From the National Institute of Haematology and
Immunology, Research Group of the Hungarian Academy of Sciences,
H-1113 Budapest, Daróczi u. 24, Hungary, the
§ Institute of Enzymology, Biological Research Center,
Hungarian Academy of Sciences, H-1113 Budapest, Hungary, and the
¶ Department of Genetics, University of Illinois at Chicago,
Chicago, Illinois 60612
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ABSTRACT |
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The human multidrug transporter (MDR1 or
P-glycoprotein) is an ATP-dependent cellular drug extrusion
pump, and its function involves a drug-stimulated, vanadate-inhibited
ATPase activity. In the presence of vanadate and MgATP, a nucleotide
(ADP) is trapped in MDR1, which alters the drug binding properties of
the protein. Here, we demonstrate that the rate of
vanadate-dependent nucleotide trapping by MDR1 is
significantly stimulated by the transported drug substrates in a
concentration-dependent manner closely resembling the drug
stimulation of MDR1-ATPase. Non-MDR1 substrates do not modulate,
whereas N-ethylmaleimide, a covalent inhibitor of the ATPase activity, eliminates vanadate-dependent nucleotide
trapping. A deletion in MDR1 ( amino acids 78-97), which alters the
substrate stimulation of its ATPase activity, similarly alters the drug dependence of nucleotide trapping. MDR1 variants with mutations of key
lysine residues to methionines in the N-terminal or C-terminal nucleotide binding domains (K433M, K1076M, and K433M/K1076M), which
bind but do not hydrolyze ATP, do not show nucleotide trapping either
with or without the transported drug substrates. These data indicate
that vanadate-dependent nucleotide trapping reflects a
drug-stimulated partial reaction of ATP hydrolysis by MDR1, which
involves the cooperation of the two nucleotide binding domains. The
analysis of this drug-dependent partial reaction may
significantly help to characterize the substrate recognition and the
ATP-dependent transport mechanism of the MDR1 pump
protein.
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INTRODUCTION |
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Overexpression of the human multidrug transporter (MDR11 or P-glycoprotein) is responsible for the phenomenon of multiple drug resistance in various cancer cell types. MDR1 is an integral plasma membrane protein that acts as an ATP-dependent efflux pump to reduce the intracellular concentration of diverse hydrophobic compounds (reviewed in Refs. 1-3). MDR1 belongs to the superfamily of the ATP-binding cassette (ABC) transporters and contains a tandem repeat of transmembrane domains and conserved nucleotide-binding motifs connected by a central "linker" region (1, 4, 5).
MDR1 exhibits an ATP hydrolytic activity closely related to its drug
transport function. This ATPase activity is significantly stimulated by
the transported substrate drugs but is blocked by low concentrations of
vanadate (6-10). Senior and co-workers (11-14) demonstrated that in
the presence of vanadate and MgATP, similar to the effect observed with
myosin and other related ATPases (see Ref. 15), MDR1 forms a strong
complex with a radioactively labeled nucleotide. Vanadate stops the
full catalytic cycle of MDR1, most probably by replacing inorganic
phosphate, and stabilizes a protein-trapped form of a nucleotide, which
was found to be exclusively ADP (11). Experimentally, this nucleotide
trapping can be followed by using [-32P]ATP as an
energy donor substrate, because the MDR1-trapped labeled nucleotide is
not removable by washings even in the presence of high concentrations
of MgATP and/or MgADP. A covalent MDR1 labeling occurs if the
photoaffinity analog, 8-azido-[
-32P]ATP is used in the
trapping reaction, followed by UV light treatment.
It has been documented (12) that the formation of enzyme-bound (trapped) ADP from MgATP occurs randomly at the two nucleotide binding sites of MDR1, and whereas one MDR1 molecule is capable for the binding of two MgATP molecules, the saturation stoichiometry for the trapped ADP/MDR1 is one to one. The ADP-associated form of MDR1 may represent a high energy intermediate of the protein, required for the drug-pump function (13).
In their experiments, Senior and colleagues (11-13) did not observe a modulation by the transported drug substrates either of the vanadate-dependent nucleotide trapping, or of the release of the trapped nucleotide in MDR1. In contrast, a substrate stimulation of the labeled nucleotide trapping has recently been reported in the case of MRP, another ABC transporter involved in drug resistance (16). Moreover, when studying the drug substrate interactions with MDR1, Urbatsch and Senior (17) and Dey et al. (18) found a significant inhibition of drug binding after vanadate-dependent nucleotide trapping, indicating a strong interaction between drug binding and ATP hydrolysis.
In the present paper, we demonstrate that the addition of transported drug substrates significantly increases the rate of vanadate-dependent nucleotide trapping in MDR1, when this process is studied under properly selected experimental conditions. To assay the reactions of the wild-type and mutant MDR1, the transport protein and its variants were expressed using the baculovirus-Spodoptera frugiperda (Sf9) insect cell system and characterized by analyzing their nucleotide trapping in isolated membrane preparations. The experiments presented clearly show that the vanadate-dependent nucleotide trapping in MDR1 reflects a drug-dependent partial reaction of the transport cycle, which is significantly modulated by site-directed mutations in the pump protein. Our experiments also indicate a close cooperation of the two nucleotide binding sites in the drug-dependent trapping of nucleotides.
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MATERIALS AND METHODS |
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Chemicals--
8-azido-[-32P]ATP (666 GBq/mmol)
and [
-32P]ATP (111 TBq/mmol) were obtained from ICN
Biomedicals.
MDR1 Expression and Membrane Preparation--
Sf9 cells
were cultured and infected with baculovirus vectors as described in
Ref. 19. Baculovirus transfer vectors were constructed as described
earlier (20, 21) by using the human MDR1 cDNA encoding a
protein with the following mutants: aa 78-97, K433M, K1076M, and
K433M/K1076M.
Electrophoresis and Immunoblotting-- Membranes were suspended in a disaggregation buffer (6). Samples (20 µl) were run on 6% Laemmli-type gels and electroblotted onto polyvinylidene difluoride membranes. Quantitative estimation of the expression of human MDR1 was performed using the polyclonal anti-MDR1 antibody 4077 (22), and a secondary antibody (anti-rabbit peroxidase-conjugated IgG; 20,000 × dilution, Jackson Immunoresearch), as described in Ref. 21. Horseradish peroxidase-dependent luminescence (ECL, Amersham Pharmacia Biotech) was determined by luminography and quantitated by the Bio-Rad phosphorimager system.
Measurement of ATPase Activity-- ATPase activity sensitive to vanadate was measured in isolated membranes as described in Ref. 6.
Vanadate-dependent Nucleotide Trapping--
Isolated
Sf9 cell membranes (100 µg protein) were incubated for 30 s to 10 min at 37 °C in a reaction buffer containing 50 mM Tris-KCl (pH 7.0), 0.1 mM EGTA, 2 mM MgCl2, 200 µM sodium
orthovanadate, 5 mM sodium-azide, and 1 mM
ouabain in a final volume of 50 µl, in the presence of 5-200
µM (final concentration) of Mg-8-azido-ATP or MgATP
containing 0.1-0.2 MBq of 8-azido-[-32P]ATP or
[
-32P]ATP. The reaction was stopped by the addition of
500 µl of ice-cold Tris-EGTA-MgATP-vanadate buffer (50 mM
Tris-KCl, 0.1 mM EGTA, pH 7.0, 10 mM MgATP, 200 µM sodium orthovanadate), the membranes were spun for 20 min at 4 °C at 15,000 × g, the supernatant was removed, 500 µl of Tris-EGTA-ATP-vanadate buffer were added, and the
centrifugation was repeated. In the case of [
-32P]ATP
labeling, the membranes were dissolved in electrophoresis buffer, and
activity was measured in a scintillation counter, whereas in the case
of 8-azido-[
-32P]ATP labeling, the washed pellet was
resuspended in 20 µl of Tris-EGTA buffer, placed in a drop on a
Parafilm-covered glass plate, cooled, and kept on ice. The samples were
irradiated for 10 min with a UV lamp (
max about 250 nm)
at a distance of 3 cm. Thereafter the membranes were collected in 40 µl of the electrophoresis buffer, and the samples were run on 6%
Laemmli-type gels. The proteins were electroblotted onto polyvinylidene
difluoride membranes as described above and the blots were dried and
subjected to autoradiography in a phosphorimager (Bio-Rad). The
identity of the 32P-azido-nucleotide labeled bands was
assured by immunostaining with anti-MDR1 specific antibody (antibody
4077) on the same blot.
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RESULTS |
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Nucleotide Trapping in MDR1 from Mg-8-azido-ATP--
In this set
of experiments, we examined the vanadate-dependent
nucleotide trapping in isolated Sf9 cell membranes expressing the wild-type MDR1 by using the photoaffinity ATP analog,
8-azido-[-32P]ATP. 8-Azido-ATP was reported to be an
efficient energy donor substrate for the MDR1-ATPase, although both the
Km and the Vmax values were
found to be lower for Mg-8-azido-ATP than for MgATP (23). Labeling was
performed in the presence of sodium orthovanadate as described under
"Materials and Methods" for the time
intervals and nucleotide concentrations indicated in Figs. 1 and
2. Verapamil and 5-fluorouracil (5FU)
were used as test drugs because verapamil is a well-known substrate and
activator of the MDR1 ATPase, whereas 5FU is not transported by MDR1
and does not stimulate MDR1-ATPase activity (1, 6, 24). The addition of
1 mM MgATP to the reaction medium abolished
8-azido-nucleotide trapping in MDR1, indicating a competition of MgATP
and Mg-8-azido-ATP at the specific nucleotide binding sites (data not
shown).
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Nucleotide Trapping in MDR1 from MgATP--
The physiological
energy donor for drug transport by MDR1 is MgATP; therefore, we
performed experiments similar to those described above by using
Mg[-32P]ATP. Because nucleotide cross-linking could
not be found in this case (see also Ref. 12), nucleotide trapping was
measured by counting the membrane-bound radioactivity after repeated
washings of the membranes in the presence of 200 µM
sodium orthovanadate and 10 mM MgATP at 4 °C (see
"Materials and Methods").
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Effects of Various Drugs on the Nucleotide Trapping in
MDR1--
Fig. 4 shows the detailed
examination of the effects of various drugs on the nucleotide trapping
in MDR1-expressing membranes incubated at 37 °C for 2 min with 5 µM 8-azido-[-32P]ATP (Fig.
4A), or for 1 min with 50 µM
Mg[
-32P]ATP (Fig. 4B). As shown in Fig.
4A, covalent labeling after nucleotide trapping from
8-azido-ATP was found to be significantly stimulated by the MDR1
substrate drugs verapamil, cyclosporine A (CsA), or calcein AM (CaAM),
whereas free calcein (Ca free) and 5FU, which are not MDR1 substrates
(see Refs. 1, 2, 6-8, and 24), had no effect on this phenomenon.
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Nucleotide Trapping in a Substrate Affinity Mutant of MDR1--
A
mutant form of MDR1 with a 20-amino acid deletion in its first
extracellular loop (aa 78-97 MDR1), has been shown to have a
reduced drug transport capacity (27) and a low level of ATPase activity, stimulated only by extremely high concentrations (above 200 µM) of verapamil (20). Nucleotide trapping in this mutant MDR1, expressed in Sf9 cell membranes, was followed in the
presence of 5 µM Mg-8-azido-ATP and 200 µM
vanadate for 2 min at 37 °C, and covalent labeling was achieved by
UV light treatment.
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Nucleotide Trapping in Nucleotide Binding Site Mutants of
MDR1--
In the following experiments, we examined nucleotide
trapping with 8-azido-[-32P]ATP (followed by
photo-cross-linking) in MDR1 variants in which essential lysine
residues in the Walker A motifs were mutated. These lysines were
replaced by methionines either in the N-terminal ABC domain (K433M), in
the C-terminal ABC domain (K1076M), or in both ABC domains
(K433M/K1076M). These mutant MDR1s, when expressed in Sf9 cells,
were shown to demonstrate significant 8-azido-ATP binding but no drug
transport or drug-stimulated ATPase activity (21). Because, in these
mutant proteins, nucleotide binding may also be altered at low MgATP
concentrations (21), we examined their nucleotide trapping under two
different experimental conditions.
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DISCUSSION |
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The detailed reaction mechanism of ABC transporters, which involves the hydrolysis of MgATP and a concomitant membrane transport of the relevant substrates, is still largely unknown. The discovery of a vanadate-dependent trapping of a nucleotide during the reaction cycle of the multidrug transporter MDR1 (11-14), opened new avenues for the detailed examination of its reaction mechanism. In the present experiments, we studied the effects of drug substrates on this nucleotide trapping in MDR1, as well as the modulation of this phenomenon by well characterized mutations in the protein.
In the catalytic cycle model originally suggested for MDR1 by Senior et al. (13), MgATP binding to MDR1 is followed by a conversion of this nucleotide to MgADP and Pi. Addition of vanadate blocks MDR1-ATPase probably by replacing the released inorganic phosphate and results in the formation of a trapped nucleotide, which is predominantly ADP (12, 13). Our experiments indicate that the vanadate-dependent nucleotide trapping by MDR1 most probably represents a partial reaction of the enzyme activity, the rate of which is greatly increased by the transported drug substrates. We suggest that vanadate trapping stabilizes an occluded, enzyme-bound form of the cleaved nucleotide, the formation of which shows a drug concentration dependence closely resembling the drug stimulation of the full MDR1-ATPase cycle.
Judged from MgATP binding studies (see Refs. 28 and 29) and from the MgATP dependence of the MDR1-ATPase (see Refs. 6, 10, and 25), MDR1 binds MgATP with a relatively low affinity, with a KD of about 200-500 µM. The binding of MgATP occurs randomly at the two nucleotide binding sites, and the stoichiometry of MgATP/MDR1 at saturating MgATP concentrations is 2:1 (12). Data in the literature indicate that this MgATP binding is not affected by the presence of drug substrates (1, 10, 28). Experimental data also suggest that primary drug binding by MDR1 (as measured at 4 °C) is similarly unaffected by the presence or absence of MgATP (see Refs. 17 and 30); thus, initial MgATP and drug binding seem to be independent reactions.
In contrast to MgATP binding, ATP hydrolysis requires an interaction of the protein with the transported drug substrate. Human MDR1-ATPase activity, as measured in isolated Sf9 cell membranes, is about 5-6-fold higher in the presence than in the absence of drug substrates, e.g. verapamil (the "basic" MDR1-ATPase activity is probably supported by endogenous lipid-like or other hydrophobic molecules in the membrane preparation). As we demonstrate in this report, the partial reaction of MDR1-ATPase (reflected in the MDR1 nucleotide trapping), is also strongly accelerated by the transported substrates.
In previous studies (11-13), this drug stimulation of nucleotide trapping was not observed, probably because the experiments were carried out at relatively high ATP concentrations and/or for long measurement periods. Drug substrate stimulation of the nucleotide trapping in our experiments is also restricted to early time periods and unsaturating MgATP (or 8-azido-MgATP) concentrations, that is, under conditions where the drug-dependent acceleration still significantly affects the "titration" of the relevant nucleotide trapping site. It is interesting to note that the differences between the concentration dependence and the time course of nucleotide trapping observed here for Mg-8-azido-ATP and for MgATP can be well explained by the lower Km and Vmax values of Mg-8-azido-ATP than of MgATP (17, 29): although higher MgATP concentrations are required to saturate the nucleotide binding sites, both ATP splitting and nucleotide trapping are faster with MgATP than with Mg-8-azido-ATP. In our present experiments under near-saturating conditions, the molar ratio of nucleotide trapping by MDR1 was estimated to be about 0.4-0.6, supporting the conclusion of Urbatsch et al. (12) that only one nucleotide is promoted to an occluded state during this partial reaction.
The drug substrate concentration dependence of the MDR1-ATPase activity
and that of the nucleotide trapping were found to be similar, that is,
a significant stimulation of both reactions was obtained by similar
concentrations of verapamil, vincristine, rhodamine 123, or calcein AM.
The data obtained for the deletion mutant in the first extracellular
loop of MDR1 (aa 78-97), namely that stimulation could be obtained
only with extremely high concentrations of verapamil, also strongly
suggest that substrate interactions modify the process of nucleotide
trapping similarly to that of the MDR1-ATPase activity. Still, some
substrates (such as CsA) that yield only very small stimulation of the
ATPase but strongly inhibit its verapamil stimulation (31) produced a
much greater stimulation of nucleotide trapping (the inhibition of this
reaction at higher CsA concentrations was also apparent). These data
indicate that the partial reaction of nucleotide trapping may be
efficiently promoted by some substrates that can yield only a low
turnover for the MDR1-ATPase activity, which reflects the full
catalytic cycle.
Interaction of the multidrug transporter with SH group reagents, such as NEM, preferentially occurs at the two cysteine residues in the two nucleotide binding domains (29, 32). Pretreatment of MDR1 with SH group reagents blocks MDR1-ATPase activity (29) but at low concentrations does not inhibit the primary binding of MgATP or drug substrates, as shown by the MgATP- or drug-dependent quenching of an MDR1-bound fluorescent SH-reactive probe (32). A recent communication (33) suggests that NEM may in fact increase drug binding by MDR1 under certain conditions. Because, as reported here, NEM fully blocks drug-stimulated nucleotide trapping, MgATP and primary drug substrate binding most probably occur independently from the following reaction steps, leading to ATP hydrolysis.
The mutant MDR1 proteins, in which lysines in the first (K433M), second (K1076M), or both nucleotide binding domains are replaced by methionines, were demonstrated to bind MgATP less efficiently at low MgATP concentrations (2-5 µM) but similarly to the wild-type MDR1 at concentrations above 10 µM MgATP (21). None of these MDR1 variants possess measurable ATPase activity (21); thus, a mutation in one ATP binding domain is sufficient to eliminate the catalytic reaction in the whole pump protein (see also Refs. 26 and 34). This finding was interpreted to indicate a strong cooperative interaction between the two nucleotide binding domains of MDR1. As we demonstrate here, independently of the presence or absence of drug substrates, none of these nucleotide binding site mutants perform the nucleotide trapping reaction. Thus, a mutation in one of the nucleotide binding domains eliminates nucleotide trapping entirely, showing a cooperation between the ABC domains already in this partial reaction.
Based on the above-described features, we propose that the molecular mechanism of the vectorial drug transport by MDR1 is initiated by the independent primary binding of MgATP and the drug substrate. The following conformational changes in the structure of the MDR1 protein induce a drug-dependent cleavage and a concomitant occlusion of a MgADP molecule, whereas the full catalytic cycle involves the transport of drug substrate to the external membrane surface and the full hydrolysis of ATP and the dissociation of ADP and inorganic phosphate. All of the steps after MgATP and drug binding are based on the functional cooperation of the two ATP binding domains and the appropriate drug binding domains within MDR1.
The drug-dependent ATP hydrolysis and occlusion most probably significantly alter the conformation and/or location of the drug binding site(s). As shown by Urbatsch and Senior (17), vanadate-induced nucleotide trapping significantly reduced azidopine labeling in MDR1. Recent experiments of Dey et al. (18) demonstrated the presence of two nonidentical drug-interaction sites in the MDR1 protein. In this study the C-terminal drug-recognition ("on") site was found to be significantly more sensitive to vanadate trapping of nucleotides than the N-terminal ("off") site, and ATP hydrolysis was essential for the vanadate-induced reduction of drug binding. Our experiments and these data collectively suggest that ATP hydrolysis is strongly coupled to the movement of the drug substrate from an "on" to an "off" site within the MDR1 protein, and nucleotide occlusion may coincide with the occlusion of the drug binding site(s), leading to a vectorial movement of the drug substrate.
It should be noted that the above-described stimulation of nucleotide trapping in MDR1 by the substrate drugs may also be efficiently employed for the screening of specific drug interactions with MDR1, because even those substrates (e.g. CsA) that predominantly inhibit the MDR1-ATPase activity may show a strong stimulation of nucleotide trapping. Moreover, the investigation of substrate stimulation of nucleotide trapping in other related proteins, e.g. CFTR, TAP, or MRP, may help to understand the reaction mechanism and transport characteristics of these ABC transporters.
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ACKNOWLEDGEMENTS |
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We thank Prof. M. M. Gottesman for providing the anti-MDR1 polyclonal antibodies. The technical help of Ilona Zombori and Györgyi Demeter is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported by OMFB, OTKA Research Grants T17602, T22072, F23662, F13178, and F23655; ETT (Hungary); NCI National Institutes of Health Grant R37 CA40333; and Fogarty International Grant R03 TW00586.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.
Howard Hughes International Research Scholar. To whom
correspondence should be addressed. Tel. and Fax: 36-1-372-4353;
E-mail: B.Sarkadi{at}ohvi.hu.
1 The abbreviations used are: MDR1, human multidrug resistance protein; 5FU, 5-fluorouracil; ABC, ATP-binding cassette; CsA, cyclosporine A; NEM, N-ethylmaleimide; Sf9 cells, Spodoptera frugiperda ovarian cells; aa, amino acid.
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REFERENCES |
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