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MRC Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, Ontario M5S 1A8, Canada
1Correspondence: Department of Medicine, University of Toronto, Room 7342, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. E-mail: david.clarke{at}utoronto.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: multidrug transporter • protein folding • proteasome inhibitor • endoplasmic reticulum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The intrinsic or acquired ability of tumor cells to
show resistance to multiple chemotherapeutic agents (multidrug
resistance) is a major obstacle to successful chemotherapy.
The first protein implicated in the phenomenon of multidrug resistance
was the human multidrug resistance P-glycoprotein
(P-gp),2
the product of the human MDR1 gene (2)
. Cell lines
overexpressing P-gp were initially selected for resistance to a single
cytotoxic drug, but were subsequently found to exhibit cross-resistance
to the effects of other structurally unrelated cytotoxic drugs
(3
4
5)
. Analysis of P-gp levels in tumor cells obtained
from patients showed that P-gp is expressed at biologically significant
levels in ~50% of human cancers (1
, 6
7
8)
.
P-gp also interferes in the treatment of AIDS patients with protease
inhibitors. Oral absorption and penetration of FDA-approved protease
inhibitors into the central nervous system are quite poor. This is due
to the presence of relatively high amounts of P-gp in the intestine and
at the blood–brain barrier (9
, 10)
.
There has been an intense effort to understand how P-gp is able to
confer multidrug resistance. Its physiological role is likely to
protect us from endogenous and exogenous cytotoxic compounds
(11
12
13)
. P-gp is a member of the ABC (ATP binding
cassette) superfamily of transport proteins (14)
. Its 1280
amino acids are arranged in two tandem repeats joined by a linker
region of 60 amino acids. Each repeat consists of an
NH2-terminal hydrophobic domain containing six
potential transmembrane segments, followed by a hydrophilic domain
containing a nucleotide binding fold (15
16
17)
.
An important goal is to devise strategies to `shut down' P-gp during
chemotherapy. The current protocols of using combined chemotherapy rely
to some extent on the varying affinities of each drug for P-gp. In
these mixtures, it is expected that drugs with a higher affinity for
P-gp would act as `pseudo-inhibitors', thereby allowing those with
lower affinities to enter the cell. This form of therapy has been used
successfully to treat pediatric tumors such as retinoblastoma and
neuroblastoma (6)
.
A potential novel strategy to bypass the effects of P-gp would be to
prevent P-gp from reaching the cell surface by interfering with its
maturation after synthesis in the endoplasmic reticulum. P-gp appears
to be quite susceptible to such a strategy, since it was previously
shown that its maturation can be manipulated by point mutations
(18)
and drug substrates (19)
. Many point
mutations have been identified that result in the synthesis of 150 kDa
core-glycosylated proteins as the major product (20)
.
These core-glycosylated intermediates are retained in the endoplasmic
reticulum in association with the chaperones calnexin (21)
and hsc70 (22)
and are rapidly degraded. These processing
mutants, however, can be converted to the mature 170 kDa enzyme, which
is trafficked to the cell surface if synthesis is carried out in the
presence of drug substrates (19)
. By contrast, maturation
of P-gp can be inhibited by carrying out expression in the presence of
proteasome inhibitors such as lactacystin or MG-132 (23)
.
It is generally believed that polytopic membrane proteins and
those destined to be trafficked to the cell surface are fully active
immediately after synthesis in the endoplasmic reticulum. Indeed, with
CFTR, another member of the ABC superfamily of transporters, it has
been reported that the protein is functional when still in the
endoplasmic reticulum (24)
.
In this study, we tested whether P-gp was active in the initial stages of biosynthesis. An inactive core-glycosylated P-gp intermediate would suggest that inhibition of P-gp maturation could be exploited as a potential novel method for circumventing the problem of multidrug resistance, thus resulting in the use of lower doses of chemotherapeutic drugs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. For purification purposes, a full-length MDR1 cDNA
was modified to encode for 10 histidine residues at the COOH terminus
of the protein (26)
. The sequence at the COOH terminus of
full-length P-gp, which would normally end as TKRQ, now became
TKRA(His)10LDPRQ. Construction
of Cys-less P-gp in which all cysteine residues were changed to alanine
has been described (16)
. The Cys-less mutant was also
modified to contain mutations L332C in TM6 and L975C in TM12
(27)
. A glycosylation-deficient mutant was made by
changing the codon for the three consensus glycosylation sites (N91,
N94, and N99) to code for alanine (23)
.
Purification of P-gp mutants and measurement of ATPase activity
HEK293 cells transfected with cDNAs coding for the
histidine-tagged P-gps were solubilized with 1% (w/v)
n-dodecyl-ß-D-maltoside, and the mutant P-gps were purified by
nickel-chelate chromatography using Ni-NTA spin columns (Qiagen,
Chatsworth, Calif.), as described previously (26)
. For
isolation of the processing mutants of P-gp, the concentration of
imidazole in the wash buffers was reduced from the standard 80 mM to 20
mM. Drug-stimulated ATPase activity of the isolated protein was
determined after reconstitution with lipid as described previously
(26)
.
Sulfhydryl cross-linking analysis
For each mutant P-gp, 10 (10 cm diameter) culture plates of
HEK293 cells were transfected with the mutant MDR1 cDNA. After 16 h, the media was replaced with fresh media or fresh media containing 2
µM MG-132. The cells were harvested after another 24 h and
membranes were prepared as before (28)
. The membranes were
suspended in 200 µl of Tris-buffered saline, pH 7.4. A sample of the
membrane suspension (12 µl) was mixed with 15 µl of 2x ATPase
buffer (100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM
MgCl2) containing none or 10 mM ATP.
Cross-linking was initiated by addition of 3 µl of 20 mM
Cu2+(phenanthrolene)3. The
samples were incubated for 5 min at 37°C and the reaction was stopped
by addition of 50 µl of 2x sodium dodecyl sulfate (SDS) sample
buffer [125 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS]
containing 50 mM EDTA and no reducing agent. The samples were subjected
to SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto a
sheet of nitrocellulose, and immunoblotted with a rabbit polyclonal
antibody against P-gp, followed by detection with enhanced
chemiluminescence (22)
.
TPCK-trypsin digestion
HEK293 cells were transfected with various MDR1 cDNAs and
treated with or without MG-132, as described above. Membranes were then
prepared and suspended in Tris-buffered saline. The membranes (5 mg/ml
protein) were treated for 5 min at 22°C with various amounts of
TPCK-trypsin [Sigma (St. Louis, Mo.) 12,000 BAEE units per mg], and
the reaction was stopped by addition of lima bean trypsin inhibitor
(Worthington, Freehold, N.J.) (20)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We have been able to purify the mature form of the
histidine-tagged P-gp by nickel chelate chromatography for measurement
of drug-stimulated ATPase activity. In contrast, the histidine-tagged
core-glycosylated 150 kDa protein of the processing mutants could not
be purified unless the imidazole concentration during the washes was
lowered from 80 mM to 20 mM. Under these modified conditions, the
core-glycosylated protein could be isolated in sufficient quantities
for measuring drug-stimulated ATPase activity. Accordingly, the
histidine-tagged 150 kDa protein of mutants G268V, G269V, and Y710A
were isolated (Fig. 1
, lanes 1, 3, and 5). These mutants were analyzed because the major P-gp
product was the 150 kDa core-glycosylated biosynthetic intermediate
(18
, 21)
.
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To convert the processing mutants G268V, G269V, and Y710A to the mature
enzyme, the mutants were synthesized in the presence of 10 µM
cyclosporin A to induce maturation (19)
. Cyclosporin A is
a particularly useful P-gp substrate for inducing maturation of the
processing mutants since it is relatively nontoxic to the cells, and
its very hydrophobic nature allows the compound to readily diffuse into
the cell. Isolation of the mutants by nickel-chelate chromatography
after synthesis with cyclosporin A resulted in the recovery of the
mature 170 kDa mutant P-gp (Fig. 1
, lanes 2, 4, and 6).
The isolated mature 170 kDa and core-glycosylated 150 kDa P-gps were
assayed for verapamil and vinblastine-stimulated ATPase activity.
Verapamil causes the greatest stimulation of the ATPase activity of
P-gp (29)
, whereas vinblastine-stimulated ATPase activity
correlates well the turnover number of vinblastine transport
(30)
. Figure 2
shows that the mature form of all three processing mutants exhibited
verapamil and vinblastine-stimulated ATPase activities. The mature
enzyme of mutant Y710A had activities that were similar to wild-type
P-gp. As reported previously (19)
, the mature enzyme of
mutants G268V and G269V had ~50 to 75% of the activity of wild-type
P-gp. By contrast, there was no detectable basal or drug-stimulated
ATPase activity in the 150 kDa core-glycosylated proteins from the
processing mutants. These results suggest that the core-glycosylated
enzymes of processing mutants are nonfunctional.
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ATPase activity of P-gp that is prevented from undergoing
maturation
It was possible that the 150 kDa core-glycosylated P-gp of the
processing mutants G268V, G269V, and Y710A were inactive because the
point mutations inherently caused misfolding of the protein and
subsequent retention in the endoplasmic reticulum. Therefore, another
approach for assaying the activity of the core-glycosylated P-gp is to
prevent maturation of P-gp by inhibitors. The proteasome inhibitors
lactacystin and MG-132 have been reported to inhibit maturation of the
cystic fibrosis transmembrane conductance regulator (31)
and human P-gp (23)
. A histidine-tagged Cys-less P-gp
containing an R113A mutation and grown in the presence of 2 µM MG-132
was assayed for drug-stimulated ATPase activity. The Cys-less P-gp was
used because it was active, but its rate of maturation was relatively
slower than the wild-type enzyme (16)
. This relatively
slower rate of maturation was used to advantage since it was possible
to `trap' the Cys-less P-gp as a core-glycosylated intermediate. By
contrast, the wild-type P-gp matured quite rapidly, such that a very
small amount of the core-glycosylated P-gp still matured (leakage) into
the mature enzyme even in the presence of MG-132 (data not shown). The
R113A mutation was included since it stabilizes P-gp against
proteolytic digestion during biosynthesis, and it resulted in increased
yield of enzyme (23)
. Figure 3
shows that the major product of the Cys-less(R113A) mutant in the
presence of increasing concentrations of MG-132 was the 150 kDa
core-glycosylated intermediate. At MG-132 concentrations greater than
0.63 µM, expression of the 170 kDa mature P-gp was inhibited (Fig. 3
,
lane 6). In the presence of 1.25 µM MG-132, the 150 kDa
core-glycosylated P-gp was the major product (Fig. 3
, lane 5).
Accordingly, the histidine-tagged Cys-less(R113A) P-gp grown with or
without 2 µM MG-132 was isolated by nickel-chelate chromatography. As
shown in Fig. 4
A (lane 4), the 150 kDa core glycosylated Cys-less(R113A)
P-gp could be recovered by nickel-chelate chromatography when the
mutant was grown with MG-132. By contrast, in the absence of MG-132,
there was approximately equal amounts of 150 kDa core-glycosylated and
170 kDa mature P-gps (Fig. 4A
, lane 3).
|
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Equivalent amounts of the isolated Cys-less(R113A) P-gps were
reconstituted with lipid and assayed for drug-stimulated ATPase
activity. Figure 4B
shows that P-gp expressed in the
presence of MG-132 showed no drug-stimulated ATPase activity. In the
absence of MG-132, however, P-gp exhibited 10.3- and 4.7-fold
stimulation of ATPase activity in the presence of verapamil and
vinblastine, respectively. This suggested that MG-132 likely
`trapped' the 150 kDa core-glycosylated P-gp in a conformation that
was unable to couple drug binding to ATP hydrolysis.
ATP-induced conformational changes in the transmembrane domain
The absence of drug-stimulated ATPase activity in the 150
kDa core-glycosylated P-gp suggested that the protein may not undergo
ATP-induced conformational changes. It has been shown that ATP
hydrolysis induces conformational changes in P-gp (32
, 33)
through the transmembrane (TM) domains (27
, 28)
. The
ATP-induced conformational changes in the TM domains can be detected by
disulfide bond formation between Cys332 in TM6 and Cys975 in TM12.
Cross-linking between these two cysteine residues occurs only in the
presence of an oxidant while P-gp is hydrolyzing Mg·ATP. No
cross-linking was observed in the presence of Mg·ADP, nonhydrolyzable
ATP analog (AMP-PNP) or with Mg·ATP and vanadate. Vanadate inhibits
ATP hydrolysis by P-gp (34)
.
Cys-less P-gp containing the mutations L332C and L975C was expressed
with or without MG-132 and cross-linked in the presence or absence of
Mg·ATP. Figure 5
shows that ATP induces cross-linking in mutant L332C/L975C when
expressed in the absence of MG-132. P-gp containing a disulfide bond
between TM6 and TM12 migrates with lower mobility on SDS-PAGE (Fig. 5
,
lane 4) (27
, 28)
. In the presence of MG-132, however, the
150 kDa core-glycosylated mutant L332C/L975C was not cross-linked by
oxidant in the absence or presence of ATP (Fig. 5
, lanes 6 and 8).
These results show that the 150 kDa core-glycosylated mutant did not
exhibit the same conformational changes during ATP hydrolysis as
observed in the 170 kDa mature enzyme.
|
Carbohydrate moieties of P-gp are not required for P-gp function
The results indicate that the 150 kDa core-glycosylated
intermediates isolated from processing mutants or by preventing
maturation of the core-glycosylated P-gp with MG-132 were not capable
of coupling drug binding to ATP hydrolysis. One possible explanation
was that the processing of the carbohydrate moieties of P-gp as it
traversed the Golgi apparatus was essential for function. To test this
hypothesis, residues in the consensus glycosylation sites (N91, N94,
and N99) were mutated to alanine (35)
, and a histidine tag
was attached at the COOH terminus of the protein (26)
to
facilitate purification of the mutant P-gp by nickel-chelate
chromatography (Fig. 6
A). Equivalent amounts of 170 kDa mature and 140 kDa
glycosylation-deficient P-gp were reconstituted with lipid and assayed
for drug-stimulated ATPase activity. Figure 6B
shows that
the 140 kDa glycosylation-deficient P-gp exhibited verapamil- and
vinblastine-stimulated (15.9- and 7.8-fold, respectively) ATPase
activities that were similar to the wild-type P-gp. Therefore, the
presence of carbohydrate moieties on P-gp is not essential for coupling
drug binding to ATPase activity. These results support the findings of
Schinkel et al. (35)
, who showed that the
glycosylation-deficient P-gp was able to confer drug resistance on
cells expressing this mutant P-gp.
|
TPCK-trypsin sensitivity of P-gp expressed with or without MG-132
Another plausible explanation for the lack of activity in the 150
kDa core-glycosylated intermediates of P-gp was that the presence of
point mutations or synthesis in the presence of MG-132 may `trap' the
transporter in an inactive conformation. We previously demonstrated
that P-gp processing mutants are present in the cell as partially
unfolded proteins (20)
and are ~100-fold more sensitive
to trypsin digestion compared to the 170 kDa mature enzyme.
To test whether the proteasome inhibitor MG-132 also affected the
proper folding of P-gp, we assayed for sensitivity of P-gp to trypsin
digestion. Membranes were prepared from HEK293 cells expressing
Cys-less(R113A) P-gp that were grown with or without MG-132. The
membranes were then treated with various concentration of trypsin.
Figure 7
shows that P-gp expressed in the presence of MG-132 was very sensitive
to trypsin digestion. The 150 kDa core-glycosylated intermediate was
not detected after treatment with 10 µg/ml trypsin. By contrast, the
170 kDa mature enzyme when expressed in the absence of MG-132 was
relatively resistant to trypsin. It required the use of 100-fold more
trypsin (1000 µg/ml) before it became undetectable by immunoblot
analysis. The trypsin sensitivity of the 150 kDa core-glycosylated P-gp
in the absence of MG-132 was also similar to that seen in the presence
of MG-132. These results are consistent with the explanation that the
150 kDa core-glycosylated P-gps are inactive because they are
structurally different from the 170 kDa mature enzyme and that MG-132
prevents P-gp from folding into the mature trypsin-resistant P-gp.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Expression of P-gp processing mutants in the
presence of drug substrate, however, induced the transporter to adopt a
trypsin-resistant structure that was trafficked to the cell surface and
exhibited drug-stimulated ATPase activity. It appeared that drug
substrates affected biosynthesis of P-gp by inducing superfolding of
the transmembrane domains (20)
.
Structural differences between mature and core-glycosylated proteins
have also been reported for the cystic fibrosis conductance regulator
(CFTR), another member of the ABC superfamily of transporters. The most
common mutation in cystic fibrosis is a deletion of F508 (
F508)
(36)
. The mutation causes the CFTR protein to be retained
inside the cell instead of being at its proper location on the cell
surface (37)
. Recently, it was shown by protease digestion
studies that the structure of F508 CFTR was different from the
mature wild-type CFTR (38)
. It was interesting that the
proteolytic digestion pattern of F508 CFTR resembled that of the
immature core-glycosylated wild-type CFTR. This suggests that, as with
point mutations in P-gp, the F508 processing mutation in CFTR does
not significantly alter the structure of CFTR, but interferes with the
folding steps that occur after synthesis of the protein. This is
supported by the finding that F508 CFTR can be induced to fold
properly and be correctly targeted to the plasma membrane if synthesis
is carried out at reduced temperatures (39)
or in the
presence of chemical chaperones such as glycerol, trimethylamine
N-oxide, or deuterated water (40
, 41)
.
Maturation of CFTR and P-gp can be inhibited by proteasome inhibitors.
Expression of wild-type CFTR in the presence of MG-132 completely
blocks maturation of the protein (31)
. This study also
reported that MG-132 slowed maturation of P-gp. The authors also did
not observe any accumulation of a 130 kDa P-gp breakdown product in the
presence of MG-132. This is contrary to our findings (20
, 23)
, and likely reflects differences in the cell lines used.
There is, however, a difference between CFTR and P-gp in that the
F508 CFTR core-glycosylated and immature wild-type CFTR proteins are
functional. Patch-clamp studies of the endoplasmic reticulum of cells
expressing F508 and wild-type CFTR suggest that core-glycosylated
proteins form chloride channels that are normally regulated
(24)
. This suggests that the structural requirements to
form a regulated channel may not be as stringent as those required to
form a drug transporter.
It is not clear how proteasome inhibitors interfere with maturation of
CFTR and P-gp. One possibility is that inhibition of a proteasome by
inhibitors such as MG-132 could have pleiotropic effects on the cell
metabolism as a result of accumulation of undegraded proteins. In CFTR,
it has been shown that maturation is an ATP-dependent process
(42)
. Another possibility is that the proteasome directly
plays a role in the folding process by acting as a potential chaperone.
Since most of the P-gp and CFTR molecules are accessible from the
cytoplasm during synthesis in the endoplasmic reticulum, they could be
in direct contact with proteasomes. Recent evidence from studies of
yeast Ste6p processing mutants supports the notion that the proteasome
may have such a role (43)
. The Ste6p protein is a yeast
ABC transporter that transports a mating factor. Loayza et al.
(43)
reported that overexpression of the Hrd2p proteasome
subunit in yeast promoted the appearance of functional Ste6p processing
mutants at the cell surface. Thus, the proteasome could play a dual
role in the folding of ABC transporter; they could promote folding of
the proteins as well as mediate degradation of incorrectly folded
proteins (44
, 45)
.
Studies of human P-gp have identified several steps that prevent
maturation of the enzyme; the steps are summarized in Fig. 8
. Some mutations such as G341C and Q347C (23
, 46)
expose a
proteolytic site in the first extracellular loop that result in
degradation of the protein during or immediately after synthesis. These
mutants can be rescued, however, by mutating the protease-sensitive
site (23)
. Another type of folding defect is that caused
by point mutations that result in misprocessing of P-gp. In these
mutations, the P-gp accumulates as inactive, partially folded
intermediates that do not seem to be able to overcome a barrier to
further folding. In the presence of drug substrates, however, these
barriers are removed and the protein folds correctly and is delivered
to the cell surface in an active form (19)
. By contrast,
expression in the presence of proteasome inhibitors blocks proper
folding and maturation of P-gp. These findings suggest there are
several steps during P-gp synthesis and maturation that could be
potential targets for inhibition during chemotherapy. Potential
strategies would be to identify a pharmacological agent(s) that causes
exposure of hypersensitive-protease sites such as R113 (Fig. 8
, step
1), that can induce the same alterations as in the processing mutants
(e.g., G268V, Fig. 8
, step 2), or that blocks protein folding steps as
seen with protease inhibitors (Fig. 8
, step 3). Learning how to
manipulate such steps in the endoplasmic reticulum may also contribute
to strategies to treat other diseases that involve defective protein
folding and/or trafficking (47
48
49)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication February 8, 1999. Accepted for publication July 6, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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