Effects of ATP depletion and phosphate analogueson P-glycoprotein conformation in live cells Katalin Goda1, Henrietta Nagy1, Eugene Mechetner2, Maurizio Cianfriglia3and Ga´bor Szabo´ Jr1
Trang 1Effects of ATP depletion and phosphate analogues
on P-glycoprotein conformation in live cells
Katalin Goda1, Henrietta Nagy1, Eugene Mechetner2, Maurizio Cianfriglia3and Ga´bor Szabo´ Jr1
1
Department of Biophysics and Cell Biology, University of Debrecen, Hungary;2Chemicon International, Inc., Temecula, CA, USA;
3
Laboratorio di Immunologia, Istituto Superiore di Sanita`, Rome, Italy
P-glycoprotein (Pgp), a membrane pump often responsible
for the multidrug resistance of cancer cells, undergoes
con-formational changes in the presence of
substrates/modula-tors, or upon ATP depletion, reflected by its enhanced
reactivity with the UIC2 monoclonal antibody When the
UIC2-shift was elicited by certain modulators (e.g
cyclo-sporin A or vinblastine, but not with verapamil or
Tween 80), the subsequent binding of other monoclonal
anti-Pgp Ig sharing epitopes with UIC2 (e.g MM12.10) was
abolished [Nagy, H., Goda, K., Arceci, R., Cianfriglia, M.,
Mechetner, E & Szabo´ Jr, G (2001) Eur J Biochem 268,
2416–2420] To further study the relationship between
UIC2-shift and the suppression of MM12.10 binding, we
compared, on live cells, how ATP depletion and treatment of
cells with phosphate analogues (sodium orthovanadate,
beryllium fluoride and fluoro-aluminate) that trap
nucleo-tides at the catalytic site, affect the two phenomena Similarly
to modulators or ATP depleting agents, all the phosphate
analogues increased daunorubicin accumulation in
Pgp-expressing cells Prelabeling of ATP depleted cells with UIC2 completely abolished the subsequent binding of MM12.10,
in accordance with the enhanced binding of the first mAb Vanadate and beryllium fluoride, but not fluoro-aluminate, reversed the effect of cyclosporin A, preventing UIC2 binding and allowing for labeling of cells with MM12.10 Thus, changes in UIC2 reactivity are accompanied by complementary changes in MM12.10 binding also in re-sponse to direct modulation of the ATP-binding site, con-firming that conformational changes intrinsic to the catalytic cycle are reflected by both UIC2-related phenomena These data also fit a model where the UIC2 epitope is available for antibody binding throughout the catalytic cycle including the step of ATP binding, to become unavailable only in the catalytic transition state
Keywords: P-glycoprotein; multidrug resistance; UIC2; MM12.10; conformation
P-glycoprotein (Pgp) is an integral plasma membrane
protein that functions as an ATP-dependent efflux pump
for a broad range of lipophylic or amphiphylic compounds
[1–3] Expression of this, and related pumps on cancer cells,
renders them resistant to a wide range of cytotoxic
compounds, causing multidrug resistance (mdr) [1–3] Based
on the structure of its two ATP binding sites and the
mechanism of ATP hydrolysis, Pgp belongs to the ATP
Binding Cassette (ABC) family of transport ATPases [4,5]
The molecule is comprised of two homologous halves, each
containing an ATP binding site characterized by Walker A
and B sequence motifs, and six transmembrane segments
The two halves of Pgp are connected by a linker peptide
( 75 amino acids) composed of charged amino-acid
residues with several phosphorylation sites [1,6] As no
difference was found in resistance between cells transfected
with wild-type and phosphorylation defective forms of the human Pgp, the role of phosphorylation sites is not clearly understood [7]
Pgp interacts directly with its substrates, probably within the cell membrane, and transports them out reducing their intracellular concentration [1,8] A number of compounds, often referred to as modulators (reversing agents, chemo-sensitizers), are capable of decreasing or eliminating mdr by preventing Pgp-mediated substrate export [1,9]
The protein exhibits a substrate-stimulated ATPase activity, suggesting that ATP hydrolysis and drug transport are intimately linked [10] Phosphate (Pi) analogues, e.g vanadate (Vi), beryllium fluoride (BeFx; the exact compo-sition of the complex is unknown) and fluoro-aluminate (AlF4) are potent inhibitors of Pgp ATPase activity [10–15] The characteristics of their inhibitory effect are similar in general, as it is due to trapping of nucleotides at the catalytic site in a noncovalent but tenaciously bound form [10–15] However, BeFx and Vitrap only ADP, while AlF4 traps both ATP and ADP, when Pgp molecules are preincubated with MgATP [11,12] PPiprotects effectively against BeFx inhibition of Pgp by competing with BeFx, whereas PPihad
no effect on inhibition by Vi [12] These data were interpreted to suggest that Pianalogues may trap Pgp at different steps of the catalytic process [13]
The Vi-trapped intermediate of Pgp (i.e PgpÆMg– ADPÆVi) is thought to be equivalent to the PgpÆMg–ADPÆPi complex, which represents the catalytic transition-state in the normal reaction pathway [14,15] The formation of
Correspondence to G Szabo´ Jr, Department of Biophysics
and Cell Biology, University of Debrecen, PO Box 39,
H-4012 Debrecen, Hungary.
Fax/Tel.: + 36 52 412 623,
E-mail: szabog@jaguar.dote.hu
Abbreviations: Pgp, P-glycoprotein; mdr, multidrug resistance;
ABC, ATP Binding Cassette; V i , vanadate; BeF x , beryllium
fluoride; AlF 4 , fluoro-aluminate; CsA, cyclosporin A, FITC,
fluorescein-5-isothiocyanate.
(Received 6 December 2001, revised 18 March 2002,
accepted 12 April 2002)
Trang 2Pgp Mg–ADP Viintermediate occurs randomly at one of
the two nucleotide-binding sites of Pgp, but not
simulta-neously [14–16] It has been demonstrated in photoaffinity
labeling experiments that the affinity of Pgp to different
substrates is decreased after Vitrapping, supporting the idea
that during ATP hydrolysis Pgp undergoes conformational
changes also affecting the drug binding site [17]
Conformational changes of Pgp upon its catalytic cycle
can be detected by measuring the binding of certain
monoclonal antibodies (mAbs; UIC2 [18], MC57 [19]) It
was previously shown that reactivity of the UIC2 mAb with
Pgp is increased in the presence of substrates/modulators or
ATP depleting agents, or when both nucleotide-binding
sites are inactivated by mutations [18] The upshift of UIC2
binding in the presence of substrates/modulators is applied
as an indicator of the expression of functional Pgp
molecules in clinical tissue samples [20] Changes in the
proteolysis profile of Pgp were also detected in the presence
of nucleotides alone or ATP with Vi, indicative of
conformational changes propagating to the extracellular
domains of the pump upon its interaction with these
nucleotides [21]
We have recently shown [22] that drug transport-related
conformational changes of Pgp can also be detected via
mAb competition involving UIC2 Using the
UIC2-MM12.10 mAb pair, we have described that cyclosporin A
(CsA), vinblastine, and valinomycin (and several other
drugs; N Nagy, K Goda, F Fenyvesi & G Szabo´ Jr,
unpublished data)
preincubation of the cells with UIC2 completely suppresses
the subsequent binding of MM12.10 In contrast, UIC2
mAb added at saturating concentration decreased the extent
of MM12.10 labeling only mildly (up to 40%) without
drug treatments, or when the cells were preincubated with
verapamil or Tween-80 (and other drugs; N Nagy, K
Goda, F Fenyvesi & G Szabo´ Jr, unpublished data)
conformational state characterized by enhanced UIC2/
MM12.10 mAb competition (i.e complete suppression of
MM12.10 labeling after UIC2 binding) may be intrinsic to
the normal catalytic cycle, or, alternatively, the CsA-type
drugs may induce a special conformation adopted by Pgp
only in the presence of these drugs The first possibility
would be confirmed if the same conformational state could
be induced by nonsubstrate agents interfering with the
ATPase cycle To study this question, we compared the
effects of Pianalogues (Vi, BeFx, AlF4), trapping nucleotides
at the catalytic site of Pgp, and of ATP depletion, on
UIC2-shift and UIC2/MM12.10 competition Accurate molecular
interpretation of the changes in mAb binding are expected
to help to visualize the conformational steps during the
catalytic cycle
M A T E R I A L S A N D M E T H O D S
Cell lines
The human mdr1-transfected NIH 3T3 (NIH 3T3 MDR1
G185) mouse fibroblast cells [23] and the drug sensitive
human epidermoid carcinoma cell line KB-3-1 and its
multidrug resistant relative KB-V1 [24], obtained by
vinblastine selection, were used The cell lines (obtained
from M Gottesman, NIH, Bethesda, USA) were grown as
monolayer cultures at 37°C in an incubator containing 5%
CO2 and maintained by regular passage in Dulbecco’s minimal essential medium (supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine and
25 lgÆmL)1 gentamycin) KB-V1 and NIH 3T3 MDR1 cells were cultured in the presence of 180 nMvinblastine or
670 nM doxorubicin, respectively Cells were trypsinized 2–3 days prior to the experiments and maintained without vinblastine or doxorubicin until use The cells were occa-sionally checked for mycoplasma
culture rapid detection system with a 3H-labeled DNA probe from General-Probe Inc and were found to be negative
Chemicals The cells were treated with the modulators or other agents at the following concentrations: 100 lM verapamil, 100–
500 lM Na-orthovanadate (from a 13.5-mM translucent stock solution freshly prepared in distilled water [25]), fluoro-aluminate (AlF4; prepared from 1 mM AlCl3 and
5 mMNaF), beryllium fluoride (BeFx; prepared from 2 mM BeSO4 plus 10 mM NaF) [11], 10 lM cyclosporin A, 50–
100 lM vinblastine or 10 lM valinomycin Intracellular ATP was depleted by 5 lM oligomycin or 5 mM sodium azide applied together with 5 mM2-deoxy-D-glucose The optimal concentration of Pianalogues (Vi, BeFxand AlF4) and ATP depleting agents were pretitrated in daunorubicin accumulation experiments The concentrations of the chemicals applied increased steady-state daunorubicin accumulation to the approximate level reached in Pgp– parental cells and did not significantly increase the ratio of dead cells, as assessed by propidium iodide exclusion All of the above agents were from Sigma–Aldrich (Budapest) Cell culture media and supplements were also from Sigma Fluorescein isothiocyanate (FITC) was purchased from Molecular Probes (Eugene, OR, USA) All the other chemicals used in the experiments were of analytical grade, from Sigma The MRK16 (Cancer Chemotherapy Center, Tokyo, Japan), UIC2, 4E3, MC57, MM12.10 and MM8.15 anti-Pgp mAb preparations were > 97% pure by SDS/ PAGE The FITC conjugates of MM12.10 and UIC2 were prepared as described previously [26,27]
Flow cytometric assays Nearly confluent monolayers of cells were harvested by 2–3 min trypsin treatment [0.05% trypsin and 0.02% EDTA in NaCl/Pi (pH 7.4)] and washed twice with NaCl/Pi before antibody labeling The mAbs MRK16, MC57, MM8.15, MM12.10 (8 lgÆmL)1) and 10 lgÆmL)1
of UIC2 and 4E3 were applied The antibody competi-tion test was performed as follows: 106 cells in 1 mL NaCl/Pi supplemented with 8 mM glucose were preincu-bated in the absence or presence of different drugs/ modulators at 37°C for 20 min, then the first mAb, UIC2 was added, without washing the cells After further
30 min incubation at 37°C, the FITC-conjugated secon-dary mAb MM12.10 was added (again without washing the cells) and incubation followed at 37°C for another
30 min The extent of competition between mAbs UIC2 and FITC-MM12.10 were expressed as Rcompetition, the difference of mean fluorescence intensities of cell-bound FITC-MM12.10, in the absence and in the presence of
Trang 3UIC2, divided by the fluorescence intensity obtained in
the absence of UIC2
In the case of the UIC2 shift assay [18], the cells were
pretreated with drugs, labeled first with UIC2 then with the
secondary antibody [FITC-conjugated goat anti-(mouse
IgG2a) Ig, F/P ¼ 4.3, from Sigma) on ice in 100 lL of
NaCl/Pifor 45 min The samples were washed twice after
staining and resuspended in 500 lL of NaCl/Pi for flow
cytometric analysis
Daunorubicin and calcein accumulation was measured as
described previously [28,29] Briefly, the cells were
preincu-bated with modulators for 10 min or with Pianalogues (Vi,
BeFxor AlF4) for 20 min and then with daunorubicin or
calcein-AM for further 40 min, at 1 and 0.5 lM final
concentration, respectively All the incubations were carried
out at 37°C The cells were washed and the samples stored
on ice until their measurement The mean cellular
fluores-cence in each sample was determined using a modified
Becton Dickinson FACStar Plus flow cytometer (Mountain
View, CA, USA) equipped with an argon ion laser
(Spectra-Physics Inc Mountain View, CA, USA) Dead cells stained
with propidium iodide were excluded from the analysis
Fluorescence signals were collected in logarithmic mode and
the cytofluorimetric data were analyzed by the FLOWIN
software (written by M Emri & L Balkay, University of
Debrecen, Positron Emission Tomography Center,
Hungary)
R E S U L T S
As ATP depletion is known to increase UIC2 reactivity
(UIC2-shift assay, [18]), we investigated if treatment of
cells with oligomycin, or sodium azide together with
2-deoxy-D-glucose, elicit a similar effect on UIC2/
MM12.10 competition Rcompetition(i.e the relative decrease
of FITC-MM12.10 labeling after UIC2 pretreatment, see
Materials and methods) was used to characterize the effect
of the treatments In ATP-depleted cells, UIC2 completely
abolished MM12.10 labeling similarly to CsA treated cells,
as shown by the Rcompetition 1 v alues in Fig 1 Thus, ATP
depletion affects UIC2 binding and UIC2/MM12.10 com-petition in a parallel manner
Pi analogues are often used for blocking Pgp ATPase activity in plasma membrane vesicles [10], and it was recently shown that Vialso affects UIC2 binding in living cells [30] We compared the effects of CsA and Pianalogues (Vi, AlF4, BeFx) on Pgp function, measuring the cellular accumulation of different Pgp substrates As demonstrated
in Fig 2, treatments with Pianalogues restored steady-state daunorubicin levels in NIH 3T3 MDR1 cells, similarly to the effect of CsA applied at a concentration that completely inhibits the pump Vi treatment also increased calcein accumulation of Pgp+ cells, albeit to a lesser degree ( twofold, as opposed to the 30-fold increase in intracel-lular calcein levels observed after CsA treatment; data not shown) As the addition of Pianalogues did not influence significantly either the daunorubicin or calcein uptake of the parental cells (NIH 3T3 cells; Fig 2B), their effect must be Pgp-specific
The consequences of Vi, BeFx and AlF4 treatments on UIC2 binding were also examined It was shown previously
by Druley et al [30] that Viprevents UIC2 binding even on vinblastine-treated cells We have found that BeFx pretreat-ment also decreased the reactivity of UIC2 with cell surface Pgp molecules (data not shown), in contrast with CsA treatment or ATP depletion that increased UIC2 binding in accordance with what was previously shown by Mechetner
et al [18] Vi and BeFx also suppressed the effect of substrates/modulators (verapamil, CsA, vinblastine) on UIC2 binding when the cells were incubated in the simultaneous presence of Vior BeFx, and any of the above agents The order of treatments with substrates/modulators
vs Vior BeFxseemed to be indifferent as it is shown in the case of CsA and Pianalogues in Fig 3 In contrast to Viand BeFx, AlF4 did not affect the binding of UIC2 despite its inhibitory effect on Pgp function (compare Figs 2 and 3)
Fig 1 Effect of ATP depletion on UIC2-MM12.10 mAbcompetition in
NIH 3T3 MDR1 cells Cellular ATP production was inhibited by
30 min pretreatment of cells with 5 l M oligomycin or 5 m M sodium
azide applied together with 5 m M 2-deoxy- D -glucose Labeling with
mAbs was carried out and R competition was calculated as described in
Materials and methods Means of three independent experiments are
shown (± SEM).
Fig 2 Effect of phosphate analogues and cyclosporin A (CsA) on the accumulation of daunorubicin into Pgp+(NIH 3T3 MDRl) and Pgp– (NIH 3T3) cells The cells were preincubated in the presence of phos-phate analogues (V i , BeF x , AlF 4 ), or 10 lm CsA for 20 min, then 1 lm daunorubicin was added for 40 min Solid line: daunorubicin only; bold line: 10 l M CsA; empty triangles: 500 l M V i ; black triangles: BeF x ; empty squares: AlF 4 treatment Preparation of phosphate ana-logues is described in Materials and methods.
Trang 4The reactivity of several other anti-Pgp mAbs (MRK16,
4E3, MM.12.10, MC57, MM.8.17) was not affected by
either Vi or substrate/modulator treatment (data not
shown)
The effects of Pianalogue treatments on UIC2/MM12.10
competition are shown in Fig 4 The degree of the UIC2/
MM12.10 competition (i.e the suppression of MM12.10
binding after UIC2) correlates with the level of UIC2
binding upon the same treatment (compare Figs 3 and 4)
D I S C U S S I O N
The UIC2-shift [18] and the mAb competition phenomenon
involving UIC2 and MM12.10 mAbs [22] were affected by
pharmacological modulation of the ATPase cycle in a
parallel manner Near the catalytic transition state stabilized
by Vi or BeFx, Pgp apparently undergoes a global
conformational change involving the extracellular loops,
manifest in the drastic decrease of its affinity to UIC2 ATP
depletion increased UIC2 reactivity as expected [18], preventing subsequent MM12.10 binding AlF4that prob-ably freezes the catalytic cycle at an earlier phase, trapping unhydrolysed ATP [11], inhibited transport function with-out preventing UIC2 binding
In our experiments, different Pianalogues affected Pgp function and conformation in live cells As the nucleotide-binding domains of Pgp do not seem to be accessible from the extracellular surface of the cell [31], our results suggest that Pianalogues penetrate the cell membrane, despite of their negative charge
Vi can increase phosphorylation of Pgp at its linker peptide region [32] similar to phorbol esters or phosphatase inhibitors (e.g ocadaic acid) However, the latter agents do not affect or decrease drug accumulation [33,34], while in our experiments, Visignificantly inhibited drug transport In addition, other Pi analogues, e.g BeFx and AlF4 also inhibited Pgp mediated drug transport Thus, the Vieffect seems to be independent of the phosporylation state of the pump and is best interpreted assuming that Vitrapping of Pgp also occurs in live cells
Coupling of Pgp-mediated drug transport and ATP hydrolysis are generally interpreted in terms of ligand-induced ATPase activation and concomitant transitions between substrate-binding and substrate-releasing conform-ational states The consecutive steps of the catalytic cycle appear to be discriminated by the changing affinity of Pgp
to the UIC2 mAb [18,30] Reactivity of Pgp with UIC2 is
Fig 4 Effect of phosphate analogues on UIC2/MM12.10 mAb competition in NIH 3T3 MDR1 cells Cells were preincubated for
20 min in the presence or absence of phosphate analogues (V i , BeF x , AlF 4 ), followed by the addition or omission of 10 l M CsA for an additional 30 min and finally labeled with UIC2 and MM12.10 mAbs (upper panel) Incubations with CsA and phosphate analogues were also carried out in the reverse order (lower panel) Preparation
of phosphate analogues, labeling with mAbs and calculation of
R competition values are as described in Materials and methods Means of three independent experiments are shown (± SEM).
Fig 3 Flow-cytometric fluorescence intensity distribution histograms
demonstrating UIC2-shift assays performed on NIH 3T3 MDR1 cells.
Cells were preincubated for 20 min in the presence or absence of
phosphate analogues, then 10 l M CsA was added and the cells were
further incubated for 30 min and finally labeled with UIC2 mAb In
parallel experiments, incubations with CsA and phosphate analogues
were carried out in the reverse order Phosphate analogues and CsA
were continuously present till the end of labeling with UIC2 UIC2
binding was visualized by indirect immunofluorescence Solid line: no
CsA and phosphate analogue treatment; bold line: 10 l M CsA; empty
triangles: 10 l M CsA + 500 l M V i; black triangles: 10 l M CsA +
BeF x ; empty squares: 10 l M CsA + AlF 4 treatment.
Trang 5increased in the presence of substrates/modulators or ATP
depleting agents, and in double Walker A and B mutants
incapable of ATP binding and hydrolysis [18,35] These
findings may be interpreted suggesting that changes in the
UIC2 reactivity of Pgp are brought about by nucleotide
binding and release [18,30], or assuming that the hydrolysis
of ATP is the key element in driving Pgp from
UIC2-reactive conformation to a nonUIC2-reactive state We favor the
latter interpretation considering the following data
Previ-ous studies demonstrated that linker region mutants capable
of binding ATP but unable to hydrolyze it, are recognized
by UIC2 similarly to the functional molecules that have
been preincubated by Pgp substrates [36], suggesting that
changes in the availability of the UIC2 epitope cannot be
explained by the step of ATP binding itself [35] This
conclusion was further confirmed by the following
obser-vations Mutation restricted to the N-terminal Walker B
motif abolished ATP binding at both ATP sites, while
mutation of the C-terminal motif did not affect ATP
binding at the N-terminal site [35] Interestingly, both of the
above mutants were recognized by UIC2, implying that
ATP binding per se does not affect the availability of the
UIC2 epitope In our experiments, Vi(in agreement with
[30]) and BeFxtreatment induced a significant decrease of
UIC2 binding, despite the presence of substrates or
modulators, while AlF4, known to trap both ATP and
ADP produced upon hydrolysis [11], inhibited drug
pumping similarly to Viand BeFxbut did not affect UIC2
binding (Figs 3 and 4) These data also support a model
where the UIC2 epitope is available for antibody binding
throughout the catalytic cycle including the step of ATP
binding, to become unavailable only in the catalytic
transition state ATP hydrolysis could cover the energy
expenses of a global conformational change effecting a
decreased affinity for substrates, as shown in
vanadate-trapping experiments [17]
As both substrate/modulator category identified on the
basis of UIC2/MM1210 mAb binding include agents that
stimulate, and others that inhibit, ATPase activity [37–39],
the two classes of substrates/modulators may not be
distinguished based on the overall catalytic rate achieved
by Pgp in their presence The investigated conformational
effect of CsA (or vinblastine) manifest at the externally
located UIC2-binding epitopes is apparently reproduced by
ATP depletion and can be thwarted
confirming that the state elicited by CsA-type substrates/
modulators is part of the normal catalytic cycle This latter
conformational state must be present for periods long
enough to be recognized by UIC2 in a way that leads to
mAb competition In the presence of verapamil-like agents,
this state may be by-passed or its duration may be
diminished
As UIC2 binding and UIC2/MM12.10 competition
could be influenced by nonsubstrate agents in a parallel
manner, the two phenomena are, in the case of the CsA-type
modulators, negative replicas of each other Thus, the
epitopes opening up upon UIC2-shift are eventually titrated
back by MM12.10, without modulator treatment A
practical implication of this finding is that the often variable
functional modulation of Pgp by modulators (e.g CsA) in
the UIC2-shift assay can be substituted by the consecutive
application of UIC2 and MM12.10 for the labeling of Pgp+
cells, in the absence of modulators
A C K N O W L E D G E M E N T S This work was financially supported by OTKA funding T 032563 and the research grant of the Hungarian Academy of Sciences AKP 98-83 3,3 This publication was also sponsored by the research grant of the Ministry of Public Health ETT T01/103 and the OMFB grant 02692/
2000 M C is in part supported by grant 502 from Istituto Superiore di Sanita`, Rome, Italy The technical assistance of Eniko¨ Pa´sztor is gratefully acknowledged.
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