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Molecular and Cellular Biology, February 1999, p. 1450-1459, Vol. 19, No. 2
0270-7306/99/$00.00+0
Studies of Human MDR1-MDR2 Chimeras Demonstrate the
Functional Exchangeability of a Major Transmembrane Segment of the
Multidrug Transporter and Phosphatidylcholine Flippase
Yi
Zhou,1
Michael
M.
Gottesman,2 and
Ira
Pastan1,*
Laboratory of Molecular
Biology1 and
Laboratory of Cell
Biology,2 National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892
Received 31 July 1998/Returned for modification 23 September
1998/Accepted 6 November 1998
 |
ABSTRACT |
P-glycoprotein (P-gp), encoded by the MDR1 gene, is a
plasma membrane transporter which effluxes a large number of
structurally nonrelated hydrophobic compounds. The molecular basis of
the broad substrate recognition of P-gp is not well understood. Despite the 78% amino acid sequence identity of the MDR1 and
MDR2 transporter, MDR2, which has been identified as a
phosphatidylcholine transporter, does not transport most MDR1
substrates. The structural and functional differences between MDR1 and
MDR2 provide an opportunity to identify the residues essential for the
broad substrate spectrum of MDR1. Using an approach involving
exchanging homologous segments of MDR1 and MDR2 and site-directed
mutagenesis, we have demonstrated that MDR1 residues Q330, V331, and
L332 in transmembrane domain 6 are sufficient to allow an MDR2 backbone
in the N-terminal half of P-gp to transport several MDR1 substrates,
including bisantrene, colchicine, vinblastine, and rhodamine-123. These
studies help define some residues important for multidrug transport and
indicate the close functional relationship between the multidrug
transporter (MDR1) and phosphatidylcholine flippase (MDR2).
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INTRODUCTION |
Expression of the multidrug
transporter P-glycoprotein (P-gp) is one of the major causes of
multidrug resistance in cancer cells. P-gp is a 170-kDa membrane
protein encoded by the MDR1 gene in humans. Based on its sequence and
domain organization, P-gp is classified as a member of ATP binding
cassette superfamily; it consists of two symmetrical halves, each half
containing six transmembrane (TM) domains and a cytoplasmic nucleotide
binding domain. Although most members of the ABC transporter family
have stringent substrate specificities, P-gp recognizes many compounds, including anthracyclines (e.g., doxorubicin and daunomycin),
vinca alkaloids (e.g., vincristine and vinblastine), antibiotics
(e.g., actinomycin D), circular and toxic peptides (e.g., valinomycin and gramicidin), and relatively noncytotoxic agents such as verapamil, azidopine, quinidine, and cyclosporin A. These P-gp substrates have
no common chemical structure. They are all
low-molecular-weight nonanionic hydrophobic or amphipathic
compounds (11).
Although a detailed understanding of the molecular basis of P-gp
substrate specificity must await high-resolution three-dimensional protein structure analysis, much information can be obtained through mutational studies aimed at analyzing the regions of homology and
nonhomology between MDR molecules from the different species. Two MDR
homologs, MDR1 and MDR2, have been identified in humans. Despite MDR2
having a 78% overall amino acid sequence identity and a domain
organization predicted to be the same as MDR1 (32), MDR2
protein is a phosphatidylcholine transporter which recognizes some
other phospholipids (30, 31). However, the MDR2 transporter does not confer drug resistance in a broad spectrum. Photoaffinity labeling experiments have shown that the inability of the mouse mdr2
transporter to confer resistance to specific drugs is associated with
reduced binding of these drugs to the mdr2 protein (4). Studies of MDR1-MDR2 chimeric proteins show that exchanging ATP binding
domains between MDR1 and MDR2 results in few changes in the function of
MDR1; however, exchanging the homologous segments containing a few TM
regions abrogates the capacity of the MDR1 transporter to confer
multidrug resistance (3, 7, 34). The loss of multidrug
transporter function in the chimeric P-gp can be partially restored
through selectively changing a few MDR2 residues back to MDR1 residues,
indicating that not all of the nonidentical residues contribute equally
to the differences in transport function between MDR1 and MDR2
(7). Since MDR1 and MDR2 proteins have a similar domain
structure and they both transport hydrophobic and amphipathic
substrates, their substrate preferences are most likely determined by
some of the nonidentical residues within the MDR protein transmembrane
domains. Identification of these residues will help reveal the
molecular basis of the specific interaction between P-gp and its substrates.
In this work, we have focused on the residues around TM domain 6 (TM6)
of MDR1 which are conserved in MDR1 P-gp from different species.
Photoaffinity labeling experiments have suggested that this region
directly interacts with the substrates of MDR1 (1, 2, 12, 13,
25). In mouse P-gp, replacing TM5 and TM6 with the homologous
mdr2 segment is sufficient to abolish the colchicine and doxorubicin
resistance conferred by mdr1 (3). The overall strategy of
our work was to construct an inactive MDR1-MDR2 chimera and use it as a
framework to reconstruct molecules with MDR1-like function by
selectively replacing MDR2 residues with MDR1 residues. Our results
indicate that Q330, V331, and L332 in TM6 of MDR1 P-gp are crucial for
multidrug transporter activity and that with the exception of these
residues, the amino-terminal half of the MDR2 backbone, including the
first six TM domains, can support multidrug transport.
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MATERIALS AND METHODS |
Mutagenesis and vector construction.
A silent G-A
substitution at bp 1185 was first introduced into MDR2 to create an
EcoRI site at the same position as in MDR1. The MDR1-MDR2
chimera was constructed by exchanging homologous NsiI/EcoRI digestion fragments. Two sets of
mutations, (i) N330Q, A331V, and M332L and (ii) V364E, D367K, N372K,
K374S, and E380K, were generated by a PCR mutagenesis method
(17). MDR1, MDR1-MDR2(307-394), and
MDR1-MDR2(307-394)QVL, as well as MDR1-MDR2(307-394)EKKSK, were inserted into the pHa retroviral vector at SacII
and XhoI sites (27).
Chimera MDR2(1-394)-MDR1 was constructed in the vaccinia virus
pTM1 transient expression vector (9) downstream of the T7 promoter and the 5'-untranslated region, which included an internal ribosomal entry site, of the encephalomyocarditis virus as previously described (28). To obtain optimum expression, RNA
transcription has to start from the NcoI site at the end of
the 5'-untranslated region of encephalomyocarditis virus. The
NcoI site in MDR2 was created at the ATG start codon by the
PCR mutagenesis method. A three-way ligation was performed to link MDR2
(NcoI/SalI, a PCR product), MDR2
(SalI/EcoRI), and pTM1MDR1
(NcoI/EcoRI). In order to detect the expression
of the chimeric protein MDR2(1-394)-MDR1 on the cell surface, a
FLAG epitope containing octopeptide DYKDDDDK (Kodak) was inserted in
the first extracellular loop between residues F98 and G99 of MDR2. The
same FLAG epitope was also previously inserted in wild-type MDR1
between N94 and R95. MDR1-FLAG had identical characteristics to MDR1
based on fluorescence-activated cell sorter (FACS) analysis and
iodoarylazidoprazosin (IAAP) photoaffinity labeling (17a). A
SacII site was also created by using PCR in the
pTM1MDR2(1-394)-MDR1 chimera 37 bp before the MDR2 starting codon.
Two sets of mutations, (i) N330Q, A331V, and M332L and (ii) V364E,
D367K, N372K, K374S, and E380K were introduced into MDR2(1-394)-MDR1 by replacing the
BamHI/EcoRI fragment with the corresponding
fragment of MDR1-MDR2(307-394)QVL, or
MDR1-MDR2(307-394)EKKSK. The SacII/XhoI
fragment containing the entire MDR2(1-394)-MDR1 or
MDR2(1-394)-MDR1QVL was inserted into the
SacII/XhoI site of the pHa retroviral vector.
MDR1NAM was created by introducing mutations Q330N, V331A, and L332M
into MDR1 by PCR mutagenesis. All PCR products were
sequenced to confirm that only the desired mutations were introduced.
Cell culture and DNA transfection.
Both transient and stable
protein expression approaches were employed in this work. Transient
expression was based on the method developed by Moss and colleagues
(9, 10, 26). cDNA was transcribed by this method from a T7
promoter by T7 RNA polymerase, which was expressed by a modified
recombinant vaccinia virus (MVA). The cotransfection-infection
procedure was performed as described previously by Ramachandra et al.
(28). Briefly, 15 µg of DNA was mixed with 45 µl of
Lipofectin (Life Technology, Inc.) in 3.5 ml of OptiMem medium and
allowed to sit undisturbed at room temperature for 30 min. The mixture
was then added to a 75-mm2 flask preplated with 1.5 × 106 HeLa cells the night before. MVA was also added to the
flask at 108 PFU/flask. After 4 h of incubation at
32°C, 12 ml of minimal essential medium containing 10% fetal bovine
serum (FBS) was added. The cells were cultured at 32°C for another
18 h before the functional analysis.
Stable expression of MDR1 and its mutants were achieved by using a
well-established pHa retroviral vector whose promoter is
in the long
terminal repeat of Harvey murine sarcoma virus (
27).
First,
10 µg of pHaMDR1(V185) or mutant DNA was cotransfected
with 0.5 µg
of pcDNA3 (Invitrogen) into NIH 3T3 cells by the calcium
phosphate
transfection method. pcDNA3 confers G418 resistance
on the transfected
cells through expression of
neo. After selection
for 7 days
with 750 µg of G418 per ml, the surviving cells were
pooled and
labeled with anti-human MDR1 monoclonal antibody MRK-16
(
14)
at a concentration of 5 µg/5 × 10
5 cell in a volume
of 200 µl at 4°C for 30 min. MRK-16-positive
cells were isolated by
using magnetic cell sorting with a miniMACS
apparatus (Miltenyi Biotec
GmbH). The MRK-16-positive cells were
collected and expanded to
10
7. The aliquots of the expanded cells were stored in
liquid nitrogen
or analyzed either by a cytotoxicity assay or
FACS.
FACS analysis of P-gp cell surface expression and fluorescent
drug accumulation.
The cell surface expression of MDR1 P-gp or its
mutants was detected by using MDR1 monoclonal antibody MRK-16
(14) or, in some cases, M2 monoclonal antibody raised
against the FLAG epitope (Kodak). About 2 × 105 to
3 × 105 cells were incubated in 200 µl of
phosphate-buffered saline (PBS) containing 1% bovine serum albumin and
5 µg of MRK-16 or M2 antibody at 4°C for 30 min. After two washes
with ice-cold PBS, the cells were further incubated with fluorescein
isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin G2a (IgG2a)
or IgG1 monoclonal antibody (PharMingen) at 4°C for 45 min. The cells
were then washed and analyzed with a FACSort apparatus equipped with
Cellquest program (Becton Dickinson). The fluorescence intensity at the
FL1 channel was plotted to compare the cell surface expression of MDR1
or its mutants.
In a fluorogenic substrate accumulation assay, 3 × 10
5 cells were incubated in 2 ml of phenol red-free Iscove
medium (Life
Technology, Inc.) which contained either 1 µg of
bisantrene per
ml, 3 µM daunomycin, or 0.5 µM rhodamine-123. After
incubation
at 37°C for 60 min, the cells were centrifuged,
resuspended in
ice-cold PBS, and analyzed by FACS. In some cases, after
incubation
with 0.5 µM rhodamine-123, the cells were incubated at
37°C in
plain Iscove medium for 40 min before being analyzed by
FACS.
Cell toxicity assay.
The multidrug transporter activity of
MDR1 or its mutants was also examined by using a cytotoxicity assay.
Control NIH 3T3 cells resistant to G418 and NIH 3T3 cell populations
expressing MDR1 or its mutants (positive for MRK-16 labeling, isolated
by magnetic cell sorting) were plated at 103 cells/well
in 96-well plates. On the second day, the cells were cultured in
selective medium containing bisantrene, colchicine, daunomycin, or
vincristine at a concentrations of 0, 0.3, 1, 3, 10, 30, 100, 300, or
1,000 ng/ml. After 72 h of incubation at 37°C, cell viability
was determined with reagent WST-1 (an MTT analog from Boehringer
Mannheim) according to the manufacturer's instruction. Triplicates of
each sample were analyzed in this assay. The 50% inhibitory
concentration (IC50) was defined as the drug concentration
required for 50% inhibition of the maximum WST-1 staining. The
percentage of drug transporter activity of the MDR1 mutants versus the
activity of wild-type MDR1 was calculated as 100 × (IC50 [mutant] 
IC50
[neo])/(IC50 [MDR1] IC50 [neo]).
Photoaffinity labeling with [125I]IAAP.
Aliquots of 5 × 105 HeLa cells transiently expressing
MDR1, MDR1NAM, MDR2(1-394)-MDR1, MDR2(1-394)-MDR1QVL, and
MDR2(1-394)-MDR1EKKSK were washed with ice-cold PBS and
resuspended in 100 µl of PBS containing 7 nM [125I]IAAP
(Amersham) with or without 5 µM cyclosporin A or vinblastine at the
indicated concentrations. The cell suspensions were incubated in the
dark at room temperature for 60 min, followed by cross-linking under UV
light at 366 nm for 30 min on ice. The cells were then washed once with
ice-cold PBS, and the cell pellets were subjected to
immunoprecipitation with anti-P-gp polyclonal antiserum PEPG
-13 as described previously (1, 2). The immunoprecipited samples were divided into two aliquots. One aliquot was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and used
to detect labeled radioactivity; the other aliquot was subjected to
immunoblotting analysis with C219 monoclonal antibody.
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RESULTS |
Sequence alignment of MDR1 and MDR2 between residues 307 and
394.
A previous mutational study demonstrated that a mouse
mdr1-mdr2 chimera containing TM5 and TM6 of mdr2 does not confer
resistance to doxorubicin or colchicine (3), indicating that
some of the replaced MDR1 residues are pivotal for multidrug
transporter activity. To determine which residues within this region
are essential for the multidrug transporter activity, we aligned the
amino acid sequences of 10 mammalian MDR proteins (human MDR1 and MDR2;
mouse mdr1, mdr2, and mdr3; rat mdr1 and mdr2; and Chinese hamster
mdr1, mdr2, and mdr3) by using the Wisconsin Package software (Genetics Computer Group, Inc.). An alignment of the segment between residues 307 and 394 is shown in Fig. 1. Within this
segment, 19 residues differ between human MDR1 and MDR2 P-gp
(underlined in the MDR1 segment); 12 of these residues are conserved
among the multidrug transporters (numbered). Based on an MDR1 TM
organization model (5, 18, 19), these 12 residues are
distributed as follows: two in the extracellular loop connecting TM5
and TM6 (T318 and S327), four in TM6 (Q330, V331, L332, and S351), and
six in the cytoplasmic region following TM6 and preceding the
N-terminal nucleotide binding domain (E353, E364, K367, K372, S374, and
K380).
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FIG. 1.
Amino acid sequence alignment of mammalian MDR proteins
between residues 307 and 394. The residues that differ between MDR1 and
MDR2 are underlined. The residues in MDR1 differing from MDR2 but
conserved in mdr1 and mdr3 are numbered with their position in MDR1.
The residues studied in this work are in boldface. MDR prefixes: h,
human; r, rat; ch, Chinese hamster; m, mouse.
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Functional analysis of MDR1-MDR2(307-394),
MDR1-MDR2(307-394)QVL, and
MDR1-MDR2(307-394)EKKSK.
Our strategy to identify the
essential MDR1 residues was to restore the multidrug transporter
activity in MDR1-MDR2 chimeras by reintroducing selected MDR1 residues.
The initial study was focused on the chimeric protein
MDR1-MDR2(307-394). Two sets of mutations, (i) Q, V, and L at
positions 330, 331, and 332 in TM6 or (ii) E, K, K, S, and K at
positions 365, 367, 372, 374, and 380, respectively, within the
cytoplasmic region, were introduced into MDR1-MDR2(307-394) to
create MDR1-MDR2(307-394)QVL and
MDR1-MDR2(307-394)EKKSK (Fig. 2).
Functional characterization of the chimeras was performed with stably
transfected NIH 3T3 cells. Retroviral vectors containing the gene
encoding MDR1(V185), a well-characterized mutant of MDR1 (6,
28), or the MDR1-MDR2 chimera were cotransfected with pcDNA3, which confers G418 resistance. After G418 selection, the surviving cell population was pooled and incubated with monoclonal antibody MRK-16. The MRK-16-positive cells were isolated by a magnetic
cell sorting method and used for functional studies.
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FIG. 2.
Diagram of the chimeric MDR proteins. The FLAG epitope
was inserted in MDR2 between F98 and G99. Open boxes, MDR2 segment;
shaded boxes, MDR1 segment.
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Two approaches were used to compare the multidrug transporter activity
of P-gp and its mutants. The first approach was to
test the ability of
MDR1-MDR2 chimeras to confer multidrug resistance
in stably transfected
NIH 3T3 cells. In this assay, the cells
were exposed to cytotoxic drugs
by using bisantrene, colchicine,
daunomycin, or vincristine at
various concentrations. The surviving
cells were detected with the MTT
analog WST-1. This assay scores
the viable cells by detecting the
enzymatic activity of functional
mitochondria. As summarized in Table
1, MDR1-MDR2(307-394) conferred
low-level bisantrene resistance compared to MDR1(V185), but it
did not
confer resistance to colchicine, daunomycin, and vincristine.
Reintroducing MDR1 residues Q330, V331, and L332 into TM6 increased
resistance to bisantrene and partially restored resistance to
colchicine, daunomycin, and vincristine. Reintroducing E364, K367,
K372, S374, and K380 into the cytoplasmic loop did not improve
drug
resistance. Similar results were also obtained by a direct
cell colony
formation assay (data not shown).
The second approach involved analyzing the transporter activity by a
fluorogenic substrate accumulation assay. This assay
takes advantage of
the fact that some P-gp substrates have intrinsic
fluorescence; the
accumulation of these substrates can be determined
by measuring the
intracellular fluorescence by FACS analysis.
Thus, this assay directly
measures P-gp transporter activity.
In this experiment we used
fluorescent substrates rhodamine-123
and bisantrene. As shown in Fig.
3, the cells expressing
MDR1-MDR2(307-394)QVL
accumulated less bisantrene and
rhodamine-123 than the cells expressing
MDR1-MDR2(307-394) or
MDR1-MDR2(307-394)EKKSK. These results indicate
that
reintroducing MDR1 residues Q330, V331, and L332 into TM6
of
MDR1-MDR2(307-394) partially restored the transporter activity
for
bisantrene and rhodamine-123 but that reintroducing E364,
K367, K372,
S374, and K380 into the cytoplasmic region did not
produce similar
effects. Identical results were also obtained
in an analysis of
transiently expressed MDR1-MDR2 chimeras and
wild-type MDR1 (data not
shown).
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FIG. 3.
Expression and function of MDR1-MDR2 chimeras after
stable transfection. (A) The cell surface expression of MDR1,
MDR1-MDR2(307-394), and its mutants on transfected 3T3 cells was
determined by using MRK-16 antibody labeling and FACS analysis. The
transporter activity of MDR1, MDR1-MDR2(307-394), and its mutants
was analyzed by using a fluorescent substrate accumulation assay. The
3T3 cells expressing MDR1 or its mutants were incubated in medium
containing 1 µg of bisantrene per ml (B) or 0.5 µM rhodamine-123
(C) at 37°C for 60 min. The fluorescence intensity, representing the
intracellular accumulated substrate, was detected by FACS. Key: light
gray areas, 3T3/neo; dark gray areas, 3T3/MDR1(V185); dotted line,
3T3/MDR1-MDR2(307-394); heavy line, 3T3/MDR1
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The difference in drug transport could be due to a difference in the
amount of P-gp on the cell surface. Therefore, we compared
the cell
surface expression of MDR1 with the MDR1-MDR2 chimeras
by using MRK-16.
The results indicate that over 90% of the sorted
cells in the mass
cell population are MRK-16 positive. MDR1(V185),
MDR1-MDR2(307-394), and MDR1-MDR2(307-394)EKKSK were
expressed
on the cell surface at similar levels, but the expression of
MDR1-MDR2(307-394)QVL
was slightly less (Fig.
3). This difference
may partially contribute
to the lower transporter activity observed in
the cells expressing
MDR1-MDR2(307-394)QVL. In summary, both the
cytotoxicity assay
and the fluorescent substrate accumulation assays
indicated that
within the region containing residues 307 to 394, Q330,
V331,
and L332 in TM6 play a crucial role in sustaining multidrug
transport
activity, while S351, K367, K372, S374, and K380 in the
cytoplasmic
loop are less
important.
Functional analysis of MDR2(1-394)QVL-MDR1 and
MDR2(1-394)EKKSK-MDR1.
To further investigate the
significance of Q330, V331, and L332 in multidrug transporter activity,
we examined the effects of these mutations in an MDR1-MDR2 chimera
containing the entire MDR2 N-terminal transmembrane region (TM1-6;
residues 1 to 394). Two sets of mutations, (i) N330Q, A331V, and M332L
and (ii) V364E, D367K, N372K, K374S, and E380K, were introduced
into MDR2(1-394)-MDR1 to generate MDR2(1-394)-MDR1QVL
and MDR2(1-394)-MDR1EKKSK (Fig. 2). Since it was possible
that the MRK-16 epitope would be missing in these MDR2-MDR1 chimeras, a
FLAG epitope was inserted into the first extracellular loop of both
chimeric and wild-type MDR1 to evaluate cell surface expression.
MDR2(1-394)-MDR1 and MDR2(1-394)-MDR1QVL were
stably expressed in 3T3 cells. After G418 selection and magnetic
cell sorting
as described above, the mass population of MRK-16-positive
cells
was subjected to cytotoxicity assays. The results showed that
MDR2(1-394)-MDR1 conferred low resistance to bisantrene and no
resistance to colchicine, daunomycin, and vincristine. In comparison,
MDR2(1-394)-MDR1QVL conferred increased resistance to
bisantrene
and partial resistance to vincristine and colchicine; no
resistance
to daunomycin was observed (Table
2). The amounts of MDR1(V185),
MDR2(1-394)-MDR1, and MDR2(1-394)-MDR1QVL proteins expressed
on
the cell surface were compared by labeling with monoclonal antibody
raised against the FLAG epitope, followed by FACS analysis.
MDR2(1-394)-MDR1QVL
was expressed at a level comparable
to wild-type MDR1, but MDR2(1-394)-MDR1
was expressed at a
somewhat lower level (data not shown).
To correct for the expression difference between MDR2(1-394)-MDR1
and MDR2(1-394)-MDR1QVL in 3T3 cells, we transiently expressed
both proteins, as well as MDR2(1-394)-MDR1EKKSK and MDR1, in HeLa
cells by using a vaccinia virus-T7 RNA polymerase system. In comparison
with other protein expression systems, the vaccinia virus-T7 RNA
polymerase system enables one to achieve a high transfection efficiency
and to produce large amounts of recombinant protein within a short
period of time. The protein expressed on the cell surface was
labeled
by using a monoclonal antibody raised against the FLAG
epitope and then
detected by FACS. As shown in Fig.
4, the
cells
transfected with MDR1 or its mutants were separated into two
populations.
One population of cells was minimally fluorescent and thus
similar
to the cells transfected with the pTM1 vector, indicating that
these cells did not express the MDR1-FLAG epitope. The other cell
population was more fluorescent and equivalent, indicating that
the
cell surface expression of MDR2(1-394)-MDR1QVL and
MDR2(1-394)-MDR1EKKSE
was at a level similar to wild-type P-gp.
However, the expression
of MDR2(1-394)-MDR1 was still
slightly less than that of the other
proteins.
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FIG. 4.
Expression and function analysis of transiently
expressed MDR2(1-394)-MDR1 and its mutants. MDR2(1-394)-MDR1,
MDR2(1-394)/MDR1QVL and MDR2(1-394)/MDR1EKKSK were
transiently expressed in HeLa cells. The cell surface-expressed protein
was labeled by using a monoclonal antibody raised against the FLAG
epitope followed by FACS analysis. Transporter activity of
MDR2(1-394)-MDR1QVL and MDR2(1-394)-MDR1EKKSK was
analyzed by a fluorescent substrate accumulation assay. The HeLa cells
transfected with MDR1 or its mutants were incubated in medium
containing 1 µg of bisantrene per ml, 0.5 µM rhodamine-123, or 3 µM daunomycin at 37°C for 60 min. The cells incubated with
rhodamine-123 were further incubated in rhodamine-123-free medium at
37°C for another 45 min. The fluorescence intensity, representing the
intracellular accumulated substrate, was detected by FACS. To better
show the difference between two cell populations, a zoom-in version of
the FACS result is used for the bisantrene and daunomycin panels.
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The transport activities of transiently expressed
MDR2(1-394)-MDR1QVL and MDR2(1-394)-MDR1EKKSK were
determined by the fluorogenic
substrate accumulation assay.
Rhodamine-123, bisantrene, and daunomycin
were used in this experiment.
As shown in Fig.
4, the cells transfected
with P-gp showed two
populations. One population of cells, overlapping
with the cells
transfected with pTM1 vector, exhibited a high
fluorescence intensity,
indicating a lack of multidrug transporter
activity. The second cell
population had less fluorescence and
was unique to the cells
transfected with MDR1 or its mutants,
indicating that the reduced
fluorescence was associated with overexpression
of MDR activity.
MDR2(1-394)-MDR1QVL exhibited efflux activity
for bisantrene
and rhodamine-123 but not for daunomycin.
MDR2(1-394)-MDR1EKKSK
showed low-level efflux activity
for bisantrene but no efflux
activity for rhodamine-123 or daunomycin.
This result suggests
that substituting residues Q330, V331, and L332 in
MDR2(1-394)-MDR1
can improve multidrug transporter activity in
MDR1-MDR2 chimeras,
though the transporter efficiency varies with the
substrate
tested.
Previous studies showed that a mouse mdr2(1-394)-mdr1 chimera does
not confer doxorubicin and colchicine resistance in a stable
transfected cell line (
3). We obtained similar results with
human P-gp chimeras. However, with either the stable or the transient
expression systems, we were not able to express this protein on
the
cell surface at a level equivalent to the wild-type P-gp or
MDR2(1-394)-MDR1QVL. The low-level expression of
MDR2(1-394)-MDR1
observed in this experiment may be related to an
inhibitory effect
of the MDR2 coding sequence on protein expression,
since full-length
MDR2 P-gp is also expressed at a lower level than
MDR1 in either
stable or transient expression systems (
4,
35). Because MDR2(1-394)-MDR1
was expressed at a lower level
than MDR2(1-394)-MDR1QVL, it is
difficult to directly compare
their transport activities. However,
the importance of Q330, V331, and
L332 in MDR2(1-394)-MDR1 can
be demonstrated through comparison of
the transporter activities
of MDR2(1-394)-MDR1QVL and
MDR2(1-394)-MDR1EKKSK.
Expression and functional analysis of MDR1NAM.
To confirm
the significance of Q330, V331, and L332 in TM6, we generated a MDR1
mutant containing the mutations Q330N, V331A, and L332M. This MDR1
mutant, MDR1NAM, was transiently expressed in HeLa cells by using a
vaccinia virus-T7 expression system as described above. The cell
surface expression of MDR1 and MDR1NAM were compared by labeling
intact cells with P-gp monoclonal antibody MRK-16. The multidrug
transporter activity of MDR1NAM was compared with MDR1 by using a
fluorogenic substrate accumulation assay. Bisantrene, rhodamine-123,
and daunomycin were tested. We found that although MDR1NAM was
expressed on the cell surface as much as the wild-type MDR1, its
capacity to efflux daunomycin was almost lost and the activity to
efflux bisantrene, calcein AM, and rhodamine-123 were also
significantly reduced but not abrogated (Fig.
5). Similar results were also obtained
with 3T3 cells that stably expressed MDR1NAM. Compared to
MDR1(V185), MDR1NAM conferred 49% as much resistance to bisantrene
and 22% as much resistance to daunomycin. These results suggest that
Q330, V331, and L332 are quantitatively important for overall multidrug
transporter activity of MDR1.
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|
FIG. 5.
Functional analysis of MDR1NAM. MDR1NAM and MDR1
were transiently expressed in HeLa cells. The cell surface expression
of each protein was compared by MRK-16 labeling followed by FACS
analysis. The transport activity of transiently expressed MDR1NAM
was analyzed by fluorescent substrate accumulation assay at a final
concentration of 1 µM bisantrene, 0.5 µM rhodamine-123, or 3 µM
daunomycin. A zoom-in version of the FACS result is used for the
bisantrene panel.
|
|
Photoaffinity labeling of MDR1, MDR2(1-394)-MDR1,
MDR2(1-394)-MDR1 QVL, and MDR2(1-394)-MDR1
EKKSK.
To determine whether the change of multidrug
transporter activity of MDR1-MDR2 chimeras was related to a change in
their ability to interact with substrate, we performed
photoaffinity labeling experiments with IAAP. The HeLa cells
transiently expressing MDR1, MDR1NAM,
MDR1-MDR2(1-394), MDR1-MDR2(1-394)QVL, or
MDR1-MDR2(1-394)EKKSK were incubated with 7 nM IAAP, a
concentration below the Km of wild-type
MDR1, in the presence or absence of 5 µM cyclosporin A. After
photoaffinity labeling, P-gp was immunoprecipitated and separated by
SDS-PAGE. The radioactive label on P-gp was measured by using the STORM
system (Fig. 6A). We found that MDR1 and
all of the MDR1-MDR2 chimeras could be labeled with IAAP and that this
IAAP labeling was blocked by the MDR1 reversing-agent cyclosporin A. After normalization to the relative amount of P-gp with Western blotting analysis, we calculated a relative IAAP labeling efficiency for each sample. These data showed that as compared to MDR1 (100%), the relative specific IAAP labeling efficiencies of MDR1NAM,
MDR2(1-394)-MDR1, MDR2(1-394)-MDR1QVL, or
MDR2(1-394)-MDR1EKKSK were 68, 37, 85, or 90%, respectively
(Fig. 6B). Like mouse mdr2 (4), human MDR2 was only
nonspecifically photoaffinity labeled at low level with IAAP (data not
shown). Using IAAP labeling as an indicator, we also determined
whether the MDR1-MDR2 chimeras could interact with the P-gp
substrate vinblastine by determining if vinblastine would competitively
block IAAP labeling. We found that IAAP labeling of
MDR2(1-394)-MDR1 was blocked by vinblastine in a
dose-dependent manner (Fig. 7).
Furthermore, MDR2(1-394)-MDR1QVL and
MDR2(1-394)-MDR1EKKSK were even more sensitive than MDR1 to a
low concentration of vinblastine. These results indicate a relatively
normal interaction between vinblastine and MDR1-MDR2 chimeras.
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|
FIG. 6.
IAAP photoaffinity labeling of MDR1, MDR1NAM,
MDR2(1-394)-MDR1, MDR2(1-394)-MDR1QVL, and
MDR2(1-394)/MDR1EKKSK. A total of 500,000 HeLa cells
transiently expressing MDR1 and its mutants were incubated with 7 nM
[125I]IAAP in 100 µl of PBS with or without 5 µM
cyclosporine A at room temperature for 60 min, followed by
cross-linking with UV at 366 nm for 30 min on ice. (A) The P-gps were
immunoprecipitated with P-gp polyclonal antibody PEPG-13 and
analyzed by SDS-PAGE. (B) The radioactivity associated with the P-gp
band was measured and quantitated by using the STORM system. After
normalization to the relative amount of P-gp in each sample, the
relative IAAP specific labeling efficiency is shown. The relative IAAP
specific labeling efficiency of MDR1 P-gp is shown as 100%.
|
|
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|
FIG. 7.
IAAP photoaffinity labeling in the presence of various
concentrations of vinblastine. IAAP labeling was carried out as
described above in the presence of vinblastine at concentrations of 0, 0.1, 0.3, 1, and 3 µM. After immunoprecipitation and SDS-PAGE
separation, the radioactivity associated with P-gp was measured and
quantitated by using the STORM system. The normalized IAAP labeling is
shown. The relative IAAP labeling in the absence of vinblastine is
shown as 100%.
|
|
Our previous work demonstrated that
cis-fluopentixol
stimulates IAAP photoaffinity labeling of MDR1 through binding to
the
second drug-binding site (
8). To determine whether MDR1
residues
in TM6 are involved in forming the second drug-binding site,
we
performed IAAP labeling of various MDR1-MDR2 chimeras in the
presence
or absence of
cis-fluopentixol. We found that
cis-fluopentixol
stimulated IAAP labeling of
MDR1-MDR2(307-394)QVL and MDR1-MDR2(307-394)EKKSK
but not of
MDR2(1-394)-MDR1QVL or MDR2(1-394)-MDR1EKKSK (data
not
shown). These results suggest that MDR1 N-terminal residues
1 to 307 are essential for the second drug (
cis-fluopentixol)
binding
site.
| |
DISCUSSION |
Determination of the residues in P-gp essential for the transport
of specific drugs offers the promise of defining substrate binding
domains in this energy-dependent multidrug transporter. Using an
approach involving exchanging homologous segments of MDR1 and MDR2 and
site-directed mutagenesis, we demonstrated that MDR1 residues Q330,
V331, and L332 in TM6 were essential for multidrug transporter
activity. Substituting Q330, V331, and L332 with MDR2 residues could
significantly impair MDR1 activity, while the triple mutation N330Q
A331V M332L was sufficient to allow the MDR2 TM domain (residues 307 to
394 or residues 1 to 394) in the N-terminal half of P-gp to support
multidrug transporter activity. These results suggest structural and
functional similarity between the multidrug transporter and
phosphatidylcholine flippase.
Identification of Q330, V331, and L332 as the critical residues for
the multidrug transporter activity.
To identify the essential
residues for MDR1 broad substrate specificity, we focused on the
residues conserved only among MDR multidrug transporters, while
omitting residues conserved in MDR1 and MDR2. The latter conserved
residues are assumed to be structurally or functionally important for
both multidrug transporter and phosphatidylcholine flippase, but
they do not determine the broad multidrug recognition of P-gp.
Construction of MDR1-MDR2 chimeras allowed us to investigate the
role of some unique residues for multidrug transporter and to determine
the residues responsible for the functional differences between the
multidrug transporter and phosphatidylcholine flippase.
Identification of Q330, V331, and L332 as the critical residues for the
multidrug transporter was based on two lines of evidence.
One is that
the substitutions Q330N, V331A, and L332M in MDR1
significantly
decreased overall MDR1 multidrug transporter activity.
The other is
that substituting N330Q, A331V, and M332L in the
MDR2 region of either
MDR2(307-394)-MDR1 or MDR2(1-394)-MDR1 partially
restored the
lost MDR1 function in the MDR1-MDR2 chimeras. Even
though the restored
activity was somewhat less than the activity
of MDR1, depending on the
individual substrate tested, the effects
of Q330, V331, and L332 on the
MDR1-MDR2 chimeric proteins are
significant, considering they
represent less than 16% (3/19) of
the differences between MDR1 and
MDR2 within the region from TM5
to TM6 (307 to 394), and 2.5% (3/121)
of the differences within
the region from TM1 to TM6 (1 to 394). The
ability to restore
the multidrug transport activity of MDR1-MDR2
chimeras is unique
to Q330, V331, and L332, since residues E364, K367,
K372, S374,
and K380 within the cytoplasmic region did not produce
similar
effects. In addition, the introduction of either residues E364,
K367, K372, S374, and K380 or residues T318, L322, G324, and S327
into
MDR1-MDR2(307-394)QVL did not further improve the multidrug
transport activity, implying that these residues may not significantly
contribute to drug transport (data not shown). It should be pointed
out
that N-Q and M-L have very similar chemical properties and
that they
are often interchangeable among proteins with the same
biological
function. Therefore, it is possible that the functional
difference made
by mutations N-Q, A-V, and M-L is due to their
unique structural
location rather than to their chemical
differences.
Mutations at residues 330, 331, and 332 produce quantitative
effects on multidrug transport activity.
Although Q330, V331, and
L332 are important for the broad substrate spectrum of P-gp,
these results do not exclude the possibility that other
residues, including the residues conserved in both MDR1 and MDR2, are
essential for the interaction with P-gp substrates. In fact, both
MDR1-MDR2(307-394) and MDR2(1-394)-MDR1 have residual ability
to efflux bisantrene but not enough to support multidrug transport
unless Q330, V331, and L332 are present. On the other hand,
residues Q330, V331, and L332 are not absolutely necessary for
multidrug transporter activity. Substituting Q330, V331, and L332
with MDR2 residues in an MDR1 backbone decreased but did not abolish
overall multidrug transport activity. These results suggest that
multiple factors may be involved in sustaining multidrug transporter
activity, and each factor can function independently. In addition,
earlier mutational studies showed that replacing Q330, V331, or L332
individually with Ala or Cys has no significant effects on MDR activity
(20-22). However, by simultaneously replacing Q330, V331,
and L332 with Ala, we observed an overall reduced MDR1 activity similar
to MDR1NAM (data not shown). Thus, the triple mutation Q330N,
V331A, and L332M appeared to have a synergistic effect. Similar
observations have also been made recently in studies on substitutions
of nonconserved MDR1 residues in TM12 (15). We do not know
whether all three residues are important, or whether any two of them
might have the same effect. Taken together, we believe that the effects
of Q330, V331, and L332 on multidrug transport are quantitatively significant.
Mutations at residues 330, 331, and 332 may cause a change in P-gp
conformation.
It is not yet clear why Q330, V331, and L332 are
important for multidrug transporter activity. In an IAAP photoaffinity
labeling experiment, we observed that all the MDR1-MDR2 chimeras were
labeled by IAAP and that this IAAP labeling was blocked by the P-gp
inhibitor cyclosporin A. Although the restoration or loss of MDR1
activity in MDR1-MDR2 chimeras roughly paralleled the increase or
decrease in IAAP binding, the changes in IAAP labeling are not
quantitatively correlated with the changes in multidrug transporter
activity. Furthermore, we found that vinblastine blocked IAAP labeling
of all the MDR1-MDR2 chimeras in a dose-dependent manner, even though MDR2(1-394)-MDR1 and MDR2(1-394)-MDR1EKKSK displayed
little ability to efflux [3H]vinblastine (data not
shown). This finding is similar to a previous observation that a
nonfunctional MDR1 mutant carrying a mutation in TM6 displays rather
normal drug-binding affinity. In that case the mutated residue, S344,
is conserved in both MDR1 and MDR2 (20).
Taken together, these results suggest that Q330, V331, and L332 may be
involved in the interaction between MDR1 and its substrates
but perhaps
not by directly binding substrates. Based on the finding
that
substitutions of residues 330, 331, and 332 in TM6 did not
fully
eliminate MDR1 function, we speculate that these three residues
are
structurally important; mutations that occurred at these positions
may
cause a conformational change which not only affects MDR1
substrate
interaction but also changes other unknown steps in
the drug transport
process. This hypothesis is plausible in light
of a recent study which
demonstrates that movement between TM6
and TM12 is essential for
drug-stimulated ATP hydrolysis and drug
transport and that interaction
between L332 and L975 is involved
in this process (
22). In a
recent study, we found that in the
absence of P-gp substrate,
MDR1NAM bound to UIC2, a conformation-sensitive
monoclonal antibody
against P-gp (
23,
24), less well than
wild-type MDR1, but
that this difference was eliminated by the
presence of a P-gp substrate
(
35). Since the UIC2 epitope depends
on a particular P-gp
conformation (
24), the decreased level
of UIC2 recognition
may indicate a conformational change in MDR1
caused by mutations Q330N,
V331A, and
L332M.
Implications for mechanism of action of P-gp.
The multidrug
transporter has been suggested to be a "hydrophobic vacuum
cleaner" (29) or a "multidrug flippase"
(16), owing to its apparent ability to remove drugs directly
from the lipid bilayer. This concept was reenforced by the discovery
that the closely related MDR2 gene product is indeed a
phosphatidylcholine flippase (31) and that MDR1 is also able
to translocate short-chain phospholipids (33). The results
presented here show that the entire amino-terminal TM domain from MDR2
can support multidrug transport with substitution of only three amino
acid residues, suggesting that structural differences between MDR1 and
MDR2 in the N-terminal TM region are much smaller than the
approximately 30% differences observed in their amino acid sequences.
This structural similarity provides further evidence for the essential
mechanistic similarities of these two transporters. Whether this
reflects some association of hydrophobic drug substrates of the P-gp
transporter with membrane lipid or the direct recognition of drugs
intercalated within the plasma membrane remains to be determined. It
would be interesting to determine if MDR2 residues in MDR1 can confer phosphatidylcholine flippase activity on MDR1. However, this assay is
technically difficult, and we have not been able to perform it.
The ability of the N-terminal TM domain of MDR2 to support multidrug
transport is even more surprising in light of previous
results that
segments of MDR2 in the amino-terminal TM region
cannot support MDR1
function even when MDR1 TM6 is preserved (
3,
7). The
current results suggest that MDR2 residues can function
in the
context of other MDR2 residues, but not of MDR1 residues,
in the
amino-terminal TM domain. The simplest interpretation of
these results
is that the amino-terminal TM domain of MDR2 forms
a structure capable
of supporting MDR1 function if and only if
interaction between MDR2
residues is possible. Thus, the requirement
for the folding of
the amino-terminal TM domains of MDR1 and MDR2
would appear to be more
MDR type specific than the requirement
for interaction of the amino-
and carboxyl-terminal TM domain
of the P-gp, except perhaps in
TM6.
The results reported here provide important information for
understanding how drugs and inhibitors interact with P-gp and
the
molecular basis of the difference between the multidrug transporter
and
phosphatidylcholine flippase. These data will help in the
design
of new drugs to reverse drug resistance caused by P-gp.
The ability to
dramatically modify the substrate specificity of
P-gp by
substitution of a few amino acid residues also allows
us to design
transporters with special properties for use as selectable
markers in human gene therapy (
11).
| |
ACKNOWLEDGMENTS |
We are grateful to Christine Hrycyna for providing pTM1
MDR1(FLAG) plasmid and to Bernard Moss for providing the MVA
vaccinia virus.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Molecular Biology, National Cancer Institute, Building 37, Room 4E16, National Institutes of Health Bethesda, MD 20892. Phone: (301) 496-4797. Fax: (301) 402-1344. E-mail: pasta{at}helix.nih.gov.
| |
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Molecular and Cellular Biology, February 1999, p. 1450-1459, Vol. 19, No. 2
0270-7306/99/$00.00+0
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Abstract
Introduction
