(Received for publication, December 12, 1994)
From the
A single amino acid substitution, Gly
Val,
in the human P-glycoprotein (Pgp) was previously shown to cause an
altered pattern of drug resistance in cell lines transfected with the
MDR1 cDNA carrying this mutation. To further define the function of
amino acid 185 in the Pgp, the wild-type and the mutant Val
Pgps were expressed in Sf9 insect cells, and their biochemical
properties were compared. Verapamil- and colchicine-stimulated ATPase
activities were markedly increased with concomitant increase in
affinity for these compounds with Gly
Val
substitution in the Pgp. However, the vinblastine-stimulated ATPase
activities of the wild-type and Val
Pgps were nearly
identical. Because transport substrate-induced ATP hydrolysis is
generally thought to reflect transport function, these data suggest
that colchicine and verapamil are transported at an increased rate with
Gly
Val substitution in the Pgp. These results
also indicate that amino acid 185 is involved in verapamil and
colchicine, but not in vinblastine, binding/transport. Kinetic analyses
indicate that cyclosporin A, an inhibitor of Pgp, binds to the
verapamil and vinblastine binding/transport site(s) in the Pgp. Taken
together, the results presented herein reveal that the verapamil and
vinblastine binding/transport site(s) are in close proximity and that
the cyclosporin A binding site spans the common region of these two
drug binding/transport site(s) in the Pgp molecule.
Tumor cells undergoing chemotherapy often develop resistance to
a wide variety of anti-neoplastic drugs in the phenomenon known as
multidrug resistance (MDR)()(1) . Multidrug
resistant tumor cells frequently express large quantities of a
130-170-kDa membrane glycoprotein, referred to as P-glycoprotein
(Pgp), encoded by the MDR1 gene(2) . Evidence suggests that the
human Pgp acts as an energy-dependent transporter that extrudes from
cells a spectrum of compounds and drugs with diverse structure and
function. The human Pgp is a 1280-amino acid polypeptide, with
secondary structure predicted to have 12 transmembrane segments and 2
ATP binding domains on the cytoplasmic side of the molecule. The human
Pgp shares considerable sequence and structure homology with an
ever-growing list of ATP-binding cassette transport
proteins(3) .
Direct interaction of photoaffinity analogs of chemotherapeutic agents such as colchicine(4) , Adriamycin(5) , and vinblastine (6) with the Pgp has been reported. Mapping of the drug binding site(s) in Pgp using photoaffinity analogs has indicated that these site(s) are within or in close proximity to the putative transmembrane segments of the molecule (7) . However, the transmembrane segments and amino acid residues of the Pgp involved in substrate binding and transport have not been precisely identified. An understanding of the molecular interactions of drugs with the Pgp would greatly facilitate our ability to modulate its function. An approach most commonly employed toward this end by several laboratories is site-directed mutagenesis in combination with analyses of relative drug resistance in cells(8, 9) . Although the information obtained from these studies is valuable for understanding the overall process of MDR, these assays do not accurately reflect the transport function of the Pgp.
Recently, Scarborough and co-workers (10) have demonstrated a drug-stimulated ATPase activity of the human Pgp expressed in cultured Sf9 insect cells. Several laboratories have subsequently shown the drug-stimulated ATPase activity in mammalian cells constitutively expressing the Pgp(11, 12, 13) . Most importantly, the ability of a variety of compounds to stimulate the Pgp ATPase activity has recently been directly correlated with their transport by the Pgp(14) . Thus, measurements of the ATPase function of the Pgp may be used to explore the possible molecular mechanism by which the Pgp mediates drug transport.
Cells selected in vitro against any single drug from a diverse group of lipophilic
cytotoxic compounds usually develop cross-resistance to other drugs in
the group. Some multidrug-resistant cell lines are significantly more
resistant to the drug used in their selection than to the other
drugs(15, 16) . Further studies have suggested that a
single amino acid substitution, Gly
Val, in the
human Pgp was responsible for the preferential resistance of KB
epidermoid carcinoma cells to colchicine (15, 16, 34) . However, it is not known how
the ATPase function of the Pgp is modulated by the Val substitution at
amino acid 185 in the polypeptide. In this paper, I report the
expression of wild-type Pgp and compare its functional properties with
that of the Val
Pgp. The results reported here suggest
that the amino acid at position 185 in the Pgp is involved in
colchicine and verapamil but not in vinblastine binding/transport. The
results presented herein also indicate the propinquity of
binding/transport site(s) of verapamil, vinblastine, and cyclosporin A
in the human Pgp.
To ensure that these recombinant baculoviruses contained the wild-type MDR1 cDNA, their DNA was amplified by polymerase chain reaction according to the protocol provided by the manufacturer (Invitrogen), cloned into M13 bacteriophage vector, and sequenced around the region that codes for the amino acid 185 in the Pgp. The results clearly indicated that these recombinant baculoviruses were the wild-type MDR1 BVs (data not shown).
Preparation of recombinant baculovirus
carrying the human MDR1 cDNA with Val substitution in the
expressed protein, termed in this paper as Val
MDR1 BV,
was previously described by Germann et al.(20) .
Figure 1:
Western blot analysis of membranes
isolated from Sf9 cells infected with wild-type MDR1 BV and Val MDR1 BV. The Sf9 insect cells were infected with wild-type MDR1
BV 9 and 10 and Val
MDR1 BV and incubated at 27 °C
for 3 days. The membranes were prepared as described, and equal amounts
of membrane protein (20 µg/lane) were subjected to SDS-PAGE and
Western blot analysis using C-219 antibody as described under
``Experimental Procedures.'' Lanes1, 4, 7, 10, membranes from wild-type MDR1 BV
9-infected Sf9 cells; lanes2, 5, 8, 11, membranes from wild-type MDR1 BV 10-infected
Sf9 cells; lanes3, 6, 9, 12, membranes from Val
MDR1 BV-infected Sf9
cells. Only the
130-kDa region of the blot is shown. No
antibody-reactive material was seen elsewhere in the
blots.
Analyses of several batches of membrane
preparations indicated that the amounts of Pgp expressed were variable.
Since the objective of the present study was to measure the functional
differences between the wild-type and the Val Pgps,
quantitative estimation of the amount of Pgp protein in these membranes
was essential. Although Pgps separated on SDS-PAGE gels were stained
with Coomassie Blue and silver, quantitation could not be performed
because several non-Pgp protein bands were also observed in this region
of the gels. The C219 monoclonal antibody-reactive hexapeptide epitope (27) is identical in the wild-type and Val
Pgps.
Thus, immunoblots developed using the C219 antibody as the primary
antibody were used for quantitating the Pgps in equal amounts of
several membrane preparations (Fig. 1). The amount of
Val
Pgp arbitrarily was considered 1 absorbance unit, and
the amounts of wild-type Pgps were calculated as multiples of the
amount of Val
Pgp. For example, the amount of Val
Pgp in lane3 was considered 1 absorbance unit,
and the amounts of Pgp in lanes1 and 2 were
expressed relative to the amounts of Pgp in lane3.
Thus, the average values of 4 determinations indicated that the
wild-type Pgp 9 and 10 were 1.89 and 1.58 times the amount of
Val
Pgp (data not shown). These quantitative measurements
would not distinguish between functionally active and inactive forms of
the Pgps (see ``Discussion'').
Figure 2:
Effect of verapamil on the ATPase activity
of the wild-type and Val Pgps. The wild-type Pgp 9-
(
), wild-type Pgp 10- (
), and Val
Pgp-
containing membranes were incubated with increasing concentrations of
verapamil and assayed for ATPase activity as described under
``Experimental Procedures.'' Each data point was the average
of duplicate determinations.
Colchicine stimulated
ATPase activity of the wild-type Pgp 9 and 10, and at 400
µM, these activities reached a maximum of about 8 nmol of
P released per min per unit of Pgp (Fig. 3). A K
value of 163 ± 18 µM was
calculated from the double-reciprocal plots of five different
determinations. The Val
Pgp exhibited a maximal ATPase
activity in the presence of colchicine of about 25 nmol of P
released per min per unit of Pgp, with a K
of approximately 67 ± 9 µM (five
determinations). These results suggest that replacement of Gly
with Val in the Pgp increases the affinity for colchicine by more
than 2-fold.
Figure 3:
Effect of colchicine on the ATPase
activity of the wild-type and Val Pgps. The wild-type Pgp
9- (
), wild-type Pgp 10- (
), and Val
Pgp-
(
) containing membranes were incubated with increasing
concentrations of colchicine and assayed for ATPase activity as
described under ``Experimental Procedures.'' Each data point
was the average of duplicate
determinations.
Vinblastine markedly increased the rate of ATP
hydrolysis by the wild-type and Val Pgps (Fig. 4).
Both forms of Pgp exhibited nearly identical maximum ATPase activity of
approximately 13 nmol of P
released per min per unit of Pgp
in the presence of vinblastine. The K
for
vinblastine calculated from double-reciprocal plots of four
determinations was 0.45 ± 0.06 µM. These data
suggest that mutation at amino acid 185 in the Pgp polypeptide does not
alter the affinity for vinblastine.
Figure 4:
Effect of vinblastine on the ATPase
activity of the wild-type and Val Pgps. The wild-type Pgp
9- (
), wild-type Pgp 10- (
), and Val
Pgp-
(
) containing membranes were incubated with increasing
concentrations of vinblastine and assayed for ATPase activity as
described under ``Experimental Procedures.'' Each data point
was the average of duplicate
determinations.
Although the above data suggest
that the mutation Gly
Val in the Pgp alters the
affinity for verapamil and colchicine, it does not rule out the
possibility that the affinities for ATP binding are responsible for
these results. However, measurements using increasing concentrations of
Mg
ATP on the drug-stimulated Pgp ATPase activities yielded
identical K
values of 0.62 mM for
Mg
ATP with both Pgp forms (data not shown). These results
indicate that the mutation at amino acid 185 in the Pgp does not affect
nucleotide binding.
We have shown recently that cyclosporin A
inhibits verapamil- and vinblastine-activated Val Pgp
ATPase activities in a competitive manner, with K
values of approximately 20 nM(22) . In the
present study, the interaction of cyclosporin A with the wild-type Pgp
in the presence of the above drugs was analyzed (Fig. 5).
Verapamil- and vinblastine-stimulated wild-type Pgp ATPase activities
were inhibited by cyclosporin A, and the double-reciprocal plots
suggested that the inhibition was competitive (Fig. 5, insets). The K
for cyclosporin A in the
presence of both of these drugs was 150 ± 10 nM (four
determinations). Thus, the affinity for cyclosporin A was increased by
more than seven times with Gly
Val mutation in the
Pgp molecule.
Figure 5:
Kinetics of inhibitions of the verapamil-
and vinblastine-stimulated wild-type Pgp ATPase activity by cyclosporin
A. The verapamil (panelA) or vinblastine (panelB) concentration was varied, with constant cyclosporin A
concentrations of 0 (), 0.2 (
), and 0.4 µM (
). Insets, double-reciprocal plots of the
data.
The colchicine-induced mutation in the MDR1 gene bestows upon
transfected cell lines a selective resistance to this
drug(15, 16, 34) . Although the biochemical
properties of the Val Pgp are known mainly through the
work from the laboratories of Gottesman (20) and
Scarborough(10, 21, 22) , it is not known how
the Gly
to Val mutation alters the ATPase function of the
Pgp. The results presented here indicate that the wild-type human Pgp
expressed in Sf9 cells exhibits drug-stimulated ATPase activity.
To
critically examine any functional differences between the wild-type and
Val Pgps, it is necessary to present the drug-stimulated
ATPase activity data obtained with equal amounts of Pgp protein in
these membranes. Although the densitometric analysis employed to
quantitate the Pgps in the membranes is reliable, it does not
discriminate functional from nonfunctional Pgp proteins in the membrane
preparations. Several functional photoaffinity ATP and drug analogs are
available, which may prove to be valuable in quantitating the amounts
of Pgp in the membrane
preparations(28, 29, 30) . However, it is not
clear how the Gly
Val mutation regulates the
interactions of these photoaffinity analogs with the Pgp. Thus, to
minimize this uncertainty, two isolates of wild-type MDR1 BVs, which
express different amounts of the wild-type Pgps, were employed in the
present study. As shown in Fig. 2Fig. 3Fig. 4, the
drug-stimulated ATPase activities of the two wild-type Pgps were nearly
identical, indicating that the densitometric quantitation of Pgps
employed is dependable. In addition, the kinetic parameters K
, K
, and K
, which do not depend on the enzyme
concentration, are suitable for describing the functionally different
Pgp preparations.
The results presented in Fig. 2and Fig. 3indicate that the interactions of Pgp with verapamil and
colchicine are affected by the mutation at amino acid 185 in the
polypeptide. The distinctly different activation profiles of Pgp ATPase
and K values for these drugs suggest that
replacement of Gly
with Val leads to higher affinity
interactions with the Pgp. Importantly, the increase in
colchicine-induced Val
Pgp ATPase activity compared with
that of the wild-type Pgp directly correlates with the increase in drug
resistance imparted by the mutation Gly
to Val in the Pgp
molecule(15, 16) . Homolya et al. (14) have recently demonstrated that only those compounds that
stimulate ATP hydrolysis are transported by the Pgp. Furthermore,
solubilized transport ATPases display ATP hydrolysis-coupled
conformational changes, substrate binding, and dissociation, indicating
the complete catalytic cycle (31, 32, 33) .
Thus, the Pgp ATPase activity induced by a drug is a direct measure of
its transport.
The vinblastine-stimulated ATPase activity profiles
and the K values obtained with the wild-type and
Val
Pgps are nearly identical, indicating that
Gly
is not involved in vinblastine binding/transport. In
contrast, the relative drug resistance of transfected cell lines
suggested that Gly
Val substitution in the Pgp
slightly increases the sensitivity to
vinblastine(15, 16) . These data would be consistent
with the hypothesis that there may be an additional non-Pgp-mediated
resistance mechanism for vinblastine action, which could account for
these observations(15) .
We have shown recently that there
are two classes of substrates for Pgp. The first group interacts with
the Pgp and elicits ATPase activity, implying that these drugs are
transported by the Pgp. The second class of compounds, which include
cyclosporins, interact with Pgp with high affinity but fail to elicit
ATP hydrolysis. However, the cyclosporins act as competitive inhibitors
of transport substrate-induced ATP hydrolysis(22) . The data
shown in Fig. 5indicate that cyclosporin A also inhibits
verapamil- and vinblastine-stimulated wild-type Pgp ATPase activities
in a competitive manner. The simple interpretation of these
observations is that cyclosporin A binds to the verapamil and
vinblastine binding/transport site(s). Interestingly, the affinity for
cyclosporin A increased markedly with Gly
Val
substitution, suggesting that amino acid 185 is involved in the
cyclosporin A interactions with the Pgp.
These results may bear in an important way on the issue of number and location of drug binding/transport sites in the Pgp molecule. There may be two drug binding/transport sites in the Pgp that are distinguishable by the presence or absence of amino acid 185, one site to which both verapamil and colchicine bind and a second to which vinblastine alone binds. Because the verapamil- and vinblastine-stimulated Pgp ATPase activities were inhibited by cyclosporin A, the verapamil and vinblastine binding/transport sites are probably in close proximity, and cyclosporin A binds to the common region of both of these sites. Alternatively, amino acid 185 is not in the drug binding/transport site(s), but mutations at this residue bring about conformational changes that impose different constraints on the Pgp, leading to altered patterns of affinity and ATPase activity with different drugs. Further studies aimed at characterizing the drug binding/transport site(s) of the Pgp should enable us to better define the mechanism of MDR in tumor cells.