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J Biol Chem, Vol. 273, Issue 27, 16631-16634, July 3, 1998
From the Human P-glycoprotein (P-gp), an
ATP-dependent efflux pump responsible for cross-resistance
of human cancers to a variety of lipophilic compounds, is composed of
two homologous halves, each containing six transmembrane domains and an
ATP-binding/utilization domain. To determine whether each site can
hydrolyze ATP simultaneously, we used an orthovanadate (Vi)-induced
ADP-trapping technique (P-gp·MgADP·Vi). In analogy with other
ATPases, a photochemical peptide bond cleavage reaction occurs within
the Walker A nucleotide binding domain consensus sequence
(GX4GK(T/S)) when the molecule is trapped with Vi in
an inhibited catalytic transition state (P-gp·MgADP·Vi) and
incubated in the presence of ultraviolet light. Upon reconstitution into proteoliposomes, histidine-tagged purified P-gp from
baculovirus-infected insect cells had drug-stimulated ATPase activity.
Reconstituted P-gp was incubated with either ATP or 8-azido-ATP in the
presence or absence of Vi under ultraviolet (365 nm) light on ice for
60 min. The resultant products were separated by SDS-polyacrylamide gel
electrophoresis and subjected to immunoblotting with seven different
human P-gp-specific antibodies covering the entire length of the
molecule. Little to no degradation of P-gp was observed in the absence
of Vi. In the presence of Vi, products of approximately 28, 47, 94, and
110 kDa were obtained, consistent with predicted molecular weights from
cleavage at either of the ATP sites but not both sites. An additional
Vi-dependent cleavage site was detected at or near the
trypsin site in the linker region of P-gp. These results suggest that
both the amino- and carboxyl-terminal ATP sites can hydrolyze ATP.
However, there is no evidence that ATP can be hydrolyzed simultaneously
by both sites.
One of the main causes of broad-based cellular resistance to a
wide variety of cytotoxic agents in cancer cells is expression of a
170-kDa plasma membrane polypeptide known as the multidrug transporter
or P-glycoprotein (P-gp),1
encoded by the MDR1 gene in humans (1, 2). This 1280-amino acid plasma membrane-associated glycoprotein is composed of two homologous halves, each containing six transmembrane domains and one
ATP site. P-gp acts as an ATP-dependent efflux pump for
chemotherapeutic agents and other drugs (3). The precise mechanism of
action of P-gp, however, remains unknown. ATP binding and hydrolysis are essential for the proper functioning of P-gp. It has been previously demonstrated that each ATP site in P-gp can hydrolyze ATP
and that both sites must be intact to retain activity of the transporter (4, 5). These data suggested a model of P-gp action in
which the ATP sites alternate and do not hydrolyze ATP simultaneously
(6, 7).
To determine whether both sites hydrolyze ATP simultaneously, we used
orthovanadate (Vi), a phosphate analog that stabilizes the inhibited
catalytic transition state of P-gp (P-gp·MgADP·Vi), mimicking the
physiological state in which MgADP and phosphate are bound and
subsequently released (8). Upon incubation with Vi and ATP, only one
cycle of hydrolysis occurs as a result of the stabilization of the
inhibitory complex (9). When this complex is irradiated with
ultraviolet light, a photochemical reaction occurs modifying the amino
acid in the third position within the Walker A nucleotide binding
domain consensus sequence (GX4GK(T/S)) (10)
followed by cleavage of the peptide bond (11). This technique has been
successfully used to study the mechanism of action of myosin-ATPase
(12, 13), adenylate kinase (14), and most recently, the
F1-ATPase from rat liver mitochondria (15). The studies
described here represent the first use of this technique in the study
of an ATP-binding cassette (ABC) transporter. The results indicate that
ATP hydrolysis occurs within one or the other ATP site but could not be
detected in both simultaneously.
Expression and Purification of Wild-type P-gp Containing a
C-terminal 6-Histidine Tag (P-gp·H6)--
Recombinant
baculovirus encoding wild-type P-gp containing a six-histidine tag at
the C terminus (BV-MDR1(H6)) was used to infect
Trichoplusia ni (High FiveTM) cells (Invitrogen,
San Diego, CA) as described (16). P-gp·H6 was purified by
metal affinity chromatography as described (16). Protein concentration
of the purified material was determined by the Amido Black 10B protein
assay (17). Approximately 300 µg of purified protein was obtained
from 20 mg of crude membrane protein prepared from 2 × 108 cells.
Preparation of Sodium Orthovanadate--
Sodium orthovanadate
was freshly prepared in water and heated at 100 °C for 3 min,
vortexed, and cooled to room temperature. The concentration of the
stock solution was determined spectrophotometrically at
A268 (molar extinction coefficient, 2925 M Measurement of Vi-sensitive ATPase Activity in Proteoliposomes
Reconstituted with Purified P-gp·H6--
Vi-sensitive
ATPase activity in purified P-gp·H6 preparations was
performed as described (16, 18).
Photochemical Cleavage of Purified
P-gp·H6--
Purified P-gp·H6 (4 µl;
~1.4 µg) was first diluted 25-fold to a final volume of 100 µl to
form proteoliposomes in 50 mM MOPS·KOH (pH 7.2), 125 mM KCl, 5 mM MgCl2 in the presence
and absence of 600 µM sodium orthovanadate in 12 × 75-mm glass test tubes and allowed to incubate at room temperature for
3 min. Verapamil (30 µM) was subsequently added to all
samples from a 3 mM stock made in Me2SO, and
the tubes were allowed to stand at room temperature for an additional 3 min. Subsequently, 2.5 mM ATP was added, and the reaction
mixture was immediately transferred to ice. In reactions where
photoactivation was induced by UV light, the samples were transferred
to a 96-well flat-bottom cluster and were placed under a 365-nm
ultraviolet (UV) lamp (Black-Ray lamp from UVP, Upland, CA) on aluminum
foil-covered ice under subdued light conditions. The samples were
irradiated for 40 min covered with a glass plate at a distance of 1.3 cm and for an additional 20 min uncovered at a distance of 1 cm. For
samples containing 8-azido-ATP, the reaction was pre-irradiated for 10 min on ice prior to addition of drug and Vi.
SDS-Polyacrylamide Gel Electrophoresis (PAGE) and Immunoblot
Analysis--
Samples were prepared in 1× Laemmli sample buffer (19)
and allowed to incubate at room temperature for 30 min prior to
electrophoresis. SDS-PAGE was performed (19) using 8, 8-16, and
4-20% Tris/glycine gels (Novex, San Diego, CA) followed by
immunoblotting as described (16). Immunoreactive bands were visualized
by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).
Antibodies--
Monoclonal anti-P-gp antibody C219 (Centocor,
Malvern, PA) (20) was used at a 1:2000 dilution. Human specific
anti-P-gp polyclonal antibodies 4007 and 4077 (21) were used at
dilutions of 1:1000 and 1:3000, respectively. Polyclonal human specific anti-P-gp antibodies PEPG-13, PEPG-2, and PEPG-7 were used at dilutions
of 1:3000, and PEPG-12 was used at a dilution of 1:1000 (22).
Mild Trypsin Digestion of Purified
P-gp·H6--
Purified P-gp·H6 (1.4 µg)
was diluted in a total volume of 100 µl in 50 mM
Tris·HCl (pH 8.0). Modified trypsin (Promega) was added at a ratio of
30:1 protein:trypsin (0.046 µg). The reaction was carried out for 5 min at 37 °C. Subsequently, a 5-fold excess of trypsin inhibitor was
added followed by Laemmli sample buffer.
Human P-gp is a 1280-amino acid protein with two homologous halves
functionally connected by a flexible linker region. Each half contains
six hydrophobic transmembrane regions implicated in the binding of
substrates and inhibitors based on photoaffinity labeling studies and
the behavior of mutant transporters and a highly conserved ATP
binding/utilization domain (1). Through mutational analysis, it has
been demonstrated that both sites are essential for function since
disruption of either nucleotide binding domain results in an inactive
protein (23-25). Biochemical analyses using P-gp from Chinese hamster
ovary cells have revealed that each ATP site is capable of hydrolyzing
ATP (4).
In this study, we sought to determine whether both ATP sites of human
P-gp were acting independently and hydrolyzing ATP simultaneously or if
cross-talk exists between the two sites that allows for only one
hydrolysis event to occur at a time. This alternating catalytic site
model of ATP hydrolysis was originally suggested by Senior and
colleagues (6) in studies that demonstrated that 1 mol of
Mg2+-8-azido-ADP was bound per mol of hamster P-gp. This
hypothesis has been supported by experiments involving chemical
modification of one ATP site that prevented vanadate trapping at the
other site (5).
Model for Photooxidative Peptide Bond Cleavage of Human
P-gp--
To assess directly whether ATP hydrolysis can occur at both
sites simultaneously, we made further use of sodium Vi, a phosphate analog that is photochemically active. Irradiation with UV light at 365 nm results in specific oxidations of protein side chains within
Vi-trapped species and in peptide bond cleavage. The mechanism of
photocleavage for myosin, which involves a seryl radical intermediate, has been determined by Grammer et al. (11). ATPases form a
MgADP·Vi·enzyme inhibitory complex, which upon irradiation results
in peptide bond cleavage at the third position within the Walker A
motif (GX4GKT). This has been shown directly for
myosin (serine) (12, 13), F1-ATP synthase (alanine) (15),
and adenylate kinase (proline) (14), and flagellar ATPase dynein (26).
Human P-gp has a serine residue at the third position in both
nucleotide binding domains.
COMMUNICATION
Mechanism of Action of Human P-glycoprotein ATPase Activity
PHOTOCHEMICAL CLEAVAGE DURING A CATALYTIC TRANSITION STATE USING
ORTHOVANADATE REVEALS CROSS-TALK BETWEEN THE TWO ATP SITES*
§,
,,
,,§§
Laboratory of Cell Biology and
¶ Laboratory of Molecular Biology, Division of Basic Sciences,
National Cancer Institute, National Institutes of Health, Bethesda,
Maryland 20892 and ** Department of Biological Chemistry, The Johns
Hopkins University School of Medicine, Baltimore, Maryland 21205
ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References
INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References
1 cm2). Vi prepared in this
manner may consist of varying amounts of monomeric vanadate and its
oligomers, di-, tetra-, and pentavanadate.
RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References
Schematic Representation of Potential UV-induced Vanadate Cleavage Sites in Human P-gp-- A schematic diagram of the potential vanadate cleavage sites in human P-gp is shown in Fig. 1. The cleavage products are identified using antibodies specific for P-gp. The epitopes for these various antibodies are shown in Fig. 1F. If simultaneous hydrolysis occurs at both ATP sites, three fragments would be generated (Fig. 1D) of approximately 47, 71, and 23 kDa. If hydrolysis occurs only at the N-terminal site (Fig. 1B), only 47- and 94-kDa fragments would be produced as a result of the UV-induced cleavage reaction in the presence of Vi. Conversely, if hydrolysis only occurs at the C-terminal ATP site (Fig. 1C), only fragments of predicted molecular masses of 118 and 23 kDa would be generated. If both sites were active but not in the same molecule, 47-, 94-, 118-, and 23-kDa fragments would be predicted. If nucleotide-independent cleavage at or near the trypsin site of P-gp occurs, the molecule would also be cleaved into two peptides of 80 and 60 kDa representing the N-terminal and C-terminal halves of the protein, respectively (Fig. 1E).
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Purified P-gp Retains Vi-sensitive Drug-stimulated ATPase Activity-- To facilitate identification of the UV-induced vanadate cleavage products and to eliminate other interfering ATPases, we used wild-type P-gp containing a six-histidine tag at the C terminus (P-gp·H6) purified from insect cells using metal affinity chromatography as described under "Experimental Procedures" (16). Using a rapid dilution method to reconstitute the protein into proteoliposomes, Vi-sensitive drug-stimulated ATPase activity of P-gp·H6 was confirmed (16). The reconstituted protein demonstrated high specific activity (5.8 µmol/min/mg of protein) in the presence of 30 µM verapamil. This method reconstitutes approximately 20% of the starting material, 50% of which has ATPase activity (16). This yield of functional P-gp was used to calculate the specific activity of the protein.
UV-induced Vi Cleavage of the Human P-gp·H6 Polypeptide Chain-- We performed the UV-induced vanadate cleavage reactions under the same optimal conditions as in the ATPase activity assay described above, except that MOPS buffer was substituted for Tris because it is known that Tris buffers result in less efficient photocleavage because of the formation of stable Tris·Vi complexes (28). P-gp ATPase activity is comparable in either Tris or MOPS buffer (data not shown). Purified P-gp·H6 was reconstituted into proteoliposomes by rapid dilution either in the presence or absence of Vi. To start the formation of the MgADP·Vi·P-gp complex, 2.5 mM ATP was added, and the samples were immediately irradiated on ice for a total of 60 min and subjected to SDS-PAGE and immunoblot analysis as described under "Experimental Procedures."
Photooxidative Peptide Bond Cleavage at the ATP Sites of Human P-gp Is Vi- and UV-dependent-- Peptide bond cleavage occurs only in the presence of Vi and UV irradiation (Fig. 2, lanes 3). UV irradiation alone generated little or no cleavage products (Fig. 2, lanes 2). Additionally, little or no cleavage was observed in the absence of UV light either in the presence or absence of Vi (Fig. 2, lanes 1 and 4). Because both the amount of functional P-gp·H6 in the reaction (200 ng) and the efficiency of the cleavage reaction were extremely low, we could not visualize the bands by Coomassie Brilliant Blue or silver stain nor could we generate enough of each fragment for N-terminal sequencing. However, using a variety of human P-gp-specific antibodies that recognize different regions of the molecule, we were able to clearly identify the cleavage products (a), (b), (c), and (d) but not (e) (Fig. 1F). Because of the hydrophobic nature of the peptides generated, there was a slight disparity between the predicted molecular weights of the products (Fig. 1) and the apparent molecular weights as determined by SDS-PAGE. The crucial results are, however, that we detect the higher molecular weight fragments (b and c) but not fragment (e) (Fig. 1), arguing against simultaneous hydrolysis and cleavage at both ATP sites. The absence of this fragment does not prove unequivocally that it is not being formed in amounts below the level of detectability. We do not believe that it is present but migrating anomalously because we could not detect this fragment in several gel systems with different antibodies.
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Vi-induced Cleavage of Human P-gp Near the Trypsin-sensitive Site in the Linker Region-- In our experiments, two additional bands migrating at approximately 80 and 60 kDa are apparent (Figs. 2 and 3). The 80-kDa band cross-reacts with 4077 (Figs. 2A and 3B), PEPG-13 (Figs. 2C and 3C), and PEPG-7 (Fig. 2B), and the 60-kDa band is preferentially recognized by PEPG-12 (Figs. 2D and 3E) and 4007 (data not shown). Both bands were recognized by PEPG-2 (Fig. 3D). Because the 80-kDa peptide is recognized well by 4077, PEPG-7, and PEPG-9 directed against amino acids 348-419 (data not shown), it is unlikely that this fragment represents fragment (e) (Fig. 1), the product of UV-induced Vi peptide bond cleavage at both ATP sites in the same P-gp molecule. Because both bands were recognized by PEPG-2, the cleavage site must necessarily reside between amino acids 637 and 712. Conversely, PEPG-12 and 4007 preferentially recognize the C-terminal half although PEPG-12 can under certain conditions weakly recognize some fragments containing the N-terminal half. Under the conditions used in this experiment, C219 does not detect these fragments (Fig. 3A). Importantly, the linker region of human P-gp, defined as the peptide segment between amino acids 633 and 709, is known to be sensitive to cleavage by mild trypsin digestion (27). Taken together, these results suggest that the two bands most likely represent the N- and C-terminal halves of P-gp produced by vanadate cleavage at or near this lysine/arginine-rich trypsin-sensitive region of P-gp, as has been previously observed for myosin (12). Additionally, upon overexposure of these immunoblots (data not shown), the natural degradation products generated during manipulation of the untreated samples for electrophoretic analysis are apparent and migrate in the same positions as the Vi-induced 80- and 60-kDa products, lending credence to the argument that these bands represent the two halves of the protein.
To further confirm the identity of these fragments representing the N- and C-terminal halves of P-gp·H6, the purified protein was treated mildly with trypsin, followed by SDS-PAGE, immunoblotting, and probing with human P-gp-specific antibodies (Fig. 4). PEPG-2 (Fig. 4A) recognizes both halves of the protein whereas 4077 (Fig. 4B) recognizes the N-terminal half and 4007 preferentially recognizes the C-terminal half (Fig. 4C). Migration positions are similar to those observed in the cleavage reactions shown in Figs. 2 and 3.
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Mechanism of ATP Hydrolysis of Human P-gp-- In this study, we have demonstrated that both ATP sites of human P-glycoprotein are capable of hydrolyzing ATP because we generate peptide products accounting for cleavage at both active sites. We have no evidence, however, for any detectable amount of the 71 kDa (e) double cleavage fragment under the reaction conditions tested and with any of the antibodies used. This fragment along with the (a) and (d) fragments would have been generated if simultaneous hydrolysis was occurring at both of the ATP sites. Our data suggest that cleavage can occur only at one site at a time and that the two sites are functionally interdependent. These data do not demonstrate, however, that the catalytic sites alternate with equal efficiency but only that they are unlikely to act simultaneously.
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FOOTNOTES |
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* 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 postdoctoral fellowship from The Jane Coffin Childs Memorial Fund for Medical Research.
Present address: Canji, Inc., 3030 Science Park Rd., San
Diego, CA 92121.
Supported in part by a grant from NIDDK, NIH (to
P. L. P.).
§§ To whom correspondence should be addressed: Laboratory of Cell Biology, Bldg. 37, Rm. 1A-09, NCI, National Institutes of Health, 37 Convent Dr. MSC 4255, Bethesda, Md 20892-4255. Tel.: 301-496-1530; Fax: 301-402-0450; E-mail: mgottesman{at}nih.gov.
1 The abbreviations used are: P-gp, P-glycoprotein; P-gp·H6, human P-glycoprotein containing a 6-histidine tag at the C terminus of the protein; Vi, sodium orthovanadate; UV, ultraviolet light at 365 nm; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; aa, amino acid.
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REFERENCES |
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References
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Vi/
