The remaining [3H]daunomycin associ-ated with the membranes corresponded to nonspecific binding at sites other than the ABCG2R482G protein.. Screening nucleotides for propensity to modify
Trang 1by the multidrug transporter ABCG2?
Christopher A McDevitt1, Emily Crowley1, Gemma Hobbs1, Kate J Starr2, Ian D Kerr2and Richard Callaghan1
1 Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, UK
2 Centre for Biochemistry and Cell Biology, School of Biomedical Sciences, University of Nottingham, UK
Resistance to chemotherapy presents a continuing and
significant obstacle in the treatment of both solid
tumours and haematological malignancies One of the
most prevalent primary cellular defence mechanisms
against chemotherapeutic agents is the
membrane-bound transporter [1] The defining feature of these
transporters is their ability to interact with a broad
range of structurally unrelated compounds, a property
that has led them to be described as ‘multidrug
trans-porters’ [2–4] The resistant phenotype is conferred by
the reduction in cytoplasmic concentrations of
chemo-therapeutic drugs to levels below that required for
cytotoxicity Resistance to chemotherapy has been
attributed to the expression of three ‘multidrug
trans-porters’, all members of the ATP binding cassette
(ABC) superfamily, designated as ABCB1, ABCC1
and ABCG2 Specifically, ABCG2 has been implicated
in clinical multidrug resistance in acute myeloid leu-kaemia [5–8] However, although ABCB1 and ABCC1 have been extensively characterized, there are many unresolved issues relating to the basic biochemistry of ABCG2
ABCG2 is a 72 kDa integral membrane protein con-sisting of six transmembrane helices and an amino terminal nucleotide binding domain (NBD) [9–11] It is described as being a ‘half-transporter’ as the canonical ABC transporter typically consists of two transmem-brane domains (TMDs) and two NBDs Furthermore, the topological organization of ABCG2 is distinct from ABCB1 and ABCC1, as NBD is N-terminal to TMD [9] To date, there are no high-resolution structures available for any of the eukaryotic ABC
Keywords
ABC transporter; chemotherapy; membrane
protein; multidrug-resistance; power-stroke
Correspondence
R Callaghan, Nuffield Department of Clinical
Laboratory Sciences, John Radcliffe
Hospital, University of Oxford, Oxford OX3
9DU, UK
Fax: +44 1865 221 834
Tel: +44 1865 221 110
E-mail: richard.callaghan@ndcls.ox.ac.uk
(Received 28 May 2008, revised 24 June
2008, accepted 27 June 2008)
doi:10.1111/j.1742-4658.2008.06578.x
ABCG2 confers resistance to cancer cells by mediating the ATP-dependent outward efflux of chemotherapeutic compounds Recent studies have indi-cated that the protein contains a number of interconnected drug binding sites The present investigation examines the coupling of drug binding to ATP hydrolysis Initial drug binding to the protein requires a high-affinity interaction with the drug binding site, followed by transition and reorien-tation to the low-affinity state to enable dissociation at the extracellular face [3H]Daunomycin binding to the ABCG2R482G isoform was examined
in the nucleotide-bound and post-hydrolytic conformations Binding of [3H]daunomycin was displaced by ATP analogues, indicating transition to
a low-affinity conformation prior to hydrolysis The low-affinity state was observed to be retained immediately post-hydrolysis Therefore, the dissoci-ation of phosphate and⁄ or ADP is likely to be responsible for resetting
of the transporter The data indicate that, like ABCB1 and ABCC1, the ‘power stroke’ for translocation in ABCG2R482G is the binding of nucleotide
Abbreviations
ABC, ATP binding cassette; ATP-c-S, adenosine 5¢-[c-thio]-triphosphate; NBD, nucleotide binding domain; TMD, transmembrane domain; TNP-ATP, 2¢,3¢-O-(2,4,6-trinitrophenyl) adenosine 5¢-triphosphate.
Trang 2transporters, although an 18 A˚ structure of ABCG2
was obtained using electron microscopy [12] This
report indicated that soluble purified ABCG2
dis-played a propensity to form a higher order oligomer, a
tetramer of dimers, which is consistent with the
obser-vations of higher order oligomeric species in cell
membranes [13] Although the precise molecular
composition remains controversial, there is a growing
weight of evidence favouring a higher order structure
[12–16]
ABCG2 displays distinct, but not exclusive,
sub-strate specificity compared with other multidrug
trans-porters In particular, the protein confers resistance to
the anticancer drugs mitoxantrone [17], methotrexate
[18] and the camptothecins [19] Although early
cellu-lar studies failed to generate a consensus for the
sub-strate profile, the discrepancies were attributed to a
mutation generated during long-term selection in the
presence of anticancer drugs Selection in mitoxantrone
produced R482G or R482T point mutations that
pres-ent considerably broader substrate selectivity [20,21]
For example, the R482G isoform is a gain-of-function
mutation which mediates the transport of doxorubicin,
daunomycin and rhodamine 123, whereas it has a loss
of function with respect to methotrexate transport
Recent investigations have demonstrated that
ABCG2R482G, like other multidrug transporters,
con-tains more than one drug binding site In addition, the
binding sites are linked by both negative and positive
heterotropic allostery In a departure from the drug–
protein interactions with ABCB1, the R482G isoform
also contains multiple sites of interaction for a single
drug (daunomycin), which can manifest as homotropic
allostery [22] The latter has been observed for the
bacterial half-transporter LmrA, but not for any
eukaryotic ABC protein [23]
The translocation of drugs across the plasma
mem-brane requires that the drug binding event(s) in TMD
is intrinsically coupled to the catalytic cycle within
NBDs The best evidence for an interaction between
the two domains is the ability of numerous substrates
and modulators of ABCG2 (and the R482G isoform)
to stimulate the rate of ATP hydrolysis [21,24,25],
albeit to a lesser degree than that commonly
encoun-tered with ABCB1 The translocation event requires
that the drug binding sites switch from the initial
high-affinity, inward-facing configuration to an
outward-facing, low-affinity configuration to facilitate
dissociation [26] Originally, the impetus for the switch
in binding site affinity and orientation was thought to
be the energy produced by nucleotide hydrolysis In
the case of ABCB1, this was revised through the
obser-vations that nucleotide binding in the absence of
hydrolysis could cause the conformational alteration (reviewed in [27,28]) The low-affinity conformation of drug binding sites in ABC multidrug efflux pumps is assumed to correspond to the outward-facing confor-mation The energy produced by the hydrolysis of ATP is harnessed for the resetting of the transporter
to the initial high-affinity, inward-facing configuration Similar results were also obtained for ABCC1 Thus, the eukaryotic multidrug transporters are thought to mediate drug translocation through a ‘power stroke’ which is obtained by the binding of nucleotide
The focus of the present investigation was to ascer-tain whether the binding of nucleotide to ABCG2R482G was the power stroke required to switch the configura-tion of the drug binding site(s) This hypothesis was examined using a direct measure of drug binding to the protein, which was trapped in both pre- and post-nucleotide hydrolytic conformations
Results
Characteristics of drug binding to ABCG2R482G -containing membranes
The expression of ABCG2R482G has previously been established in High-5 insect cells using recombinant baculovirus [22] [3H]Daunomycin (300–350 nm) bound
to the membranes with a total binding capacity of
107 ± 13 pmolÆmg)1, which was significantly reduced following the addition of a large molar excess of doxoru-bicin (30 lm) The remaining [3H]daunomycin associ-ated with the membranes corresponded to nonspecific binding at sites other than the ABCG2R482G protein This fraction corresponded to 37 ± 8 pmolÆmg)1, and therefore the specific binding component in the mem-branes was 70 pmolÆmg)1 The dissociation constant for [3H]daunomycin binding to ABCG2R482G has previ-ously been estimated as 98 nm [22], and all subsequent binding assays in this study were conducted with 300–
350 nm of the radioligand There was no detectable dis-placement of [3H]daunomycin binding to membranes that did not express ABCG2R482G(data not shown)
A heterologous drug displacement assay was under-taken with ABCG2R482G-containing membranes to characterize the potency of the drug–protein interaction Figure 1A demonstrates that doxorubicin is able to dis-place 90 ± 2% of the specific binding component of [3H]daunomycin Moreover, the potency to displace [3H]daunomycin binding is IC50= 1.73 ± 0.51 mm (n = 9), which is in good agreement with the value previously described [22] Thus, High-5 insect cell membranes provide a specific method to examine the drug binding characteristics of ABCG2R482G
Trang 3Characteristics of nucleotide binding to purified
ABCG2R482G
Photolabelling of ABCG2R482Gby [a32P]azido-ATP was
used to characterize the interaction of nucleotides with
the transporter As shown in Fig 2A, [a32P]azido-ATP
binds to ABCG2R482G in a dose-dependent manner
Unfortunately, commercial preparations of the photo-active nucleotide do not attain sufficiently high concentrations to enable complete saturation of binding However, the binding isotherm in Fig 2A provides an estimate of the binding affinity for [a32P]azido-ATP as
KD= 201 ± 80 lm This affinity is similar to the value obtained for ATP binding to ABCB1 [29] The ability of nucleotides to displace binding is shown in Fig 2B, with values normalized to the amount bound in the absence
of added nucleotide Neither ADP nor AMP altered the photolabelling of [a32P]azido-ATP bound, whereas the ATP analogues adenosine 5¢-[c-thio]-triphosphate (ATP-c-S) and 2¢,3¢-O-(2,4,6-trinitrophenyl) adenosine
Fig 1 Heterologous displacement of [ 3 H]daunomycin binding to
ABCG2 R482G by doxorubicin (A) ABCG2 R482G -containing insect cell
membranes (20 lg) were incubated with [3H]daunomycin (300 n M )
in the presence or absence of varying concentrations of doxorubicin
(1 n M to 300 l M ) Incubations were performed at 20 C for a period
of 120 min to ensure that equilibrium had been reached Unbound
[ 3 H]daunomycin was removed using a rapid filtration assay, and the
amount of bound radioligand was determined by liquid scintillation
counting Values refer to the mean ± SEM of at least three
inde-pendent membrane preparations, and the dose–response curve
was fitted using nonlinear least-squares regression (B) A series of
nucleotides was examined for their propensity to displace the
bind-ing of [3H]daunomycin (300 n M ) to ABCG2R482Gcontaining High-5
insect cell membranes (20 lg) The radioligand was incubated with
the ABCG2 R482G -containing membranes in the presence of 10 m M
nucleotide The only exception was the ATP analogue TNP-ATP,
which was used at a concentration of 0.6 m M The amount of
[ 3 H]daunomycin bound to the membranes in the absence of
nucleo-tide was assigned a value of unity, and all other data were
expressed as a fraction of this Values correspond to the mean ±
SEM of three independent membrane preparations.
Fig 2 The binding of nucleotides and analogues to ABCG2R482G (A) Purified ABCG2 R482G (0.25 lg) was photolabelled with [a 32 P]azido-ATP (3–300 l M ) as described in Materials and methods Labelled protein was visualized and quantified by autoradiography
of SDS-PAGE analysis The amount of bound protein was plotted
as a function of nucleotide concentration, and the data were fitted with the Langmuir binding isotherm using nonlinear least-squares regression (B) Photoaffinity labelling of purified ABCG2 R482G
(0.25 lg) was undertaken using a fixed concentration (30 l M ) of [a 32 P]azido-ATP in the presence or absence of ADP (10 m M ), AMP (10 m M ), ATP-c-S (10 m M ) or TNP-ATP (1 m M ) The intensity of labelling in the absence of excess nucleotide was assigned a value
of unity.
Trang 45¢-triphosphate (TNP-ATP) produced considerable
reductions in the amount of bound nucleotide
Screening nucleotides for propensity to modify
drug binding to ABCG2R482G
ABCG2R482G-containing membranes were incubated
with [3H]daunomycin and a series of adenine
nucleo-tides and three analogues to assess interactions A
fixed concentration of nucleotide (10 mm) was used,
apart from TNP-ATP which was administered at
0.6 mm because of its higher potency Figure 1B
dem-onstrates the ability of the nucleotides to reduce the
fraction of [3H]daunomycin bound to ABCG2R482G In
the presence of AMP, the binding of [3H]daunomycin
remained at 93 ± 4% (n = 8, P > 0.05) of that
obtained in the untreated control, and the addition of
ADP produced a marginal decrease to 80 ± 5%
(n = 4, P > 0.05) The addition of ATP produced a
statistically significant decrease (n = 4, P < 0.05) in
the amount of [3H]daunomycin bound to a value of
59 ± 9% The nonhydrolysable nucleotide, ATP-c-S,
produced an even greater decrease to 59 ± 4%
(n = 8, P < 0.05) Despite the use of a considerably
lower concentration (0.6 mm), the fluorescent and
slowly hydrolysable analogue TNP-ATP reduced the
binding to 35 ± 4% (n = 6, P < 0.05)
Binding of [3H]daunomycin to ABCG2R482Gin a
pre-hydrolysis configuration
ATP, and its nonhydrolysable analogues ATP-c-S and
TNP-ATP, reduced the degree of [3H]daunomycin
binding to ABCG2R482G, thus warranting further
examination of the effect of these nucleotide
analogues Figure 3 shows the effects of a range of
ATP-c-S concentrations on the interaction of [3
H]dau-nomycin with ABCG2R482G At the highest
concentra-tion of nucleotide, only approximately 20% of the
radioligand was bound to the protein The extent of
binding was fitted with a dose–response curve, which
generated a potency of IC50= 11.8 ± 1.6 mm for
ATP-c-S Similar analysis was undertaken for the
slowly hydrolysable analogue TNP-ATP, as shown in
Fig 4 At a concentration of 2 mm, < 10% of the
ini-tial binding of [3H]daunomycin was observed The
potency of TNP-ATP to displace [3H]daunomycin
binding was characterized by IC50= 0.27 ± 0.02 mm,
which is 44-fold greater than that of ATP-c-S
Both TNP-ATP and ATP-c-S cause a decrease in
the extent of [3H]daunomycin binding to ABCG2R482G
Given the distinct sites for binding of nucleotides and
drugs to the protein, this decrease occurs via a
nega-tive allosteric mechanism The addition of either nucle-otide analogue will effectively trap the protein in a conformation closely resembling the pre-hydrolytic state The decrease in capacity for drug binding reflects
a lower affinity interaction between [3H]daunomycin and the protein immediately prior to ATP hydrolysis
Fig 3 Heterologous displacement of [ 3 H]daunomycin binding to ABCG2R482Gby the nonhydrolysable nucleotide ATP-c-S The effect
of the nonhydrolysable ATP analogue ATP-c-S (100 l M to 20 m M )
on [ 3 H]daunomycin (300 n M ) binding to ABCG2 R482G was examined using High-5 cell membranes (20 lg) Incubations were undertaken
at 20 C for a period of 120 min, and the membrane-bound radioli-gand was harvested by vacuum filtration through a manifold The general dose–response relationship was fitted to the data (mean ± SEM, n ‡ 3) using nonlinear least-squares regression.
Fig 4 Heterologous displacement of [ 3 H]daunomycin binding to ABCG2 R482G by TNP-ATP [ 3 H]Daunomycin (300 n M ) was incubated with ABCG2R482G-containing High-5 insect cell membranes (20 lg)
in the presence or absence of varying concentrations of the fluores-cent ATP analogue TNP-ATP (10 l M to 1.2 m M ) Incubations were performed at 20 C for a period of 120 min to ensure that equilib-rium had been reached Unbound [ 3 H]daunomycin was removed using a rapid filtration assay, and the amount of bound radioligand was determined by liquid scintillation counting Values refer to the mean ± SEM of at least three independent membrane prepara-tions, and the dose–response curve was fitted using nonlinear least-squares regression.
Trang 5Binding of [3H]daunomycin to ABCG2R482Gin a
post-hydrolysis configuration
Given that the [3H]daunomycin binding site is
switched to a low-affinity configuration on nucleotide
binding, what is the consequence of ATP hydrolysis
for the drug binding sites? To address this issue,
ABCG2R482G was trapped immediately
post-hydroly-sis using sodium orthovanadate [21] The metal
oxo-anion vanadate serves as a transition state mimic,
exploiting its chemical similarity to phosphate Thus,
ATP and vanadate generate an ADP-vanadate
struc-ture mimicking the transition state for the hydrolysis
of the c-phosphate of ATP [30] Figure 5
demon-strates the effect of pre-incubation of ABCG2R482G
-containing membranes with 100 lm NaVO3 and a
series of ATP concentrations The data show a 90%
decrease in the amount of [3H]daunomycin bound to
the membranes, indicating that the capacity for
sub-strate interaction is considerably reduced The
potency for vanadate trapping to reduce [3
H]dauno-mycin binding to ABCG2R482G was 21.3 ± 3.3 mm
of nucleotide Therefore, the data demonstrate that
ABCG2R482G remains in a conformation that contains
a low-affinity binding site for [3H]daunomycin
imme-diately post-nucleotide hydrolysis
Discussion
A precise molecular mechanism for substrate
translo-cation by any ABC protein remains unresolved,
despite considerable investigation using varied approaches and recent high-resolution X-ray crystal structures Investigations with multidrug transporters, involved in conferring drug resistance in cancer cells, have provided the most information For two of the proteins, ABCB1 and ABCC1, it has been demon-strated that the binding of nucleotide imparts marked and essential conformational changes within TMDs The present study provides the first evidence that nucle-otide binding per se also plays a role in the initiation of the drug translocation process for ABCG2, despite its structurally dissimilar architecture to the aforemen-tioned transporters
A radioligand binding approach was used in the investigations and has previously been evaluated for use with the ABCG2R482Gisoform [22] The ‘gain-of-func-tion’ mutation confers resistance to the anthracycline daunomycin by transporting it out of the cytoplasm [31] A previous study has indicated that there are two allosterically coupled binding sites for daunomycin, although it is unclear whether the coupling is between the two monomers in a transporter, or between distinct dimeric units [22] Measurement of [3H]daunomycin binding provides a useful insight into the pharmacology
of the ABCG2R482G isoform, as the binding site is in communication with those for different drug substrates The initial nucleotide screen revealed that several nucleotide species were capable of modulating drug binding, thereby reaffirming the interdomain communi-cation reported for ABCG2 However, despite using relatively high concentrations, neither AMP nor ADP was capable of altering the drug–ABCG2 interaction
In the case of the monophosphate AMP, this was entirely expected as this nucleotide plays no role in the catalytic process of ABCG2, and was therefore a control for specificity of the interaction The lack of effect of the diphosphate nucleotide indicates that, following inorganic phosphate release, the ADP-bound ABCG2R482G isoform adopts a conformation capable
of supporting the binding of [3H]daunomycin
The triphosphate nucleotide ATP caused a consider-able decrease in the ability of ABCG2R482G to bind [3H]daunomycin This decrease in drug binding was also observed in the presence of the ATP analogues ATP-c-S (nonhydrolysable) and TNP-ATP (slowly hydrolysable), although the magnitude of effect with the latter was more pronounced The ATP analogues were preferred for subsequent investigations, as ATP
is an inherently unstable or reactive compound in aqueous solutions, even at the reduced temperatures employed in radioligand binding assays Detailed investigation revealed that [3H]daunomycin binding by ABCG2R482Gwas essentially abrogated in the presence
Fig 5 The binding of [3H]daunomycin to vanadate-trapped
ABCG2 R482G ABCG2 R482G was trapped in the presence of sodium
orthovanadate (100 l M ) and a series of ATP concentrations (100 l M
to 300 m M ) at 37 C for 30 min The vanadate-trapped protein was
then incubated with [ 3 H]daunomycin (300 n M ) for 120 min at 20 C.
Bound and free radioligand were separated using a rapid filtration
assay, and the former was detected using liquid scintillation
count-ing Values correspond to the mean ± SEM of at least three
inde-pendent membrane preparations, and the dose–response curve
was fitted using nonlinear least-squares regression.
Trang 6of sufficient c-S or TNP-ATP Thus, the
ATP-loaded conformation of ABCG2R482G ([E]Æ[ATP],
where ‘E’ refers to ABCG2R482G) facilitates a negative
heterotropic allosteric effect of NBDs on TMDs This
finding with ABCG2R482G is entirely consistent with
the observation for the interaction of [3H]vinblastine
with ABCB1 and for [3H]estrone-sulfate binding to
ABCC1 [32,33] Such a decrease in the affinity or
capacity of drug binding to ABCG2R482G is likely to
represent the outward-facing conformation of the
transporter, as the presence of an inward-facing drug
binding site with low affinity would preclude an
effi-cient rate of translocation A possible alternative
explanation is that, although the binding of ATP
reduces the drug binding site to low affinity, it does
not generate an outward-facing conformation
How-ever, this would require that the drug binding site
adopts an occluded inward-facing conformation to
prevent dissociation, and that reorientation occurs
fol-lowing harnessing of the energy from ATP hydrolysis
If one of the initial events in the nucleotide catalytic
cycle is responsible for the decrease in affinity (and
pre-sumably reorientation) of drug binding sites, what role
do subsequent steps play in the translocation process?
As mentioned above, the [E]Æ[ADP] conformation
appears to have returned to high affinity, and the
inter-vening steps in the catalytic cycle are responsible for
the restoration of binding capacity In order to
main-tain ABCG2R482Gin a stable post-hydrolysis
conforma-tion, we employed the vanadate trapping procedure
The data revealed that vanadate-trapped ABCG2R482G
protein ([E]Æ[ADP]Æ[Vi]) remained in a low-affinity
[3H]daunomycin binding conformation By inference,
therefore, the step in the catalytic cycle corresponding
to the release of inorganic phosphate ([Pi]) is likely to
correspond to the restoration of a high-affinity
conformation for the transporter, which is supported
by the restoration of high-affinity binding in the
ADP-bound conformation That this step of the catalytic
cycle is associated with the greatest free energy change
also makes it ideal for the mediation of drug binding
site reorientation, although the binding data cannot
unequivocally inform on the orientation of the sites,
only their affinity for interaction with drugs
The data presented here suggest that the
ABC-G2R482G isoform undergoes the following sequence of
conformational transitions:
½EH$ ½EL ½ATP $ ½EL ½ADP½Pi
$ ½EH ½ADP $ ½EH where [E]Hand [E]L correspond to the high- and
low-affinity conformations of ABCG2R482G, respectively
The sequence, based on the measurement of drug– protein binding, indicates that the binding of ATP per se is the ‘power stroke’ for drug translocation, and that energy obtained from the hydrolysis process is used to reset the transporter That ATP binding is responsible for the shift in binding affinity from high to low has now been demonstrated for all three eukaryotic multidrug efflux proteins in the ABC family
Materials and methods
Materials
[3H]Daunomycin (0.185 TBq CiÆmmol)1) was purchased from Perkin Elmer LAS (Beaconsfield, UK) and Ready Protein+ scintillation fluid was obtained from Beckman Coulter (High Wycombe, UK) Doxorubicin, sodium ortho-vanadate, ATP, ADP, AMP, ATP-c-S and TNP-ATP were purchased from Sigma (Poole, UK) GF⁄ F filters were purchased from VWR International (Lutterworth, UK) Insect Xpress medium was obtained from Cambrex (Read-ing, UK) and Ex-cell 405 medium from JRH Biosciences (Andover, UK)
Insect cell culture and membrane preparation
The Trichoplusia ni (High-5) cell line was routinely used for the expression of ABCG2R482G and maintained in shaking suspension cultures, as described previously [22] High-5 cells at a density of approximately 3· 106cellsÆmL)1 were infected with recombinant baculovirus (approximately
1· 108
plaque-forming unitsÆmL)1) at a multiplicity of infection of five After 1 h of incubation with virus, the cells were diluted to a density of 1.5· 106cellsÆmL)1 and maintained in suspension for 3 days before harvesting by centrifugation (2000 g, 10 min)
Crude membrane preparations were isolated as described previously [34], with the exception that buffers contained
20 mm Mops, pH 7.4, 200 mm NaCl and 0.25 m sucrose Briefly, cells were ruptured with four rounds of nitrogen cavitation using 6500–10 000 kPa at 4C, with a 20 min incubation between rounds Cell debris was removed by centrifugation at 2000 g for 10 min Crude membranes were isolated by ultracentrifugation at 100 000 g for 60 min at
4C Membranes were resuspended at protein concentra-tions of approximately 50 mgÆmL)1 in isolation buffer (0.25 m sucrose, 20 mm Mops, pH 7.4, containing a prote-ase inhibitor cocktail) and stored at)80 C
Radioligand binding assay
Radiolabelled drug binding assays were based on a previ-ously published technique used to investigate ABCB1 [35] Membranes (20 lg) were incubated with a radiolabel,
Trang 7[3H]daunomycin, in a total volume of 100 lL in
polypro-pylene test tubes for 120 min to ensure attainment of
bind-ing equilibrium The membranes, [3H]daunomycin and any
other drugs were incubated in hypotonic binding buffer,
comprising 50 mm Tris⁄ HCl, pH 7.4 Hypotonic buffer was
used in binding assays to ensure no intraliposomal drug
accumulation Nonspecific binding to the filters or the lipid
component of membranes was defined as the amount of
[3H]daunomycin bound in the presence of a large molar
excess (30 lm) of doxorubicin Drugs were added from
centrated stocks in dimethylsulfoxide and the solvent
con-centration was maintained at < 1% (v⁄ v) Actual
concentrations of [3H]daunomycin added to the tubes were
determined by liquid scintillation counting Unbound ligand
was separated from bound ligand through porous
glass-fibre filters (GF⁄ F) using rapid vacuum filtration on a
48-well manifold The GF⁄ F filters were pre-soaked in
wash buffer supplemented with 0.1% (w⁄ v) BSA for
10 min Samples on the filters were rinsed twice with 10 mL
of ice-cold wash buffer (50 mm Tris⁄ HCl, pH 7.4, 20 mm
MgSO4) [3H]Daunomycin bound to the filters was
mea-sured by liquid scintillation counting using Ready Protein+
scintillation fluid
Heterologous displacement assays used ABCG2R482G
-containing crude membranes incubated with a single
con-centration of [3H]daunomycin (300–350 nm) at 20C for
120 min Doxorubicin was added over the concentration
range 1 nm to 300 lm, obtained from the serial dilution of
a concentrated stock in dimethylsulfoxide All nucleotides
and analogues were added from concentrated stocks in
buf-fer containing 5 mm MgCl2, 100 mm Mops at pH 6.8 The
NaVO3 stock solution (100 mm) was treated as described
previously by Goodno [36] to remove polymeric species
Membranes were incubated with 100 lm NaVO3 in the
presence of varying concentrations of MgATP (100 lm to
300 mm) in ATPase buffer (150 mm NH4Cl, 50 mm
Tris⁄ HCl, pH 7.4, 5 mm MgSO4) The vanadate trapping
of ABCG2R482G was achieved at 37C for 30 min prior to
the binding assay, according to a previously published
procedure [21]
The amount of [3H]daunomycin bound at each
concen-tration of heterologous drug or nucleotide was expressed as
a fraction of that obtained with radiolabel alone The
fraction bound was plotted as a function of added drug
concentration, and nonlinear regression of the general
dose–response relation (Eqn 1) was used to ascertain the
potency (IC50) and degree of displacement (FD)
Binding of [a32P]azido-ATP to purified
ABCG2R482G
ABCG2R482Gwas purified using immobilized metal affinity,
anion exchange and gel filtration chromatography; full and
extensive details have been described previously [22] Binding
of nucleotide to ABCG2R482G was determined using
photo-affinity labelling with [a32P]azido-ATP Purified protein (0.25 lg) was incubated with [a32P]azido-ATP (3–300 lm) in the dark for 20 min in ATPase buffer (150 mm NH4Cl,
50 mm Tris, pH 7.4, 5 mm MgSO4, 0.02% NaN3) at 4C At this temperature, ABCG2 does not generate measurable ATP hydrolysis Samples were then irradiated with UV light (k = 265 nm, 100 W, 5 cm) for 8 min, and the samples were resolved by electrophoresis using 10% polyacrylamide gels The gels were dried, and photolabelled protein was detected
by autoradiography Where displacement of nucleotide bind-ing was examined, the [a32P]azido-ATP concentration was fixed at 30 lm Relative labelling intensities were determined using densitometric analysis of autoradiograms
Statistical analyses
Heterologous displacement assays were analysed using the dose–response relationship shown below:
B¼ Bminþ ðBmax BminÞ
1þ 10½ðlogIC 50 LÞ n ð1Þ where B is the maximal [3H]daunomycin binding, Bmax is the maximal binding, Bminis the minimum binding, IC50is the concentration of drug that leads to half-maximal bind-ing of radiolabel (nm), n is the Hill slope factor and L is log10[ligand concentration (m)] The binding capacities are expressed as a fraction of the total obtained in the absence
of drug or nucleotide
Equation (1) was fitted to the displacement data by non-linear least-squares regression using the graphpad prism 4.0 program All data are presented as the mean ± SEM
of multiple independent observations, and P < 0.05 was considered to be statistically significant
Acknowledgements
The work undertaken in this study was supported by Cancer Research UK and Medical Research Council project grants awarded to RC The authors would like
to thank TMW and DCS for critical assessment of all aspects of the project
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