(Received for publication, November 28, 1994; and in revised form, October 26, 1995)
From the
The characteristics of P-glycoprotein (MDR1), an ATP-dependent
drug extrusion pump responsible for the multidrug resistance of human
cancer, were investigated in an in vitro expression system.
The wild-type and several mutants of the human MDR1 cDNA were
engineered into recombinant baculoviruses and the mutant proteins were
expressed in Sf9 insect cells. In isolated cell membrane preparations
of the virus-infected cells the MDR1-dependent drug-stimulated ATPase
activity, and 8-azido-ATP binding to the MDR1 protein were studied. We
found that when lysines 433 and/or 1076 were replaced by methionines in
the ATP-binding domains, all these mutations abolished drug-stimulated
ATPase activity independent of the MgATP concentrations applied.
Photoaffinity labeling with 8-azido-ATP showed that the double lysine
mutant had a decreased ATP-binding affinity. In the MDR1 mutant
containing a Gly to Val replacement we found no
significant alteration in the maximum activity of the MDR1-ATPase or in
its activation by verapamil and vinblastine, and this mutation did not
modify the MgATP affinity or the 8-azido-ATP binding of the transporter
either. However, the Gly
to Val mutation significantly
increased the stimulation of the MDR1-ATPase by colchicine and
etoposide, while slightly decreasing its stimulation by vincristine.
These shifts closely correspond to the effects of this mutation on the
drug-resistance profile, as observed in tumor cells. These data
indicate that the Sf9-baculovirus expression system for MDR1 provides
an efficient tool for examining structure-function relationships and
molecular characteristics of this clinically important enzyme.
The overexpression of a 170-kDa membrane protein, termed
P-glycoprotein (Pgp) ()or multidrug resistance transporter
(MDR1), is one of the major causes of multidrug resistance of cancer
cells to chemotherapy. The human MDR1 protein actively extrudes or
interacts with a wide variety of chemically dissimilar and apparently
unrelated compounds, e.g. cytotoxic drugs (Vinca alkaloids, colchicine, epipodophyllotoxins, and anthracyclines),
calcium channel blockers (verapamil and nifedipine), calmodulin
antagonists (trifluoperazine), or cyclosporines (Gottesman and Pastan,
1993). It has been recently reported that Pgp is also capable of
interacting with a variety of small peptides and peptide derivatives
(Sharma et al., 1992; Sarkadi et al., 1994; Zhang et al., 1994) as well as hydrophobic fluorescent dyes
(Neyfakh, 1988; Homolya et al., 1993; Holló et al., 1994).
In connection with its transport activity, Pgp has been shown to possess a drug-stimulated ATPase activity in isolated plasma membrane vesicles (Sarkadi et al., 1992; Doige et al., 1992; Shimabuku et al., 1992; Al-Shawi and Senior, 1993), or when the partially purified protein was reconstituted into liposomes (Ambudkar et al., 1992; Doige et al., 1993; Shapiro and Ling, 1994; Urbatsch et al., 1994). Drug transport in vesicles containing partially purified Pgp was shown to be coupled to ATP hydrolysis (Sharom et al., 1993). These studies, in addition to earlier data indicating an energy-requirement for MDR1-dependent drug extrusion clearly demonstrate a direct coupling between drug transport and ATP hydrolysis in the functioning of this protein.
Regarding its molecular architecture, P-glycoprotein is a member of the ABC (ATP-binding cassette) family of membrane transporters, containing 1280 amino acids, which form two symmetrical homologous halves, each with six putative transmembrane domains and an intracellular nucleotide binding domain (Endicott and Ling, 1989; Higgins, 1992). Of major importance would be to learn how ATP binding and hydrolysis is coupled to drug transport within this molecule, that is how the nucleotide binding domains interact with the transmembrane regions when performing active drug extrusion. Both predicted NBDs contain the ``homology A'' and ``homology B'' consensus sequences, originally described by Walker et al.(1982). These highly conserved regions are characteristic for several ATP-binding proteins including all the known ABC transporters (Higgins, 1992), and it has been demonstrated that the intact structure of these regions of the NBDs is essential for the drug transporting activity of Pgp. In the mouse MDR1 gene, the conversion of Gly to Ala at positions 431 or 1073, or that of Lys to Arg at positions 432 or 1074 (within the homology A consensus sequences), resulted in an almost complete loss of the function of the transporter, although 8-azido-ATP binding was conserved (Azzaria et al., 1989).
Concerning the site of interaction of Pgp with its transported substrates, e.g. cytotoxic drugs or drug resistance reversing agents, photoaffinity labeling has determined that both halves of Pgp contribute to a single substrate binding site (Bruggemann et al., 1992). Photoaffinity drug labeling experiments combined with trypsin digestion, mapped two major photolabeled fragments within, or immediately COOH-terminal to the last transmembrane domains of each half of the molecule, suggesting that some regions of the drug binding sites are in close proximity to the ATP binding regions (Greenberger, 1993).
A key region in substrate
handling seems to be the cytoplasmic loop between the second and third
transmembrane -helices. A spontaneous mutation in this region from
the wild-type Gly to Val occurred at position 185 in human KB cells,
when these cells were selected in high concentrations of colchicine.
This substitution greatly increases the relative resistance to
colchicine and etoposide (Choi et al., 1988; Kioka et
al., 1989), and influences the photoaffinity labeling of Pgp with
different drugs (Safa et al., 1990; Bruggemann et
al., 1992). Recent experiments of Loo and Clarke (1994a) indicated
that a Gly-Val exchange at five different positions in the cytoplasmic
loops of Pgp resulted in a similarly increased resistance to colchicine
and adriamycin without altering resistance to vinblastine.
In order
to investigate the functional significance of the changes in the
nucleotide binding and substrate recognition regions, we expressed the
wild-type and some mutant MDR1 proteins in a baculovirus-Sf9 insect
cell expression system, and characterized the MDR1-ATPase activity and
ATP binding in isolated membrane preparations. As demonstrated earlier,
this baculovirus-insect cell expression system can be used for
producing large amounts of membrane-inserted human MDR1, which,
although underglycosylated, is antigenically and functionally similar
to its mammalian counterpart (Germann et al., 1990; Sarkadi et al., 1992; Zhang et al., 1994). In the present
experiments we used site-directed mutagenesis to alter single amino
acids of the human Pgp in the homology A consensus sequences in the
NBDs of the NH-terminal (Lys
to Met) and/or
COOH-terminal (Lys
to Met) halves, and applied the cDNA
of the spontaneous Gly
to Val substitution mutant. The
MDR1 cDNAs were engineered into baculovirus vectors, recombinant
baculoviruses were generated, and after the infection of Sf9 insect
cells with the MDR1 viruses the cell membranes were isolated. The
expression levels and the functional characteristics of the MDR1
mutants were studied by immunoblotting, by measuring vanadate-sensitive
drug-stimulated ATPase activity, and by examining the binding of
8-azido-ATP in the isolated cell membranes.
The transfer
vector pAc373-MDR1/Val was prepared as described by
Germann et al.(1990); for the preparation of transfer vector
pVL941-MDR1/Val
a similar strategy was utilized. The
transfer vector pVL1393-MDR1/Gly
was constructed by
isolating the wild-type MDR1 cDNA from plasmid pHaMDRGA (Kioka et
al., 1989). The insert was ligated as a blunt ended BstUI-XhoI fragment (containing a 10-base pair
5`-untranslated region and a 110-base pair 3`-untranslated region with
no polyadenylation site) into the SmaI site of baculovirus
transfer vector pVL1393 (Pharmingen). To obtain
pVL941-MDR1/Gly
, both pVL941-MDR1/Val
and
pVL1393-MDR1/Gly
were subjected to BglII
digestion. This enzyme cuts MDR1 cDNA at nucleotide positions 258 and
1223 but leaves both vectors unharmed. The approximate 1-kilobase pair BglII insert, originated from pVL1393-MDR1/Gly
,
was gel-purified and ligated into the gel-purified
pVL941-MDR1/Val
, which lacked the MDR1 258-1223 region.
The orientation of the fragment was checked by restriction mapping, the
presence of the Gly
codon was confirmed by sequencing the
polymerase chain reaction products generated with primers mdr01-mdr02R;
mdr01 was used as sequencing primer. Transfer vectors
pVL941-MDR1/Val
/Met
/Lys
,
pVL941-MDR1/Val
/Lys
/Met
, and
pVL941-MDR1/Val
/Met
/Met
were produced by replacing the 1177-3372 EcoRI-PstI region of MDR1 in pVL941-MDR1/Val
with those of pUCFVXMDR1/neo MK, KM, and MM, respectively. (
)
Figure 1:
A,
Immunoblot detection of human MDR1 expressed in Sf9 cells by
baculovirus. Isolated membranes of baculovirus-infected Sf9 cells were
subjected to electrophoresis and to immunoblotting with the anti-MDR1
polyclonal antibody 4077, as described under ``Materials and
Methods.'' Peroxidase-dependent luminescence on the immunoblots
was quantitated with liquid scintillation counting. The amounts of the
expressed MDR1 protein are indicated on the bottom of the
figure (V, Val; G, Gly
;
MK, Met
; KM, Met
; MM,
Met
and Met
in MDR1). B, [
-
P]8-azido-ATP labeling of wild-type
and mutant P-glycoproteins. Isolated Sf9 cell membranes were incubated
in the presence of 35 µM final concentration of
8-azido-ATP, containing [
-
P]8-azido-ATP,
and irradiated with an UV lamp. The membranes were then precipitated
with trichloroacetic acid, washed, and dissolved in electrophoresis
buffer, and run on 4-12% gradient or 6% gels. The proteins were
electroblotted, the blots dried and subjected to autoradiography (see
Materials and Methods``). C, [
-
P]8-azido-ATP, and immunolabeling of
the wild-type and the MM mutant P-glycoproteins. Samples were prepared
as described for B. 8-Azido-ATP labeling was carried out
either in the presence of 1 mM AMP (lanes 2 and 5), or 1 mM ATP (lanes 3 and 6).
The identity of the
P-azido-ATP-labeled MDR1 band was
assured by immunostaining with MDR1-specific antibody(4077) on the same
blot (lanes 1 and 4).
Fig. 1, B and C,
demonstrate photoaffinity labeling of the isolated Sf9 cell membranes
with 8-azido-ATP. The isolated membranes were labeled in the presence
of 2 mM MgCl and 35 µM [
-
P]8-azido-ATP, membrane proteins
separated by gel electrophoresis and blotted onto polyvinylidine
difluoride membranes (see ``Materials and Methods''). The
protein band corresponding to the
[
P]azido-ATP-labeled MDR1 was identified by
immunostaining with MDR1-specific antibody on the same blot (see Fig. 1C).
As demonstrated in Fig. 1B, in contrast to the -galactosidase
expressing cell membranes, an additional 8-azido-ATP-labeled band,
corresponding to the antigenically identified MDR1 protein, was
observed in the membranes expressing either the wild-type or any of the
mutant MDR1 constructs. We found that 8-azido-ATP binding and
photoaffinity labeling required the presence of Mg
(1
mM EDTA eliminated labeling). At the 8-azido-ATP concentration
applied in these experiments (35 µM), the photoaffinity
labeling of the different MDR1 proteins were not significantly
different (Fig. 1B). As shown in Fig. 1C, the addition of excess cold ATP (1
mM) abolished radioactive 8-azido-ATP labeling of the
expressed MDR1 proteins, while 1 mM AMP was ineffective in
this respect.
In the following experiments we have analyzed the
effects of different 8-azido-ATP concentrations on the level of MDR1
labeling by this photoaffinity ATP analogue. After gel electrophoresis
and immunoblotting of the labeled membranes, the MDR1 bands (identified
and the amount of MDR1 quantitated by immunostaining) were excised, and P radioactivity was measured in a liquid scintillation
counter. As shown in Fig. 2, in the case of the wild-type MDR1
and the MK and KM mutants, ATP binding had a relatively high-affinity
component between 5 and 25 µM 8-azido-ATP concentrations,
while such a component was not observable in the case of the MM mutant.
As shown also above, there was no major difference in the level of
P incorporation between the different mutant MDR1 proteins
at higher azido-ATP concentrations.
Figure 2:
8-Azido-ATP labeling of wild type and
mutant MDR1 in isolated membranes of Sf9 cells, effect of 8-azido-ATP
concentration. Isolated Sf9 cell membranes were labeled with
8-azido-ATP as described in the legend to Fig. 1. After gel
electrophoresis and immunoblotting of the labeled membranes the MDR1
bands (identified and the amount of MDR1 quantitated by immunostaining)
were excised, and P radioactivity was measured in a liquid
scintillation counter. The figure shows the mean values of two
independent experiments.
Figure 3:
Vanadate-sensitive MDR1-ATPase activity in
isolated membranes of Sf9 cells, stimulation by verapamil. ATPase
activity of the isolated Sf9 cell membranes was estimated by measuring
inorganic phosphate liberation, as described under ''Materials and
Methods.`` The data points in the figures show the mean ±
S.D. of three to five determinations for each preparation. The
differences between the ATPase activities measured in the absence and
presence of vanadate (100 µM), respectively, are plotted.
Verapamil, when indicated (darker columns), was applied in a
concentration of 20 µM. V, Val; G, Gly
; MK, Met
; KM,
Met
; MM, Met
and Met
in MDR1.
Fig. 3also presents the ATPase activity data for the isolated
Sf9 cell membranes containing the NBD mutant MDR1 proteins. In these
membranes containing amounts of MDR1 similar to that of the Gly or Val constructs (see Fig. 1A), we found that
neither the basal nor the verapamil-stimulated ATPase activities were
significantly greater than those in the
-galactosidase-infected
cell membranes. Since in different membrane preparations this
background ATPase activity was somewhat variable (see error
bars), this may mask a low level of MDR1-ATPase activity (e.g. 3-5% of the wild-type) in the NBD site mutants.
Fig. 4shows the MgATP dependence of the verapamil-stimulated
vanadate-sensitive ATPase activities of -galactosidase-infected
and various MDR1-expressing Sf9 cell membranes. Both the wild-type and
the Gly
-Val mutant MDR1 containing membranes show a
verapamil-stimulated ATPase activity which reaches maximum levels at
about 3 mM MgATP, with an apparent K
for
ATP about 0.5-0.8 mM. MgATP concentrations above 10
mM slightly inhibit this drug-stimulated ATPase. In contrast,
the
-galactosidase expressing membranes, or the membranes
containing either the KM, MK, or MM mutants of MDR1 (in Fig. 4we present only the MM construct), have no significant
vanadate-sensitive verapamil-stimulated ATPase activity in the whole
range of MgATP concentrations examined. Thus, although present in
comparable amounts in the isolated membranes and showing 8-azido-ATP
labeling, these ATP-binding site mutants have no detectable
MDR1-related ATPase activity.
Figure 4:
MgATP concentration dependence of
vanadate-sensitive ATPase activity in isolated Sf9 cell membranes.
ATPase activity of the isolated Sf9 cell membranes was estimated by
measuring inorganic phosphate liberation as described under
''Materials and Methods.`` The data points in the figures
show the mean ± S.D. of three determinations for each membrane
preparation. The differences between the ATPase activities measured in
the absence and presence of vanadate (100 µM),
respectively, are plotted. MgATP concentrations were varied as shown on
the abscissa. V, Val; G,
Gly
; MM, Met
and Met
in MDR1
As shown above, in the case of the
Gly to Val mutant we could not see a significant
difference in the maximum level of verapamil-stimulated ATPase activity
or in its MgATP concentration dependence. However, according to the
data in the literature (Choi et al., 1988; Kioka et
al., 1989) these mutants have different drug specificities. Fig. 5presents the effects of increasing concentrations of
several MDR1-interacting drugs on the vanadate-sensitive ATPase of the
two different (Gly
and Val
) MDR1-expressing
cell membranes. In this figure we present the vanadate-sensitive
MDR1-dependent ATPase activity values corrected for the amount of the
MDR1 protein in the isolated Sf9 membranes (see Fig. 1). As
shown, the verapamil-stimulation curves for the two different MDR1
proteins are similar, although slight differences can be observed. The
concentration required for 50% of maximal stimulation (AC
)
for verapamil is slightly higher in the case of the Gly
(1.8 µM) than for the Val
(1.2
µM) MDR1 construct, and the inhibition of this latter
enzyme is more pronounced at higher verapamil concentrations. In fact,
this latter difference may explain the somewhat higher apparent
AC
values calculated in the case of the Gly
form. The vinblastine activation of the two different
MDR1-ATPases are also similar: in both cases the maximum activity is
obtained at about 2 µM vinblastine, with an apparent
AC
of about 0.5 µM.
Figure 5:
Comparison of drug-stimulation of the
vanadate-sensitive MDR1-ATPase activity in Gly (A) and Val
(B) MDR1 expressing
Sf9 cell membranes. ATPase activity of the isolated Sf9 cell membranes
was estimated by measuring vanadate-sensitive inorganic phosphate
liberation as described under ''Materials and Methods.``
MDR1-ATPase activity was calculated by using the quantitative
immunoblot data as shown in Fig. 1A. The data points in
the figure show the mean ± S.D. of three to six determinations
for each membrane preparation. The respective drug concentrations were
varied as shown on the abscissa.
In contrast to
verapamil and vinblastine, the activation of the two MDR1 forms is
distinctly different by colchicine and VP-16 (etoposide). These
compounds activate the Val MDR1 at lower concentrations
and to much higher maximum levels, than the wild-type MDR1. The
AC
value for colchicine in the Val
membrane
is 15 µM, the maximum stimulation is 65% of that by
verapamil, while in the case of the Gly
MDR1 the
AC
is over 100 µM and the level of maximum
stimulation is less than 20% of that by verapamil. In the case of
etoposide, in the Val
MDR1 membranes the AC
value is 12 µM, the maximum stimulation is again 65%
of that by verapamil, while in the Gly
MDR1 membrane the
AC
value is estimated to be about 60 µM, and
the maximum stimulation level is less than 15% of that by verapamil.
For etoposide, in the Gly
MDR1 a pronounced inhibition of
the MDR1 ATPase activity is seen at above 70 µM of the
drug, while such an inhibition is found only above 100 µM etoposide in the Val
mutant. We have also observed a
difference for the activation of the MDR1-ATPase by vincristine: in
this case the wild-type form is slightly more sensitive to the drug
(the AC
is about 1.5 µM for the Gly, and
about 4 µM for the Val enzyme), although in this case the
levels of maximum activations are about the same.
P-glycoprotein, the product of the human MDR1 gene, is responsible for an ATP-dependent extrusion of numerous cytotoxic drugs from a wide variety of cancer cells, thus a thorough knowledge concerning its molecular mechanism of action would be of utmost importance for a clinical intervention. In vitro expression systems combined with functional assays of the protein may greatly facilitate such studies and may allow a deeper understanding of the structure-function connections within the protein in its natural membrane environment.
A recombinant baculovirus-Sf9 insect cell expression system for producing large amounts of human multidrug transporter has been developed by Germann et al.(1990). The major advantages of this system are that the Sf9 cells perform most of the higher eukaryotic post-translational modifications, including glycosylation and phosphorylation, and seem to insert foreign proteins into the cell membrane in a correct transmembrane orientation (O'Reilly et al., 1992). Antibodies raised against different regions of MDR1 recognized the Sf9-expressed protein, and 8-azido-ATP and specific drug binding was also retained by the molecule (Germann et al., 1990; Sarkadi et al., 1992). The molecular mass of the Sf9-expressed MDR1 is slightly smaller than in most mammalian cells (about 130-140 kDa), representing an underglycosylated form of the protein. However, previous studies indicated that the variable levels of glycosylation in various tissues has no significant effect on the function of MDR1 (Greenberger et al., 1987), and Schinkel et al.(1993) demonstrated that Pgp mutants lacking the N-glycosylation sites produced a drug resistance pattern indistinguishable from that of fully glycosylated wild-type MDR1.
In the experiments presented above we have used the
baculovirus-Sf9 cells expression system to produce wild-type and mutant
forms of the human MDR1 protein. As shown in Fig. 1, all the
expressed mutants showed electrophoretic mobilities comparable to that
of the wild-type P-glycoprotein and were recognized by a polyclonal
antibody specific for the NH-terminal half of MDR1
(antibody 4077; see Tanaka et al.(1990)). A similar antibody
recognition of all the expressed proteins could be seen by the
commercially available C219 monoclonal antibody and by the polyclonal
antibody 4007, recognizing the COOH-terminal half of MDR1 (Tanaka et al., 1990). Since our experiments showed a highly selective
and quantitative recognition of Pgp by antibody 4077 in several cell
types (Homolya et al., 1993), the quantitative assessment of
the expression levels was carried out by using this antibody and the
ECL measurements in scintillation counter (see ``Materials and
Methods'').
In the studies reported here we used baculovirus-infected Sf9 membrane preparations with roughly similar MDR1 expression levels and provide for each mutant the measured specific protein values (Fig. 1A). In each case the respective MDR1 form was produced via baculoviruses containing the MDR1 cDNA in a pVL941 virus vector. It should be noted that MDR1 expression levels were variable when using different baculovirus vectors: e.g. MDR1 in pVL1393 had an expression level of less than one-third of that in the pVL941. Still, the molecular masses, the antibody recognition, and all the functional characteristics were similar in the MDR1 preparations prepared by different baculovirus vectors.
For the
functional characterization of the mutant MDR1 forms we have studied
8-azido-ATP binding, as well as the drug-stimulated vanadate-sensitive
ATPase activity related to the expressed protein. The photoaffinity
analog, 8-azido-ATP has been successfully used to specifically label
various ATP-binding proteins, including Pgp (Cornwell et al.,
1987; Sarkadi et al., 1992; Al-Shawi et al., 1994).
In the past few years several studies have demonstrated a high activity
vanadate-sensitive, drug-stimulated ATPase, directly connected to the
presence of MDR1 in isolated membranes (Sarkadi et al., 1992;
Doige et al., 1992; Shimabuku et al., 1992; Al-Shawi
and Senior, 1993; Loo and Clarke, 1994b), or in partially purified and
reconstituted Pgp preparations (Ambudkar et al., 1992; Doige et al., 1993; Shapiro and Ling, 1994; Urbatsch et
al., 1994; Sharom et al., 1993). The MDR1-ATPase has been
reported to have a relatively low affinity for ATP, with K(MgATP) values ranging between 0.5 and 0.8 mM (Sarkadi et al., 1992; Ambudkar et al., 1992;
Urbatsch et al., 1994), in accordance with a strong effect of
ATP depletion on drug extrusion in intact cells (Endicott and Ling,
1989; Gottesman and Pastan, 1993). All the available evidence strongly
suggest that this MDR1-specific ATPase is closely coupled to the
ATP-dependent transport function of this protein. In fact, 8-azido-ATP,
until exposed to UV light, was shown to be an ATP-like substrate of
MDR1, while covalently bound 8-azido-ATP abolished further ATP
splitting by the transporter (Al-Shawi et al., 1994).
The combination of the ATP binding and hydrolysis assays is believed to provide valuable information about the interaction of genetically manipulated Pgp both with its energy-donor substrate and the transported species. In order to address these questions, the first set of mutants examined in this work was prepared to contain point mutation(s) in the predicted nucleotide binding domains.
Both of the
predicted NBDs of P-glycoprotein contain the highly conserved A and B
consensus motifs, which were originally described by Walker et
al.(1982), from sequence comparisons of a large number of
bacterial and eukaryotic ATP-binding proteins. The glycine-rich A motif
(believed to form a loop between a bend and an
helix) plays
a crucial role in ATP utilization, and the conserved lysine residue in
the Walker A sequence is thought to interact directly with one of the
phosphate groups of the ATP molecule. Mutational analysis of this A
motif in ATP-binding proteins showing ATPase activity, such as the
and
subunits of Escherichia coli F
-ATPase (Parsonage et al., 1988), and the
yeast STE6 transporter (Berkower and Michaelis, 1991), indicated that
substitution of the conserved lysine residue jeopardizes transport
activity. The experiments of Azzaria et al.(1989) demonstrated
that in mouse Pgp the replacement of these key lysines by arginines in
either one of the NBDs eliminated the drug resistance, suggesting that
both NBDs are required for the activity of the transporter, although in
this mutant ATP binding was conserved. It is interesting to note that
when the wild-type and the same double lysine-to-arginine mutant of the
mouse MDR1 were expressed in E. coli, the wild-type
MDR1-dependent drug efflux was retained by the mutant (Bibi et
al., 1993).
Unpublished experiments of Morse et
al. showed, that when mouse cells were transfected
with human MDR1 in which lysines 433 and/or 1076 were replaced by
methionines, these mutant MDR1 proteins did not confer a
multidrug-resistant phenotype. In the experiments described here we
demonstrate that when the MDR1 mutants carrying the same
lysine/methionine substitutions were expressed in Sf9 cells, each of
these point mutations abolished the drug-stimulated ATPase activity,
independent of the MgATP concentrations applied. At the same time,
specific high affinity photoaffinity labeling of MDR1 by 8-azido-ATP
(completely inhibitable by excess cold ATP) was only altered in the
double lysine to methionine (MM) mutant, and even in this case the
labeling of MDR1 was only slightly decreased at 8-azido-ATP
concentrations above 25 µM. It is to be noted that Morse et al.
observed a significant change of
8-azido-ATP binding in the mutant MDR1 proteins when examined at low
(2.5 µM) 8-azido-ATP concentrations.
The ATP concentrations producing half-maximal stimulation of the MDR1-ATPase activity (see Sarkadi et al.(1992) and Shapiro and Ling(1994), and Fig. 4of this paper), or of the MDR1-dependent drug transport (see Gottesman and Pastan (1993)) are in the range of 0.3-0.5 mM. Thus the functional role of the high-affinity ATP binding by MDR1, as seen in the azido-ATP binding experiments, as well as its alteration in the MM mutant, cannot be properly appreciated as yet. Still, our data indicate that drug-stimulated ATPase activity is absent if these highly conservative residues are altered in any of the two NBDs of MDR1, and while single NBD mutations have no major effect on ATP binding at low ATP concentrations, the double lysine to methionine mutation considerably alters this ATP binding.
In a previous study, aimed at investigating
the role of various parts of the MDR1 protein in its function, the
separately expressed NH-terminal half of the human Pgp
showed an ATPase activity comparable to that of the full-length
protein, but did not confer drug resistance when expressed in mammalian
cells (Shimabuku et al., 1992). The authors suggested that the
NH
-terminal NBD contains all the residues required to
hydrolyze ATP, without necessarily interacting with the COOH-terminal
binding site. A key problem in the studies of Shimabuku et
al.(1992) was the relatively low level of the ATPase activity in
the isolated full-length or truncated MDR1 (about 150 nmol/mg MDR1
protein) and the lack of drug-stimulation in either case. Convincing
data for the role of the two halves of MDR1 have recently been provided
by Loo and Clarke (1994b). When the two halves of MDR1 were expressed
in Sf9 cells separately, both proteins showed a low level of ATPase
activity with no substrate stimulation, while their co-expression
restored high-activity, drug-stimulated ATPase.
The data presented in this paper, in accordance with the experiments of Azzaria et al.(1989) and Loo and Clarke (1994b), strongly support the idea that the interaction of two functional NBDs are essential both for drug extrusion and drug-stimulated hydrolysis of ATP by MDR1. At the same time it is still unclear whether the concerted hydrolysis of two ATP molecules may be required to transport one molecule of drug.
Another
key issue to be addressed in the structure-function studies is the
site(s) of interaction of the multidrug resistance protein with its
transported substrates. A single point mutation in human Pgp, a
spontanous exchange of Gly to Val at position 185 (Choi et
al., 1988), was reported to result in an increased relative
resistance to colchicine and etoposide, while unchanged or slightly
reduced resistance toward vinblastine and vincristine (Choi et
al., 1988; Currier et al., 1992; Cardarelli et
al., 1995). In this study we have reproduced the Gly to Val point mutation in the baculovirus-Sf9 expression system
for MDR1. Our experiments showed no significant alteration in the
maximum activity of the MDR1-ATPase or in its activation kinetics by
verapamil and vinblastine, and this mutation did not modify the MgATP
affinity or the 8-azido-ATP binding of the transporter either. However,
the Gly
to Val mutation significantly increased the
stimulation of the MDR1-ATPase by colchicine and etoposide, while
slightly decreasing its stimulation by vincristine. These shifts
closely correspond to the effects of this mutation on the
drug-resistance profile in intact tumor cells. Moreover, the data
indicating that the Gly
to Val exchange, while increasing
colchicine extrusion and colchicine stimulation of the MDR1-ATPase
activity, reduces the binding of this drug to the MDR1 protein (Safa et al., 1990), may suggest that in the molecular mechanism of
drug extrusion, ATP splitting is required for the dissociation of the
drug from the transporter protein. Altogether the data presented in
this paper further support the usefulness of the MDR1-ATPase assay in
isolated Sf9 membranes for a functional characterization of molecular
alterations in the P-glycoprotein.
While the present paper was under
revision, a publication by U. S. Rao (1995) reported the expression and
partial characterization of the Gly to Val MDR1 mutants
in Sf9 cells. The related findings of the two reports are mostly in
accordance, although Rao (1995) obtained a higher maximum activity and
a lower K
for the Val
MDR1-ATPase
than for the Gly
protein when using verapamil. Since in
the experiments of Rao(1995) the maximal MDR1-ATPase activity/mg of
membrane protein was significantly smaller than in our studies, this
difference may have been caused by the difficulties of MDR1
quantitation at lower protein levels, as well as by the use of
different expression vectors and Sf9 cell lines.