alanine, and leucine decreased the relative resistance to
vincristine by 55-65%, while mutation to lysine essentially
eliminated resistance to this drug. The three mutations that introduced
amino acids with uncharged side chains had less effect on VP-16
resistance, decreasing the resistance to this drug by 30-40% (Table
II). The HEK293MRP1E1089K transfectants also retained some
resistance to VP-16, with a relative resistance factor of 6.5 compared
with 15.6 for transfectants expressing wild type protein. Typical
survival curves for transfectants expressing wild type MRP1 and the
mutant proteins E1089Q, E1089D, and E1089K are shown in Fig.
4. These data confirm the essential role
played by glutamate 1089 in MRP1 with respect to the ability of the
protein to confer resistance to anthracyclines and also indicate that
elimination of a negatively charged side chain at this location reduces
the efficiency with which the protein confers resistance to other
classes of natural product drugs.
Confocal Microscopy of MRP1-, MRP1E1089Q-, and
MRP1E1089K-transfected HEK293 Cells--
To determine whether the
effects of mutations in TM14 on drug resistance profiles might be
attributable in part to changes in trafficking of MRP1, we examined the
subcellular localization of MRP1E1089Q and MRP1E1089K, as well as wild
type MRP1 by confocal microscopy. Mutant and wild type MRP1 was
detected using mAbs against two different regions of the protein: mAb
QCRL-1 and mAb MRPm6, which recognize defined linear epitopes in the
linker region and near the COOH terminus of the protein, respectively
(35). As shown in Fig. 5, cells
expressing the mutant proteins showed a pattern of strong plasma
membrane staining comparable with that of cells expressing wild type
MRP1, indicating that the trafficking of both mutant proteins was
unaffected.
Transport of [3H]LTC4 and
[3H]E217
In view of the effect of mutations of glutamate 1089 in the human
protein on resistance to vincristine and VP-16 in addition to the
anthracyclines, we also examined LTC4 and
E217 Kinetic Parameters of [3H]LTC4 and
[3H]E217
The kinetics of ATP-dependent LTC4 and
E217 Transport of [3H]Estrone-3-sulfate by Wild Type and
Mutant Human Proteins--
Substitution of glutamate 1089 in human
MRP1 with glutamine dramatically decreased the ability of the protein
to confer anthracycline resistance and reduced resistance to
vincristine by ~60%. However, mutation of glutamate 1089 to lysine
completely eliminated resistance to both anthracyclines and vincristine
(Table II). One major distinction between MRP1-mediated transport of
substrates such as LTC4 and E217 Human MRP1 and murine mrp1 confer resistance to Vinca
alkaloids and epipodophyllotoxins with approximately the same
efficiency. However, despite the fact that the orthologs are relatively
highly conserved (87% identity overall), only the human protein
confers resistance to anthracyclines (31, 32). In addition, although mrp1 and MRP1 transport LTC4 with similar kinetic
parameters, they differ markedly in their ability to transport
E217 Site-directed mutagenesis studies of the P-gps have identified single
amino acid residues at various locations in the protein that influence
substrate specificity but similar information about the MRP family is
only just beginning to emerge (37). Most of the residues identified in
P-gp are located in predicted TM helices, predominantly TMs 1, 6, 11, and 12, although single proline residues in TMs 4 and 10 have also been
shown to influence substrate specificity (23). The region of MRP1/mrp1
we have analyzed includes TM helices 12-15, which because of the five
additional NH2-proximal TM helices corresponds
topologically to TM helices 7-10 of P-gp, as well as predicted CL7 of
MRP1/mrp1 corresponding to CL4 of P-gp. Although most residues
implicated to date in determining the substrate specificity of P-gp are
located in TM helices, CL4 is a cytoplasmic region where mutation of
specific glycine residues to valine has been shown to increase
resistance to colchicine and doxorubicin (26).
Four non-conservative differences between MRP1 and mrp1 are present in
CL7. However, in contrast to the results obtained from mutagenesis
studies of CL4 of P-gp, replacement of the variant residues in mrp1
with those present in MRP1 had no detectable effect on its drug
resistance profile. Furthermore, single mutations in TM helices 12-15
of the mouse protein that converted non-conservative differences
between mrp1 and MRP1 to the human sequence also had no effect, with
the exception of glutamine 1086. Conversion of this residue to
glutamate, as it is in MRP1, increased the ability of mrp1 to confer
resistance to anthracyclines to a level approximately equivalent to
that conferred by the original hybrid containing amino acids 959-1187
of the human protein. This result combined with the lack of effect of
mutating other variant amino acids strongly suggests that, within this
region, glutamate 1089 is critical for the ability of the hybrid
protein to confer anthracycline resistance. Consistent with previous
results obtained using mrp1/MRP1 hybrids, the mutation was selective
with respect to anthracycline resistance and had no effect on the
ability of mrp1 to confer resistance to vincristine and VP-16, or to
transport LTC4 and E217 The results obtained with mrp1 mutants implicated glutamate 1089 of the
human protein in the ability to confer anthracycline resistance.
However, the levels of resistance conferred by both the mrp1/MRP1
959-1187 hybrid and the mrp1Q1086E mutant protein were ~60% that of
the wild type protein (33). The fact that the hybrid and mutant mrp1
were less effective than the wild type human protein left open the
possibility that glutamate 1089 may contribute to, but not be essential
for, resistance to this class of drugs. However, the results obtained
with MRP1 mutants confirmed that glutamate 1089 was essential for
anthracycline resistance. They also revealed that mutation of this
residue, in contrast to the comparable mrp1 mutations, also affected
resistance to other drugs. Mutation of glutamate 1089 to glutamine
essentially eliminated anthracycline resistance, confirming the crucial
role of this residue. However, unlike the reciprocal mutation in the murine protein, which had no effect on resistance to other types of
drugs, the MRP1E1089Q mutation also decreased resistance to vincristine
and VP-16, although to a lesser extent than the anthracyclines. This
was observed to a similar degree with all of the mutations that
eliminated a negative charge in the amino acid side chain. The
difference in the effect of the mrp1Q1086E and MRP1E1089Q mutations
raises the interesting possibility that the introduction of glutamate
1089 in MRP1 may have been a critical event in acquisition of the
ability to confer resistance to anthracyclines, and that the protein
has evolved so that this residue now has a more general role in drug
binding and/or transport. Whether or not this is the case should become
apparent as the drug resistance profiles and sequences of MRP1
orthologs in other species become available.
Studies of P-gp mutants of histidine 61 in predicted TM1 suggested that
the size of the side chain of the residue influenced substrate
specificity. It was observed that substitution by an amino acid with a
small side chain increased relative resistance to vinblastine while
introduction of a large side chain increased resistance to smaller
substrates including colchicine, VP-16 and doxorubicin (28). We
observed no differences between the mutant proteins, in which various
neutral amino acids had been substituted for glutamate 1089, suggesting
that a negative charge in the side chain was the predominant feature
influencing the drug resistance profile of the wild type protein. This
was supported by the results obtained by substitution of glutamate 1089 with aspartate, which resulted in no detectable change in resistance
profile, and by conversion to lysine, which essentially eliminated
resistance to both anthracyclines and vincristine, and substantially
decreased resistance to VP-16. Thus, the drug resistance profile of
MRP1 appears primarily dependent on the charge rather than size or hydrophobicity of the residue at position 1089. The relatively pronounced effect that the introduction of a positive charge at this
location has on resistance to anthracyclines and vincristine may be
attributable to the fact that the former are strongly and the latter is
weakly cationic at physiological pH while VP-16 is uncharged.
The effect of the MRP1Q1089K mutation on the ability of the protein to
confer resistance to all of the drugs tested suggested either that the
overall transport activity of the protein was diminished or that the
mutation may have adversely affected trafficking causing retention of
the protein in intracellular membranes. This mutation results not only
in a net gain of two positive charges in predicted TM helix 14, but
also in the introduction of adjacent lysine residues (Fig. 1), creating
the possibility that charge repulsion might contribute to an alteration
in the conformation of this region of the protein. However, confocal
microscopy revealed no evidence of a trafficking defect that might be
indicative of misfolding of the protein. In addition, the protein
retained full activity with respect to the transport of
LTC4 and E217 Several previous studies have demonstrated that, under appropriate
conditions, drugs such as vincristine and possibly doxorubicin act as
competitive inhibitors of LTC4 transport, suggesting that they bind to common or mutually exclusive sites on the protein. In some
cases, the presence of GSH has been shown to markedly increase the
inhibitory potency of these drugs (15, 16, 20). The results of
experiments described here together with earlier competition studies,
indicate that at least some of the residues within these sites that are
critical for the binding of unmodified drugs differ from those required
for binding and transport of conjugates such as LTC4 and
E217-estradiol 17-(
-D-glucuronide)
(E217
G). Furthermore, mutation Q1086D conferred the same
phenotype as mutation Q1086E while the mutation Q1086N did not
detectably alter the drug resistance profile of mrp1, suggesting that
an anionic side chain was required for anthracycline resistance. To
confirm the importance of MRP1 E1089 for conferring resistance to
anthracyclines, we mutated this residue to Gln, Asp, Ala, Leu, and Lys
in the human protein. The mutation E1089D showed the same phenotype as
MRP1, while the E1089Q substitution markedly decreased resistance to
anthracyclines without affecting LTC4 and
E217
G transport. Conversion of Glu-1089 to Asn, Ala, or
Leu had a similar effect on resistance to anthracyclines, while conversion to a positive amino acid, Lys, completely eliminated resistance to anthracyclines and vincristine without affecting transport of LTC4, E217
G, and the
GSH-dependent substrate, estrone-3-sulfate. These results
demonstrate that an acidic amino acid residue at position 1089 in
predicted TM14 of MRP1 is critical for the ability of the protein to
confer drug resistance particularly to the anthracyclines, but is not
essential for its ability to transport conjugated organic anions
such as LTC4 and E217
G.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-estradiol 17-(
-D-glucuronide)
(E217
G) far less efficiently than MRP1, despite the fact
that both proteins transport leukotriene C4
(LTC4) with similar kinetic parameters (31, 32).
G. These studies also identified the region from
amino acid 959 to 1187 of MRP1 as being particularly important for
anthracycline resistance (33). In the present study, we have used this
information to guide the design of point mutations in mrp1 in which
variant amino acids in the region between residues 955 and 1184 have
been replaced with the corresponding amino acid from MRP1 and vice
versa. These mutant human and murine proteins were then stably
expressed in human embryonic kidney (HEK293) cells and the
transfectants characterized with respect to their drug resistance
profiles and their ability to transport LTC4, E217
G, and, in the case of the human protein,
estrone-3-sulfate. The results of these studies identify glutamate 1089 in MRP1 as being critical for the ability to confer resistance to
anthracyclines. In contrast, mutations at this location had no
detectable effect on the ability to transport either LTC4
or E217
G and no effect on the GSH-stimulated transport
of estrone-3-sulfate.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G (44 Ci/mmol) and
[6,7-3H]estrone-3-sulfate (53 Ci/mmol) from PerkinElmer
Life Sciences. Doxorubicin HCl, etoposide (VP-16), and
vincristine sulfate were obtained from Sigma, and epirubicin from ICN
Biomedicals (Irvine, CA).
G, and Estrone-3-sulfate
Transport by Membrane Vesicles--
Plasma membrane vesicles were
prepared as described previously, and ATP-dependent
transport of [3H]LTC4 into the inside-out
membrane vesicles was measured by a rapid filtration technique (15, 16,
33). Briefly, vesicles (2.5 µg of protein) were incubated at 23 °C
in 25 µl of transport buffer (50 mM Tris-HCl, 250 mM sucrose, 0.02% sodium azide, pH 7.4) containing 4 mM ATP, 10 mM MgCl2, and
[3H]LTC4 (50 nM, 25 nCi). At 1 min, 20-µl aliquots were removed and added to 1 ml of ice-cold
transport buffer, followed by filtration under vacuum through glass
fiber filters (type A/E, Gelman Sciences, Dorval, Quebec, Canada).
Filters were immediately washed twice with 5 ml of cold transport
buffer and then dried before the bound radioactivity was determined by
scintillation counting. All data were corrected for the amount of
[3H]LTC4 that remained bound to the filter in
the absence of vesicle protein (usually <5% of the total
radioactivity). [3H]LTC4 uptake was expressed
relative to the protein concentration of the membrane vesicles.
Km and Vmax values were
determined by measuring ATP-dependent uptake at various
concentrations of LTC4, under identical conditions,
followed by non-linear regression analyses. ATP-dependent
uptake of [3H]E217
G (400 nM,
30 nCi) was measured as described for
[3H]LTC4 except that 5 µg of vesicle
protein was used and the reaction was carried out at 37 °C for 2 min
for preliminary analyses and with various concentrations of
[3H]E217
G for 1 min to determine kinetic
parameters. ATP-dependent uptake of
[3H]estrone-3-sulfate (75 nM-16
µM) was measured as described for [3H]LTC4 except that the reaction was carried
out at 37 °C for 1 min in the presence or absence of 1 mM GSH.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices,
there are 12 amino acid differences between the human and murine
proteins and, of these, 5 are non-conservative. Each of the
non-conserved amino acids in mrp1 was replaced individually with the
corresponding residue from MRP1. An additional seven sequence
differences are present in predicted cytoplasmic residues in this
region. Of the seven, five are non-conservative and each of these was
also replaced with the corresponding amino acid from the human protein.
In addition to the single substitutions, selected double and triple
mutations were made as indicated in Table
I.
View larger version (59K):
[in a new window]
Fig. 1.
Topology of human/murine MRP1. The
figure shows the predicted topology of human and murine MRP1/mrp1 with
17 TM helices. The region spanning amino acids 959/954 to 1187/1184 and
encompassing TM helices 12-15 and CL7 is indicated by a
lighter shading and an expanded view of this
region is illustrated in the bottom half of the
figure. The amino acids shown in the expanded view are those found in
murine mrp1. Residues in open circles are
identical in the murine and human proteins. Residues that are different
between the two proteins are indicated by shaded
circles, and the amino acids present in human MRP1 at the
equivalent positions are shown to one side. Proline at position 1000 in
the extracellular loop of murine mrp1 is deleted in human MRP1.
Relative drug resistance of HEK293 cells transfected with wild type and
mutant murine mrp1
View larger version (87K):
[in a new window]
Fig. 2.
Immunoblot showing the expression
levels of the mutant murine and human MRP1 proteins. Membrane
proteins (2 µg) prepared from each cell line were separated by
SDS-polyacrylamide gel and transferred to polyvinylidene difluoride
membrane as described under "Experimental Procedures." Blots were
probed with mAb MRPr1, which reacts with both the mouse and human
proteins (34). For comparison, membrane proteins from cells expressing
the wild type mrp1/MRP1 are also shown. Under the experimental
conditions used, no endogenous MRP1 was detectable in control
HEK293PC7 transfectants (data not shown).
View larger version (24K):
[in a new window]
Fig. 3.
Resistance of stably transfected HEK293
cells to vincristine (A and B) and
doxorubicin (C and D). The
relative resistance of cells expressing wild type murine mrp1
(HEK293mrp1, ), mutant proteins
(HEK293mrp1Q1086E,
; EK293mrp1Q1086D,
;
HEK293mrp1Q1086N,
), and cells transfected with the
empty expression vector (HEK293pC7,
) was determined as
described under "Experimental Procedures."
Relative drug resistance of HEK293 cells transfected with wild type and
mutant MRP1
View larger version (30K):
[in a new window]
Fig. 4.
Resistance of stably transfected
HEK293 cells to vincristine (A and
B), doxorubicin (C and
D), and VP-16 (E and
F). The relative resistance of cells expressing
wild type human MRP1 (HEK293MRP1, ), mutant proteins
(HEK293MRP1E1089Q,
; HEK293MRP1E1089D,
;
HEK293MRP1E1089K,
), and cells transfected with the
empty expression vector (HEK293pC7,
) was determined as
described under "Experimental Procedures."
View larger version (91K):
[in a new window]
Fig. 5.
Confocal microscopy of cells expressing
wild type (HEK293MRP1) and mutant
(HEK293MRP1E1089Q; HEK293MRP1E1089K) human MRP1
proteins. Cells were grown and stained for immunofluorescence
detection of MRP1 as described under "Experimental Procedures."
MRP1 was detected using both mAb QCRL-1 (left), which
recognizes amino acids 918-924 in the cytoplasmic connector region of
MRP1, or mAb MRPm6 (right), which reacts with a cytoplasmic
COOH-terminal fragment of MRP1 (amino acids 1510-1520) (34, 35).
Location of MRP1 is indicated in green. Nuclei were stained
with propidium iodide and are shown in red.
G by Wild Type and Mutant
Proteins--
In addition to differences in the drug resistance
profiles conferred by mrp1 and MRP1, we have shown previously that the
two proteins differ with respect to their ability to transport some potential physiological substrates. For example, mrp1 and MRP1 transport LTC4 with similar efficiency, but mrp1 is a much
less efficient transporter of E217
G when compared with
the human protein (32). As in the case of resistance to anthracyclines,
studies with hybrid proteins implicated sequence differences in the
COOH-terminal one-third of MRP1 and mrp1 as being primarily responsible
for differences in the efficiency with which the two proteins transport this substrate (33). To determine whether any of the mutations of mrp1
that affected the ability to confer resistance to anthracyclines altered the efficiency with which the protein transported either LTC4 or E217
G, we examined
ATP-dependent uptake of these compounds by membrane
vesicles prepared from HEK transfectants expressing mrp1Q1086E,
mrp1Q1086D, and mrp1Q1086N. Despite the effect of the mrp1Q1086E and
mrp1Q1086D mutations on doxorubicin resistance, none of the mutations
appeared to affect transport of either LTC4 or
E217
G (Fig. 6,
A and C).
View larger version (64K):
[in a new window]
Fig. 6.
ATP-dependent
[3H]LTC4 (A and
B) and
[3H]E217 G
(C and D) uptake by membrane vesicles
prepared from HEK293 cells stably transfected with wild type/mutant
murine mrp1 (A and C) and wild
type/mutant human MRP1 (B and
D). For LTC4 transport, membrane
vesicles (2.5 µg of membrane protein) were incubated at 23 °C with
50 nM [3H]LTC4 for 1 min in
transport buffer. For transport of E217
G, membrane
vesicles (5 µg of membrane protein) were incubated at 37 °C with
400 nM [3H]E217
G for 2 min.
Transfectants tested were expressing wild type or mutant murine/human
mrp1/MRP1 proteins as indicated in the figure.
G transport by wild type and mutant human MRP1,
including MRP1, E1089Q, E1089D, and E1089K. However, none of these
mutations had any detectable influence on transport including the
E1089K mutation that significantly decreased resistance to all drugs
tested (Fig. 6, B and D).
G Transport--
Initial
transport studies revealed no effect of mutations of glutamine 1086 in
murine mrp1 or glutamate 1089 in human MRP1 on transport of either
LTC4 or E217
G. However, to determine more thoroughly whether these mutations may have altered the affinity of
mrp1 or MRP1 for these substrates, we also measured the
Km and Vmax for the wild type
and mutant proteins for both LTC4 and E217
G.
The rate of uptake by membrane vesicles was determined at a number of
substrate concentrations and a non-linear regression analysis of the
combined data was used to determine a Km and
Vmax for each protein (Fig.
7). For mrp1 and mutant mrp1Q1086E, which
were expressed at comparable levels, the Km and Vmax for LTC4 transport were
virtually identical (Km = 148 nM,
Vmax = 206 pmol mg
1
min
1 and Km = 152 nM, Vmax = 194 pmol
mg
1 min
1,
respectively) (Fig. 7A). The Km and
Vmax for E217
G uptake were also
similar for both wild type and mutant protein (mrp1,
Km = 2.4 µM,
Vmax = 43 pmol mg
1
min
1; and mrp1Q1086E, Km = 1.9 µM, Vmax = 33 pmol
mg
1 min
1) (Fig.
7B). Thus, in murine mrp1, converting glutamine 1086 to glutamate had no significant effect on the ability of the protein to
transport either LTC4 or E217
G.
View larger version (27K):
[in a new window]
Fig. 7.
Kinetics of ATP-dependent
[3H]LTC4 (A and
C),
[3H]E217 G
(B and D), and
[3H]estrone-3-sulfate (E and
F) uptake by wild type and mutant proteins. The
relative expression levels of wild type and mutant MRP1 proteins in the
membrane vesicles used for examining kinetic parameters are shown in
panel G. The proteins were determined by
immunoblotting with mAb MRPr1 as described in the legend to Fig. 2. The
numbers below the blot refer to the
relative levels of MRP1 proteins. The initial rate of
ATP-dependent [3H]LTC4
(A and C) and
[3H]E217
G (B and D)
uptake by membrane vesicles prepared from HEK293 cells transfected with
wild type or mutant murine/human proteins was measured at various
LTC4 concentrations (0.01-2 µM) for 1 min at
23 °C and at various E217
G concentrations (0.2-16
µM) for 1 min at 37 °C in transport buffer, as
described under "Experimental Procedures." The initial rate of
ATP-dependent [3H]estrone-3-sulfate uptake
was also examined at various estrone-3-sulfate concentrations
(0.075-16 µM) for 1 min at 37 °C in transport buffer
in the presence or absence of 1 mM GSH (E and
F). Data were plotted as V0
versus [S] to confirm that concentration range selected
was appropriate to observe both zero-order and first-order rate
kinetics. For LTC4 and E217
G transport, the
transfectants tested were HEKmrp1 (
),
HEKmrp1Q1086E (
) (panels A and
B), HEKMRP1 (
), and HEKMRP1E1089Q
(
) (panels C and D). For
estrone-3-sulfate transport, the transfectants tested were
HEKMRP1 (
,
), HEKMRP1E1089Q (
,
),
and HEKMRP1E1089K (
,
) (panels
E and F). Closed symbols
represent uptake in the presence of 1 mM GSH;
open symbols represent uptake in the absence of
GSH. Kinetic parameters were determined from non-linear regression
analysis of the combined data. Details of Km and
Vmax values for all three substrates are
provided under "Results."
G transport were also examined for the wild type and
mutant human proteins (Fig. 7, C and D). In these
experiments, the levels of wild type MRP1 determined by immunoblotting
and densitometry were approximately twice as high as that of the mutant
MRP1E1089Q (Fig. 7G). The Km values for
LTC4 transport obtained with vesicles containing wild type
MRP1 or mutant MRP1E1089Q were similar, 116 and 108 nM,
respectively. The Vmax values were 226 pmol
mg
1 min
1 for the
wild type protein and 128 pmol mg
1
min
1 for mutant MRP1E1089Q, which when
normalized to the level of wild type protein yielded a value of 256 pmol mg
1 min
1. For
E217
G transport, Km values determined
for wild type and mutant MRP1 were also similar, 501 and 600 nM, respectively. The Vmax values
for MRP1 or MRP1E1089Q were 337 and 176 pmol
mg
1 min
1,
respectively, and when normalized for differences in expression levels,
the Vmax values for the wild type and mutant
proteins were virtually identical (337 and 352 pmol
mg
1 min
1,
respectively). Thus, as obtained with mrp1, mutation of glutamate 1089 in the human protein did not affect transport of LTC4 and E217
G.
G, and drugs
such as vincristine and daunorubicin, is a requirement for GSH, which
may be co-transported with the unmodified drug (19-21). Recently, we
have shown that GSH can also enhance the transport of some anionic
conjugates such as estrogen-3-sulfates (36). Since transport of these
compounds is more amenable to kinetic analysis than hydrophobic drugs
such as vincristine and the anthracyclines, we used
[3H]estrone-3-sulfate to investigate whether the
MRP1E1089K mutation had affected the transport of GSH dependent
substrates. In the membrane vesicles used, immunoblotting and
densitometry indicated that the levels of mutant protein were ~70%
that of wild type MRP1. The kinetic parameters of estrone-3-sulfate
transport for membrane vesicles prepared from cells transfected with
the wild type and mutant MRP1 were then determined in the presence and absence of GSH (Fig. 7, E and F). In the presence
of GSH, a Km value of 1.1 µM was
obtained for MRP1, which was essentially identical to that determined
for mutant MRP1E1089Q or MRP1E1089K (1.0 and 1.1 µM,
respectively). The Km values for MRP1, MRP1E1089Q, and MRP1E1089K were also similar in the absence of GSH (3.5, 3.5, and
3.9 µM, respectively). The Vmax
values for transport by membrane vesicles from wild type MRP1 or
mutations MRP1E1089Q, and MRP1E1089K were 403, 191, and 289 pmol
mg
1 min
1,
respectively, in the presence of GSH and 185, 110, and 150 pmol mg
1 min
1,
respectively, in its absence. When normalized for differences in
expression levels, the Vmax values for wild type
MRP1 or mutations MRP1E1089Q and MRP1E1089K were similar (403, 382, and
413 pmol mg
1 min
1,
respectively, in the presence of GSH; 185, 222, and 214 pmol mg
1 min
1,
respectively, in the absence of GSH). These findings demonstrated that,
in the presence or absence of GSH, mutations MRP1E1089Q and MRP1E1089K
behaved in a manner very similar to that of the wild type protein.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G (32). Previously, we have taken advantage of these
differences to map regions of the human protein that contribute to its
ability to transport E217
G much more efficiently than
mrp1 and to confer resistance to anthracyclines. Analysis of the drug
resistance profiles conferred by hybrid proteins and their in
vitro transport characteristics localized residues important for
both anthracycline resistance and transport of E217
G to
the COOH-terminal third of MRP1. More detailed analyses revealed that
regions between amino acids 959-1187 and 1188-1531 both contributed
approximately equally to the ability of the human protein to transport
E217
G (33). However, the hybrid containing
MRP1-(959-1187) was approximately twice as effective as the hybrid
containing the region MRP1-(1188-1531) at conferring anthracycline
resistance (33). We have now examined the consequences of converting
all non-conservative differences in mrp1 in the region between amino
acids 955 and 1184 to the corresponding amino acid present in human
MRP1 to begin identification of specific residues involved in
conferring resistance to anthracyclines and to determine whether the
same residues also enhance the transport of E217
G.
G.
G, confirming that the effect
of the mutation was specific for the chemotherapeutic drugs tested and
did not alter the transport efficiency of these two conjugated organic
anions. The mechanistic explanation for this specificity is presently
not known. One major distinction between MRP1 mediated transport of
LTC4 and E217
G, and drugs such as
vincristine and daunorubicin, is a requirement for GSH, which may be
co-transported with the unmodified drug (19-21). We have recently
shown that GSH can also markedly enhance the efficiency with which some
conjugated organic anions such as estrone-3-sulfate are transported.
Using this substrate, it has been possible to demonstrate that GSH both
decreases the Km and increases the
Vmax for transport of the conjugated estrogen
(36). This provided the opportunity to determine whether mutations that
specifically affected drug resistance were influencing the GSH
dependence of the transport process. However, comparison of the GSH
dependence of estrone-3-sulfate transport by the both the
MRP1E1089Q, which eliminated anthracycline resistance and reduced
vincristine resistance, and the MRP1E1089K mutation, which eliminated
resistance to both anthracyclines and vincristine, revealed no kinetic
differences from the wild type protein. Thus, the effects of the
mutations at position 1089 in human MRP1 appear to be due primarily to
the structure of the substrate.
G. Very recently we have also identified a single
amino acid, mutation of which eliminates the ability of MRP1 to confer
drug resistance and to transport E217
G but leaves
LTC+ transport intact.2
Knowledge of these sites may assist in
the design of MRP1 reversing agents that spare at least some of the
physiological functions of the protein while abrogating its ability to
confer drug resistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from the National Cancer Institute of Canada with funds from the Terry Fox Run.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 in part by a Queen's University graduate fellowship.
§ Senior Scientist of Cancer Care Ontario.
¶ Stauffer Research Professor of Queen's University. To whom correspondence should be addressed: Cancer Research Laboratories, Botterell Hall, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-2979; Fax: 613-533-6830; E-mail: deeleyr@post.queensu.ca.
Published, JBC Papers in Press, January 23, 2001, DOI 10.1074/jbc.M010008200
2 Ito, K., Olsen, S. L., Qiu, W., Deeley, R. G., and Cole, S. P. C. (2001) J. Biol. Chem., in press.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MRP, multidrug
resistance protein;
P-gp, P-glycoprotein;
TM, transmembrane;
mAb, monoclonal antibody;
E217G, 17
-estradiol
17-(
-D-glucuronide);
LTC4, leukotriene
C4;
MTT, 3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
HEK, human
embryonic kidney;
CL, cytoplasmic loop.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Deeley, R. G., and Cole, S. P. C. (1997) Semin. Cancer Biol. 8, 193-204[CrossRef][Medline] [Order article via Infotrieve] |
2. | Hipfner, D. R., Deeley, R. G., and Cole, S. P. C. (1999) Biochim. Biophys. Acta 1461, 359-376[Medline] [Order article via Infotrieve] |
3. | Cole, S. P. C., Bhardwaj, G., Gerlach, J. H., Mackie, J. E., Grant, C. E., Almquist, K. C., Stewart, A. J., Kurz, E. U., Duncan, A. M., and Deeley, R. G. (1992) Science 258, 1650-1654[Medline] [Order article via Infotrieve] |
4. | Cole, S. P. C., Sparks, K. E., Fraser, K., Loe, D. W., Grant, C. E., Wilson, G. M., and Deeley, R. G. (1994) Cancer Res. 54, 5902-5910[Abstract] |
5. | Higgins, C. F., Callaghan, R., Linton, K. J., Rosenberg, M. F., and Ford, R. C. (1997) Semin. Cancer Biol. 8, 135-142[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Hipfner, D. R.,
Almquist, K. C.,
Leslie, E. M.,
Gerlach, J. H.,
Grant, C. E.,
Deeley, R. G.,
and Cole, S. P. C.
(1997)
J. Biol. Chem.
272,
23623-23630 |
7. | Borst, P., Evers, R., Kool, M., and Wijnholds, J. (1999) Biochim. Biophys. Acta 1461, 347-357[Medline] [Order article via Infotrieve] |
8. | Tusnady, G. E., Bakos, E., Varadi, A., and Sarkadi, B. (1997) FEBS Lett. 402, 1-3[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Kool, M.,
van der, Linden, M.,
de Haas, M.,
Baas, F.,
and Borst, P.
(1999)
Cancer Res.
59,
175-182 |
10. |
Bakos, E.,
Hegedus, T.,
Hollo, Z.,
Welker, E.,
Tusnady, G. E.,
Zaman, G. J.,
Flens, M. J.,
Varadi, A.,
and Sarkadi, B.
(1996)
J. Biol. Chem.
271,
12322-12326 |
11. | Schinkel, A. H. (1997) Semin. Cancer Biol. 8, 161-170[CrossRef][Medline] [Order article via Infotrieve] |
12. | Grant, C. E., Bhardwaj, G., Cole, S. P. C., and Deeley, R. G. (1998) Methods Enzymol. 292, 594-607[Medline] [Order article via Infotrieve] |
13. | Grant, C. E., Valdimarsson, G., Hipfner, D. R., Almquist, K. C., Cole, S. P. C., and Deeley, R. G. (1994) Cancer Res. 54, 357-361[Abstract] |
14. | Seelig, A., Blatter, X. L., and Wohnsland, F. (2000) Int. J. Clin. Pharmacol. Ther. 38, 111-121[Medline] [Order article via Infotrieve] |
15. |
Loe, D. W.,
Almquist, K. C.,
Deeley, R. G.,
and Cole, S. P. C.
(1996)
J. Biol. Chem.
271,
9675-9682 |
16. |
Loe, D. W.,
Almquist, K. C.,
Cole, S. P. C.,
and Deeley, R. G.
(1996)
J. Biol. Chem.
271,
9683-9689 |
17. | Keppler, D., Cui, Y., Konig, J., Leier, I., and Nies, A. (1999) Adv. Enzyme Regul. 39, 237-246[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Loe, D. W.,
Stewart, R. K.,
Massey, T. E.,
Deeley, R. G.,
and Cole, S. P. C.
(1997)
Mol. Pharmacol.
51,
1034-1041 |
19. | Ding, G. Y., Shen, T., and Center, M. S. (1999) Anticancer Res. 19, 3243-3248[Medline] [Order article via Infotrieve] |
20. | Loe, D. W., Deeley, R. G., and Cole, S. P. C. (1998) Cancer Res. 58, 5130-5136[Abstract] |
21. |
Renes, J.,
de Vries, E. G.,
Nienhuis, E. F.,
Jansen, P. L.,
and Muller, M.
(1999)
Br. J. Pharmacol.
126,
681-688 |
22. |
Loe, D. W.,
Deeley, R. G.,
and Cole, S. P. C.
(2000)
J. Pharmacol. Exp. Ther.
293,
530-538 |
23. | Ueda, K., Taguchi, Y., and Morishima, M. (1997) Semin. Cancer Biol. 8, 151-159[CrossRef][Medline] [Order article via Infotrieve] |
24. | Loo, T. W., and Clarke, D. M. (1994) Biochemistry 33, 14049-14057[Medline] [Order article via Infotrieve] |
25. |
Loo, T. W.,
and Clarke, D. M.
(1999)
J. Biol. Chem.
274,
35388-35392 |
26. |
Loo, T. W.,
and Clarke, D. M.
(1994)
J. Biol. Chem.
269,
7243-7248 |
27. |
Loo, T. W.,
and Clarke, D. M.
(1997)
J. Biol. Chem.
272,
31945-31948 |
28. | Taguchi, Y., Kino, K., Morishima, M., Komano, T., Kane, S. E., and Ueda, K. (1997) Biochemistry 36, 8883-8889[CrossRef][Medline] [Order article via Infotrieve] |
29. | Gros, P., Dhir, R., Croop, J., and Talbot, F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7289-7293[Abstract] |
30. |
Loo, T. W.,
and Clarke, D. M.
(1997)
J. Biol. Chem.
272,
20986-20989 |
31. | Stride, B. D., Valdimarsson, G., Gerlach, J. H., Wilson, G. M., Cole, S. P. C., and Deeley, R. G. (1996) Mol. Pharmacol. 49, 962-971[Abstract] |
32. |
Stride, B. D.,
Grant, C. E.,
Loe, D. W.,
Hipfner, D. R.,
Cole, S. P. C.,
and Deeley, R. G.
(1997)
Mol. Pharmacol.
52,
344-353 |
33. |
Stride, B. D.,
Cole, S. P. C.,
and Deeley, R. G.
(1999)
J. Biol. Chem.
274,
22877-22883 |
34. | Hipfner, D. R., Gao, M., Scheffer, G., Scheper, R. J., Deeley, R. G., and Cole, S. P. C. (1998) Br. J. Cancer 78, 1134-1140[Medline] [Order article via Infotrieve] |
35. |
Hipfner, D. R.,
Mao, Q.,
Qiu, W.,
Leslie, E. M.,
Gao, M.,
Deeley, R. G.,
and Cole, S. P. C.
(1999)
J. Biol. Chem.
274,
15420-15426 |
36. |
Qian, Y. M.,
Song, W. C.,
Cui, H.,
Cole, S. P. C.,
and Deeley, R. G.
(2001)
J. Biol. Chem.
276,
6404-6411 |
37. |
Ryu, S.,
Kawabe, Y.,
Nada, S.,
and Yamaguchi, A.
(2000)
J. Biol. Chem.
275,
39617-39624 |