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J. Biol. Chem., Vol. 275, Issue 29, 22597-22604, July 21, 2000
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From the Laboratory of Cell Biology, NHLBI, National Institutes of
Health, Bethesda, Maryland 20892
Received for publication, March 10, 2000
Glucocorticoid receptors must be complexed with
Hsp90 in order to bind steroids, and it has been reported that at least
three other proteins, Hop, Hsc70, and a J-domain protein (either Hsp40 or Ydj1), are required for formation of active Hsp90-steroid receptor complex. In the present study, we reinvestigated activation of stripped
steroid receptors isolated from either L cells or WCL2 cells.
Surprisingly, we found, using highly purified proteins, that only Hsp90
and Hsc70 are required for the activation of glucocorticoid receptors
in the presence of steroids; in the absence of steroids, either p23 or
molybdate are also required as reported previously. Addition of Hop or
Ydj1 had no affect on the rate or magnitude of the activation of the
stripped receptors, and quantitative Western blots confirmed that
neither Hop or Hsp40 were present in our protein preparations or in the
stripped receptors. Furthermore, a truncated recombinant Hsp70 that
does not bind Hop or Hsp40 was as effective as wild-type Hsp70 in
activating stripped receptor. Since Hsc70 does not bind directly to
Hsp90 but both proteins bind to Hop, it has been suggested that Hop
acts as a bridge between Hsp90 and Hsp70. However, we found that after
Hsc70 or Hsp90 bind directly to the stripped receptors, they are fully
reactivated by Hsp90 or Hsc70, respectively. We, therefore, conclude
that Hsp90 and Hsc70 bind independently to stripped glucocorticoid receptors and alone are sufficient to activate them to bind steroids.
A number of nuclear hormone receptors and protein kinases involved
in signal transduction occur in multi-protein complexes containing heat
shock proteins and several additional proteins (1, 2).
GR1 isolated from the cytosol
in such multi-protein complexes bind glucocorticoid, but, when the
receptors are immunoadsorbed and then stripped of Hsp90 and other
associated proteins by treatment with high salt, they no longer do so
(3, 4). Although addition of Hsp90 to the stripped receptors does not
reactivate them, they can be reactivated by treating them with rabbit
reticulocyte lysate (RRL) in the presence of ATP (3, 4). Therefore,
considerable effort has gone into determining what factors in RRL, in
addition to Hsp90, are required for reactivation of immunoadsorbed
stripped GR (5). Fractionation of RRL and reactivation of stripped GR with the various fractions revealed that, in addition to ATP, three
proteins are essential for reactivation of stripped GR: Hsp90, Hsp70,
and Hop (5). Pratt and associates (6-8) suggested that these three
proteins bind together in a complex termed the foldosome. They then
suggested that the foldosome interacts with the hormone binding domain
of GR in an ATP-dependent reaction that activates its
hormone binding activity, possibly by partially unfolding the
hormone-binding domain exposing its steroid binding site. Since Hsp70
does not directly interact with Hsp90, it was thought that Hop mediated
the binding of Hsp70 to Hsp90 in the foldosome (7, 9).
In addition to the three proteins, Hsp90, Hsp70, and Hop, that form the
foldosome, two additional proteins, p23 and Hsp40 (Ydj1), have been
reported to be involved in activating hormone binding by GR (5, 10,
11). Although Hsp90, Hsp70, and Hop alone are able to activate GR, the
activated receptors are unstable. Therefore, unless steroids are
actually present during the activation process, the receptors will not
bind steroids unless a fourth protein, p23, is present and binds to
Hsp90 to stabilize the activated receptor complex (5, 7); for reasons
that are not clear, molybdate can substitute for p23 in this
stabilization reaction (8). Although a basal level of activation can be
achieved by Hsp90, Hsp70, and Hop, Pratt and associates showed that
catalytic amounts of a J-domain protein such as Ydj1 increase the level of activation (11). J-domain proteins often act as partner proteins of
Hsp70, by facilitating the binding of substrates to Hsp70, but the
mechanism of action of J-domain proteins in facilitating activation of
steroid receptors by Hsp90, Hsp70, and Hop is unclear. BAG-1 also
modulates the ability of GR to bind hormone in a
concentration-dependent manner (12).
There are several difficulties with the foldosome model for activation
of GR. First, in contrast to Hsp70 and Hsp90, Hop apparently acts
catalytically rather than stoichiometrically in activating the
progesterone receptors and perhaps GR as well (25). Second, in contrast
to Hsp90 and possibly Hsp70, Hop is not present in the activated
steroid receptor complex (1, 14). Third, there is strong evidence that
Hsp70 as well as Hsp90 can bind directly to GR (1, 15, 16, 27), raising
questions as to whether a complex of Hsp70, Hsp90, and Hop (26) is
indeed required for activation. Finally, elimination of Sti1, the
homolog of Hop in yeast, had no effect on the formation of the
Hsp70-Hsp90-steroid receptor complex in vivo (17).
Our laboratory has been studying the properties of various Hsp70s
mutated at their active sites. Preparatory to studying the ability of
these Hsp70 mutants to participate in reactivation of stripped GR, we
reinvestigated the reactivation process using highly purified Hsc70,
Hsp90, Hop, and the J-domain protein Ydj1. To our surprise, we found
that we could reactivate the stripped GR with just highly purified
Hsp70 and Hsp90; Hop was not required, and Ydj1 reduced rather than
increased the level of activation. We conclude that Hsp70 and Hsp90
bind directly and independently to GR, and the binding of these two
proteins is all that is required to reactivate the stripped receptors
to bind steroids.
Materials--
[6,7-3H]Triamcinolone acetonide
(42.8 Ci/mmol) was obtained from NEN Life Science Products. Untreated
RRL was from Green Hectares (Oregon, WI). Protein A-Sepharose was
obtained from Amersham Pharmacia Biotech. Anti-mouse Ig horseradish
peroxidase conjugate was from Amersham Pharmacia Biotech. Complete-Mini
protease inhibitor mixture was from Roche Molecular Biochemicals. The
BuGR2 monoclonal IgG against the GR, anti-Hsp40, anti-Hsp70, and
anti-Hsp90 were from Affinity Bioreagents (Golden, CO). DS14F5
monoclonal IgG against Hop was kindly provided by David O. Toft (Mayo
Clinic, Rochester, MN). Hybridoma cells producing the FiGR monoclonal
IgG against the GR were from ATCC. ECL reagent was from Amersham
Pharmacia Biotech.
Bacterial Strains and Plasmids--
Escherichia coli
BL21/DE3 containing plasmid pET23 expressing human p23 was a gift from
Dr. David O. Toft. E. coli cells expressing human Hop were
kindly provided by Dr. David F. Smith (University of Nebraska Medical
School, Omaha, NE). Plasmid encoding 60-kDa recombinant Hsp70 was
constructed by site-directed mutagenesis using the oligonucleotide
containing the stop codon at position 607 of human hsp70.
Protein Purification--
Hsc70 was purified from bovine brain
cytosol by sequential chromatography on DE52, hydroxylapatite, and
ATP-agarose as described previously (18). Recombinant Human Hsp70 was
purified from E. coli as described previously (19). Hsp90
was purified from bovine brain cytosol as described previously (20),
except that the purified Hsp90 was passed through the hydroxylapatite
column twice to remove any contaminant Hop. Human p23 was purified from
bacterial lysate by chromatography on DE52 as described (21). Fractions containing p23 were identified by SDS-PAGE pooled, concentrated by
ammonium sulfate, dialyzed against buffer C (20 mM
imidazole, pH 7.0, 25 mM KCl, 2 mM magnesium
acetate, 2 mM ammonium sulfate, 1 mM
dithiothreitol), aliquoted, and stored at Preparation of Cytosol--
L929 mouse fibroblast cells were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum. Cells were washed three times with Hanks' balanced
saline solution and suspended in 1.5 volumes of buffer containing 10 mM Hepes, pH 7.35, 1 mM EDTA, 20 mM
sodium molybdate, and 1 tablet of Complete-Mini protease inhibitor
mixture plus 5 ml of buffer. Cells were ruptured by Dounce
homogenization and centrifuged for 30 min at 37,000 rpm to prepare the
cytosol. The supernatant was aliquoted, flash-frozen, and stored at
GR Heterocomplex Assembly--
GR was immunoadsorbed at 4 °C
from either WCL2 or L929 cell cytosol using either FiGR or BuGR2
antibodies prebound with protein A-Sepharose. GR was stripped of the
associated proteins by incubating the immune pellets with TEG buffer
(10 mM TES, pH 7.6, 50 mM NaCl, 4 mM EDTA, 10% glycerol) containing 0.5 M NaCl
and subsequent washing with TEG buffer and 10 mM Hepes, pH
7.4. Immune pellets containing GR stripped of Hsp90 and associated
proteins were incubated with 50 µl of RRL or with various mixtures of
proteins (5 µM Hsp90, 1 µM Hsp70, 1 µM Hop, 5 µM p23, and Ydj1 if necessary)
and adjusted to 50 µl with HKD buffer (10 mM Hepes, 100 mM KCl, and 5 mM dithiothreitol, pH 7.35),
containing 20 mM sodium molybdate, 5 mM ATP, 25 mM creatine phosphate, 5 mM magnesium acetate,
and 10 units/ml creatine phosphokinase. The assay mixtures were
incubated for 30 min at 25 °C with suspension of the pellets by
shaking the tubes using a rotator. At the end of the incubation, the
pellets were washed twice with 1 ml of ice-cold TEGM buffer with 5 mM dithiothreitol (TEG buffer with 20 mM sodium
molybdate) and assayed for steroid-binding capacity and, in some
experiments, for receptor-associated proteins.
Assay of Steroid Binding--
Immune pellets to be assayed for
steroid binding were incubated for 2 h at 4 °C in 100 µl of
buffer containing 10 mM Hepes, pH 7.5, 1 mM
EDTA, 20 mM molybdate plus 50 nM
[3H]triamcinolone acetonide. Samples were then washed
three times with 1 ml of TEGM buffer and counted by liquid
scintillation. The steroid binding is expressed as counts/min of
[3H]triamcinolone acetonide bound per FiGR immune pellet
from 100 µl of cytosol.
Western Blotting--
To assay GR and associated proteins,
immune pellets were resolved on 4-20% SDS-polyacrylamide gels and
transferred to nitrocellulose membranes. The membranes were probed with
2 µg/ml BuGR2 for GR, 1 µg/ml for hsp90, 1 µg/ml for Hsc70, 1 µg/ml DS14F5 mouse ascites for Hop, and 1 µg/ml of anti-Hsp40
antibody. The immunoblots were then incubated a second time with the
appropriate horseradish peroxidase-conjugated counter antibody, and
immunoreactive proteins were visualized by ECL.
In the experiments on reactivation of GR by RRL or purified
proteins, we employed essentially the same procedure used by Pratt and
associates (5) except that they activated the receptor at 30 °C and
incubated overnight with hormone, whereas we activated at 25 °C and
incubated with hormone for 2 h. However, we found no difference in
our results whether we used their procedure or our own. We
immunoadsorbed receptors from the cytosol of either WCL2 cells or L
cells using BuGR2, stripped them with 0.5 M NaCl, and then
reconstituted the heterocomplex by incubating the stripped receptors
with either RRL or purified proteins at 25 °C. We routinely obtained
80-100% restoration of steroid binding activity with RRL and about
50-60% reconstitution with pure proteins, i.e. about 60-65% of the activity obtained with RRL (Fig.
1, A and B).
Surprisingly, where our results significantly differ from those of
Pratt's laboratory is that, with both steroid receptors obtained from
WCL2 cells (Fig. 1A) and from L cells (Fig. 1B), addition of purified Hop to stripped receptor did not significantly affect restoration of steroid binding activity of the receptor by the
purified proteins. Furthermore, the activation of GR for hormone
binding occurred even in the absence of p23. As observed by Pratt's
laboratory (7, 8), when reconstitution of the stripped receptors was
carried out in the presence of steroids, it was not necessary for p23
to be present because, even though the reconstituted receptor complex
is unstable in the absence of p23, it binds steroid before its
activated conformation decays. Our results show that both with steroid
receptors obtained from WCL2 cells (Fig.
2A) and with those from L
cells (Fig. 2B), we obtained the activation of GR not only
in the absence of p23 but also in the absence of Hop. Therefore, our
results strongly suggest that Hop is not required for activation of
stripped receptors; Hsp90 and Hsc70 alone are sufficient for
reactivation of GR for hormone binding.
The Molecular Chaperones Hsp90 and Hsc70 Are Both Necessary
and Sufficient to Activate Hormone Binding by Glucocorticoid
Receptor*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. Ydj1 was purified
from E. coli as described previously (22, 23). Human Hop was
purified from E. coli as described previously (13).
70 °C. WCL2 cell cytosol was provided by Gordan Hager's
laboratory (NCI, National Institutes of Health, Bethesda, MD).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (30K):
[in a new window]
Fig. 1.
Activation of GR in the absence of Hop.
GR from WCL2 (A) or L929 (B) cell cytosol was
immunoadsorbed with FiGR-protein A-Sepharose, and Hsp90 and other
associated proteins were stripped from the immune pellets with 0.5 M sodium chloride. Salt-stripped GR immune pellet was
reactivated with 50 µl of buffer containing 1 µM Hsc70,
5 µM Hsp90, and 1 µM p23 in the presence
and absence of 1 µM Hop. The efficiency of heterocomplex
assembly and the reactivation for the steroid binding activity of GR
were determined by incubating the immune pellet with 50 nM
[3H]triamcinolone acetonide at 4 °C. Lane
1, unstripped receptor; lane 2,
stripped receptor; lane 3, stripped receptor
reactivated with RRL; lanes 4 and 5,
stripped receptor reactivated with Hsp90, Hsc70, and p23 in the absence
(lane 4) and presence (lane
5) of Hop.
View larger version (31K):
[in a new window]
Fig. 2.
Hsp90 and Hsc70 alone activate GR in
the presence of ligand. GR was immunoadsorbed from 100 µl of
WCL2 (A) or 300 µl of L929 (B) cell cytosol,
receptor-associated proteins were stripped from the immune pellets with
0.5 M NaCl, and the immune pellets were then incubated for
20 min at 25 °C with an ATP-regenerating system and steroid.
Lane 1, stripped receptor; lane
2, stripped receptor incubated with Hsp90, Hsc70, and p23;
lanes 3 and 4, stripped receptor
incubated with Hsp90 and Hsc70 in the absence and presence of
Hop.
Although our results showed that Hop is not necessary for reactivation
of the stripped GR, it still was possible that it might increase the
rate of reactivation. We, therefore, measured the rate of reactivation
using purified proteins both in the presence and absence of Hop. Our
results (Fig. 3) showed that, as
previously reported for reactivation by RRL, the rate of reactivation
of the stripped receptor by purified Hsp90, Hsc70, p23, and Hop had a
half-life of about 2 min. Furthermore, we found that the rate of
reactivation was almost identical in the absence of Hop. Therefore, Hop
is not only unnecessary for reactivation, it also does not affect the
rate of reactivation by Hps90, Hsc70, and p23.
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Although the foldosome model (1, 6) implies that approximately equal
amounts of Hop, Hsp70, and Hsp90 are required for reactivation of
stripped GR, reactivation of the stripped progesterone receptor
required much less Hop than Hsp70 and Hsp90; only a 1:1 molar ratio of
Hop to progesterone receptor was required (25). To determine whether
this level of Hop was present in the stripped GR or in the purified
Hsc70 and Hsp90 used in the reconstitution experiments, we carried out
quantitative Western blots (Fig. 4). These experiments were carried out with 80 nM active GR
plus either 1 µM Hsc70, 5 µM Hsp90, or both
present. In addition, we determined the level of Hop present in the
purified Hsc70 and Hsp90 in the absence of receptor. Before stripping,
80 nM active GR contained only about 0.4 nM Hop
(Fig. 4B, lane 1) in agreement with
other studies, suggesting that even unstripped receptors contain only a
small amount of Hop since it appears to be transiently associated with
the activated complex. Following stripping, Hop was undetectable by
Western blotting showing that the stripped receptors contained much
less than the 0.2 nM Hop standard (Fig. 4B,
lane 9). Furthermore, after Hsc70, Hsp90, or both
were added to the receptors, Hop was still undetectable nor was Hop
detectable in the purified Hsc70 or Hsp90. We also performed this
experiment using recombinant Hsp90 from baculovirus/Sf9 cells
and recombinant Hsp70 from E. coli, and again we obtained
similar levels of reactivation of the GR. Therefore, the level of Hop
present in our reactivation experiments was less than 0.25% of the
active steroid receptor and less than 0.1% of the Hsc70 and Hsp90
present, strongly suggesting that our results are not due to the
presence of catalytic amounts of Hop.
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There is evidence that Hop binds to the 10-kDa C-terminal end of Hsc70
(24). Therefore, assuming that Hsp70 and Hsc70 act similarly in this
regard, a truncated Hsp70 missing its 10-kDa C-terminal end should be
as effective as wild-type Hsp70 in the reactivation process if Hop is
not required for reactivation of GR. When we tested this
prediction, our results showed that wild-type and truncated Hsp70 were
equally effective in reactivation of GR and, furthermore, in neither
case did Hop have a significant effect on reactivation (Fig.
5). These data provide further evidence that Hop is not required for reactivation of GR.
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The 10-kDa C-terminal end of Hsc70 not only binds Hop but also the
J-domain protein Hsp40 (24). Therefore, our observation that the
truncated Hsp70 was as effective as wild-type Hsp70 in reactivating GR
suggests that, like Hop, J- domain proteins may also have little or no
effect on reactivation of GR. To investigate this question, we first
determined the level of contaminant Hdj1 present in our mixture of GR,
Hsc70, and Hsp90. Based on the quantitative Western blots in Fig.
6, we estimated that 80 nM
active GR contained less than 0.04 nM Hsp40. This small
amount of Hsp40 was removed when the receptor was stripped. However,
upon readdition of 1 µM Hsc70, 5 µM Hsp90,
or both, we found that the level of Hsp40 returned to about 0.04 nM, less than 0.1% of the active GR and less than 0.01%
of the Hsc70 and Hsp90. Since, in previous studies, the maximum effect
of the J-domain protein Ydj1 occurred at levels approximately equal to
that of active receptor and about one-tenth the level of Hsp70 and
Hsp90 (11, 24), it seems highly unlikely that the small amount of Hsp40
present in our system affected reactivation of GR. In agreement with
this view, we found that addition of 0.25 or 1 µM
Ydj1 decreased rather than increased reactivation of GR (Fig.
7). Likewise, addition of recombinant Hdj1 also did not affect the reactivation of GR (data not shown). We
find, therefore, that, like Hop, Hsp40 does not play a significant role
in reactivation of stripped GR.
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Since our results suggest that only Hsc70 and Hsp90 are required for
reactivation of GR and both Hsc70 and Hsp90 bind directly to GR, we
were interested in determining whether, following the binding of one of
these two proteins to the receptor, reactivation would occur when the
second protein bound. Such an experiment can only be carried out if,
following stripping, the receptor does not irreversibly denature while
it is being incubated with the first protein at 25 °C before the
second protein is added. Therefore, we first tested whether the
stripped receptor retains its ability to be reactivated when incubated
for various times at 25 °C. We found that, following incubation of
the stripped receptor for 1 h at 25 °C, it could still be fully
reactivated by RRL (data not shown). Similarly, incubation of the
stripped receptor for 20 min at 25 °C had no effect on its ability
to be reconstituted with purified proteins (Fig.
8, lanes 2 and
3). Therefore, the stripped receptor does not irreversibly
denature when incubated at 25 °C.
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We next incubated the stripped receptor with Hsp90 and p23 at 25 °C
in the presence and absence of ATP and then removed unbound Hsp90 and
p23. In agreement with other studies, we found that stripped receptor
with bound Hsp90 and p23 was completely inactive (data not shown).
However, when the receptor with bound Hsp90 and p23 was then incubated
with Hsc70 in the presence of ATP, it regained considerable steroid
binding activity particularly when the initial incubation with Hsp90
and p23 was carried out in the presence of ATP (Fig. 8,
lanes 4 and 5). Similar results were
obtained when the stripped receptor was first incubated with Hsc70,
unbound Hsc70 removed, and then Hsp90 and p23 added in the presence of
ATP. No activity was observed following the initial incubation with
Hsc70, but considerable activity was recovered following incubation
with Hsp90 and p23 in the presence of ATP. Interestingly, more activity
was recovered when the stripped receptor was incubated with Hsc70 in
the absence than in the presence of ATP, possibly because somewhat more
Hsc70 was initially bound to the receptor in the absence of ATP.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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In the present study, we showed that GR isolated from either WCL2 cells or L cells and stripped of their associated proteins can be reconstituted in the presence of ATP with the addition of only two proteins, Hsc70 and Hsp90. In earlier studies on reconstitution of GR, others found that, in addition to Hsc70 and Hsp90, reconstitution of stripped GR required Hop (5-8). Since Hsp70 does not bind directly to Hsp90, it was suggested that Hop linked Hsp70 to Hsp90 in a complex, the foldosome (5-9, 25), that then functioned as a unit to reconstitute glucocorticoid binding to stripped receptors (6, 7).
In the studies on Hop, Pratt and associates (5) used a bacterial extract containing recombinant Hop rather than purified Hop. Therefore, it was not clear from their data whether Hop acted stoichiometrically with Hsp70 and Hsp90, as might be expected from the foldosome model, or whether it acted at a much lower concentration. In the latter case, it is possible that, as our data suggest, Hsp70 and Hsp90 play a unique role in reconstitution of GR with Hop only playing a modulatory role. Even in this case, however, our data differ in that we were able to reconstitute GR in the complete absence of Hop and, moreover, we could not detect any significant difference in the rate of reactivation in the presence and absence of Hop. This difference from the results of Pratt and associates may be related to the fact that GR can assume multiple conformations, and this may particularly be the case for the stripped receptor. Therefore, it is possible that subtle differences in the conformation of GR may account for whether or not Hop plays a modulatory role in the reconstitution of GR by Hsp90 and Hsp70.
In this regard, the GR and progesterone receptors differ significantly in their properties following stripping of Hsp90 and associated proteins, perhaps because here, too, subtle differences occur in the conformations of the two receptors. In contrast to GR, which immediately loses its ability to bind substrate following stripping but can then be reactivated, the progesterone receptor retains its ability to bind substrate following stripping and only slowly loses this activity during incubation at 25 °C. However, once it loses its activity, in contrast to GR, it cannot be reactivated. Toft and associates (25) have shown that if Hsp90, Hsp70, Hop, and Ydj1 are present during incubation at 25 °C, the progesterone receptor does not lose its substrate binding ability. Interestingly, in these studies (25), although Hop and Ydj1 were required along with Hsp70 and Hsp90 to maintain the activity of the progesterone receptor, they were not required at stoichiometric levels equal to Hsp70 and Hsp90, as might be expected if they were components of a foldosome. Rather, they were present at much lower concentrations, about equal to the concentration of the progesterone receptor. Therefore, although the earlier data suggest that Hop and Ydj1 play a modulatory role in maintaining the activity of the stripped progesterone receptor, Hsp70 and Hsp90 appear to be the primary proteins required for maintaining its activity in agreement with our results on GR. This view is also supported by data showing that the yeast homolog of Hop, Sti1, is not required for activation of GR in vivo (17).
Based on our data showing that Hsc70 and Hsp90 bind independently to GR
and that the order of their binding has little effect on reactivation
of the stripped receptor, we propose, as shown in Fig.
9, that Hsc70 and Hsp90 bind to separate
sites on the stripped receptor and that this binding in the presence of
ATP is sufficient to reactivate the stripped receptor. Future studies using mutant Hsp70s and purified GR will be required to determine whether both ATP binding and hydrolysis by Hsc70 are required for
reconstitution of the stripped receptor.
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ACKNOWLEDGEMENTS |
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We thank Dr. Gordon Hager and Dr. Barbour Warren for the WCL2 cell cytosol; Dr. David Toft for the anti-Hop monoclonal antibody, plasmid expressing p23, and for the recombinant Hsp90; Dr. David Smith for the plasmid expressing Hop.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: NHLBI, National
Institutes of Health, 9000 Rockville Pike, 3 Center Dr., MSC 0301, Bldg. 3, Rm. B1-22, Bethesda, MD 20892-0301. Tel.: 301-496-2846; Fax:
301-402-1519; E-mail: eisenbee@nhlbi.nih.gov.
Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M002035200
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ABBREVIATIONS |
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The abbreviations used are: GR, glucocorticoid receptor; Hsp70, 70-kDa heat shock protein; Hsc70, the constitutive isoform of Hsp70; Hsp90, 90-kDa heat shock protein; Hop(p60), Hsp70 and Hsp90 organizing protein; PAGE, polyacrylamide gel electrophoresis; RRL, rabbit reticulocyte lysate; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; BuGR2, monoclconal IgG antibody against GR; FiGR, monoclonal IgG antibody that binds to the same epitope as BuGR2.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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) and absence
(
) of Hop. The efficiency of heterocomplex assembly and the
reactivation for the steroid binding activity of GR was determined by
incubating the immune pellet with 50 nM
[3H]triamcinolone acetonide at 4 °C.
