Disruption of the Glucocorticoid Receptor Assembly with Heat Shock Protein 90 by a Peptidic Antiglucocorticoid
Hai-Pascal Dao-Phan,
Pierre Formstecher and
Philippe Lefebvre
INSERM U-459, Laboratoire de Biochimie Structurale,
Faculté de Médecine Henri Warembourg, 59045 Lille,
France
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ABSTRACT
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Association of glucocorticoid (GR) and
progesterone (PR) receptors with a set of molecular chaperones,
including the 90-kDa heat shock protein (hsp90), is a dynamic process
required for proper folding and maintaining these nuclear receptors
under a transcriptionally inactive, ligand-responsive state. Mutational
studies of the chicken hsp90 complementary DNA suggested that three
regions of this protein (A, B, and Z) interact with the hormone-binding
domain of GR, whereas region A is dispensable for hsp90 binding to PR.
We found that this 69-amino acid region can be narrowed down to a
35-mer
-helical, acidic peptide, which is by itself able to inhibit
hsp90 association to GR translated in vitro. The hsp90-free
GR did not bind ligand, but was devoid of any specific DNA-binding
activity, and higher peptide concentrations specifically inhibited the
binding of activated GR to DNA. When overexpressed in cultured cells,
this peptide acted as an antiglucocorticoid and inhibited the
antiactivating protein-1 activity and the ligand-dependent nuclear
transfer of GR. None of these effects, either in vivo and
in vitro, was observed for PR. The region from residue 232
to residue 265 of hsp90 is, therefore, a domain critical for its
association to GR, an association that is a prerequisite for receptor
transcriptional activity. More importantly, these results demonstrate
that targeting specific protein/protein interaction interfaces is a
powerful means to specifically modulate nuclear receptor signaling
pathways in a ligand-independent manner.
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INTRODUCTION
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Steroid receptors exist in intact cells under two forms in the
absence of ligand. The first form is a misfolded receptor, unable to
bind ligand and thus transcriptionally inefficient. By an ATP-dependent
process involving the interaction with 70-kDa heat shock protein
(hsp70), DnaJ, and other proteins, such as the immunophilin hsp59,
misfolded receptors are assembled into a heterooligomeric complex
containing a dimer of hsp90. As a consequence, the ligand-binding form
of glucocorticoid receptor (GR) is isolated from cells grown in
hormone-free medium as a heteromeric, cytosoluble complex comprising a
receptor monomer, a dimer of hsp90, and several other protein
chaperones (1, 2, 3). The formation of this heterocomplex is required to
maintain GR, but not all steroid receptors, in a conformation
appropriate for ligand binding and to prevent GR from binding to DNA
(4, 5). Exposure of cells to glucocorticoids activates GR, a process
that converts the receptor into a nuclear, monomeric, nonligand-binding
form (6), having a high affinity for glucocorticoid response elements
(GREs). Activated receptors bind to GREs as homodimers and modulate the
transcriptional activity of hormonally regulated genes (7, 8).
Chaperoning activities of hsp90 and other heat shock proteins are,
therefore, required to maintain GR and other steroid receptors in a
poised state highly sensitive to ligand stimulation (reviewed in
9 .
Peptide interference assays showed that the hormone-binding domain of
mouse GR contains two contiguous sequences in region 574659 that are
critical for hsp90 association to GR (10, 11, 12). Conversely, mutagenesis
studies of the chicken hsp90 complementary DNA (cDNA) identified three
regions that are probably contact sites of hsp90 with GR. Two of them
are hydrophilic regions termed A (region 221290) and B (region
530581), whereas the third is a putative leucine zipper called region
Z (region 392419). Region A is required for GR/hsp90 association,
whereas deletion of region B or Z yielded nonfunctional
heterooligomeric complexes (13). Importantly, progesterone receptor
(PR) association with hsp90 does not require region A (14).
We have previously shown that antiglucocorticoids, regardless of their
chemical structures, exert their effects in intact cells by preventing
the dissociation of this heterooligomeric complex (15). We, therefore,
inferred from this set of data that interfering with the chaperone-GR
association process could have significant effects on GR
transcriptional activity. This report describes the biophysical,
biochemical, and biological characterization of a 35-mer peptide that
is a segment of region A from mouse hsp90ß. We show that this highly
charged,
-helical peptide is able to inhibit the association of GR
to hsp90 in vitro. Although the hsp90-free GR displayed most
of the properties of a fully activated receptor, it was unable to bind
DNA. Moreover, the DNA-binding activity of the activated GR was also
inhibited at high peptide concentrations, suggesting that this peptide
could interact with a region in or close to the DNA-binding domain
(DBD) of GR. Finally, we found that overexpression of this peptide in
COS cells inhibited the ligand-dependent induction of a glucocorticoid-
and progestin-responsive promoter [mouse mammary tumor virus-long
terminal repeat (MMTV-LTR)]. The anti-AP-1 activity of GR was also
inhibited in the presence of this peptide. As none of these in
vitro or in vivo effects was observed with PR, a
closely related nuclear receptor, we conclude that the region mapping
from residue 232 to residue 266 of mouse hsp90ß (mhsp90ß) is a
specific contact interface with GR, and that a specific
antiglucocorticoid activity can be obtained by targeting
protein-protein interaction interfaces.
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RESULTS
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Sequence and Structure of Region 232266 of mhsp90ß
The strong similarity between a 35-amino acid-long subdomain of
region A of eukaryotic hsp90 suggests that these sequences are highly
convergent (Fig. 1A
). The main structural features of
these sequences are their acidic character and their putative
organization into a DNA-like,
-helical structure (16). We,
therefore, synthesized the peptide corresponding to this region and
analyzed its secondary structure. The circular dichroism spectrum in
aqueous solution of the 35-mer peptide from mhsp90ß displayed maximum
ellipticity at 200 nm and minimal ellipticity at 208210 and 220 nm.
These values underscore the propensity of the hsp90 peptide to fold
into an
-helical structure under these conditions (Fig. 1B
).

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Figure 1. Sequence and Structure of the mhsp90ß Peptide
A, Similarity of the peptide from region A of mhsp90ß to sequences of
other eukaryotic hsp90s. Shaded boxes indicate conserved
charges [positive for Arg(R) or Lys (K), negative for Asp (D) or Glu
(Q)], and empty boxes indicate conserved residues.
Numbers correspond to SwissProt database coordinates. Database
retrieval and analysis were performed using the Blitz service at EMBL,
and sequences alignments were optimized using the Clustal W program
(46). Abbreviations for the amino acid residues are as follows: A, Ala;
C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ileu; K, Lys; L,
Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W,
Trp; and Y, Tyr. B, The hsp90 peptide assumes an -helical
conformation in buffered aqueous solutions. The 35-mer peptide was
solubilized at a concentration of 1 mg/ml in 0.1 M
phosphate buffer. The solution was placed into a 0.1-mm path length
cuvette, and spectra were recorded from 190290 nm by a Jobin-Yvon
Dicrograph III. They are plotted as a function of the molar
ellipticity.
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Inhibition of the Association of hsp90 with GR by the hsp90
Peptide
To assess directly whether this
-helical peptide is involved in
hsp90 binding to GR and PR, we used a peptide interference assay
designed previously to identify GR sequences interacting with hsp90
(12). Dissociation of the nonactivated GR can be monitored by molecular
sieving, and relative amounts of hsp90-associated or hsp90-free GR
generated in our system were quantified (Fig. 2A
).
Adding increasing amounts of hsp90 peptide to the in vitro
translation mix, which contains, as intact cells, about 2
µM hsp90, led to a dose-dependent conversion of the
Stokes radius of the 8-nm, heteromeric complex into a 5-nm, hsp90-free
GR (15). The ligand-binding activity of GR decreased coincidentally
with this 8 to 5 nm transition (Fig. 2B
), suggesting that GR could be
activated under these conditions (6). Hsp90-GR and hsp90-PR
interactions were in addition quantitated by coimmunoprecipitating
[35S]methionine-labeled receptors synthesized in
vitro with an anti-hsp90 antibody. As shown in Fig. 2
, the
anti-hsp90 antibody coprecipitated a significant amount of labeled GR
(Fig. 2C
) and PR (Fig. 2D
), substantiating the formation of
hsp90-receptor complexes upon translation of GR and PR messenger RNAs
in vitro. Exposure of both receptors to activating
conditions induced a sharp decrease in the association of hsp90 with GR
and PR. Adding the hsp90 peptide to the translation mix caused a strong
decline in the amount of coprecipitated GR, in proportions similar to
that observed after GR activation (Fig. 2C
, lanes PAPT). In contrast,
binding of hsp90 to PR was not affected in the presence of the hsp90
peptide (Fig. 2D
). Adding the hsp90 peptide after completion of the
translation reaction did not modulate the amount of
coimmunoprecipitated GR and PR (Fig. 2
, C and D, lanes PAAT),
demonstrating that 1) the hsp90 peptide did not induce dissociation of
preformed hsp90/GR complexes; and 2) it did not interfere with antibody
binding to its epitope. Thus, this
-helical, highly charged,
35-amino acid-long region from hsp90 can be considered as a specific
recognition interface required for the association of hsp90 to GR, but
not to PR.

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Figure 2. The hsp90 Peptide Specifically Inhibits GR Binding
to hsp90
A, Conversion from the hsp90-associated GR to the hsp90-free GR
form monitored by molecular sieving chromatography.
[35S]Methionine-labeled GR was obtained by translation
in vitro in rabbit reticulocyte lysate, thereby allowing
for the formation of nonactivated complexes that have a Stokes radius
of about 8 nm and are associated with hsp90. Exposure of these
complexes to activating conditions (50 nM dexamethasone for
4 h at 4 C and mild heating at 30 C for 30 min) led to an almost
total conversion into the 5-nm form. Adding increasing concentrations
of the hsp90 peptide to the translation mix, but not other peptides,
induced a dose-dependent conversion of the nonactivated form into the
5-nm form without exposure to activating conditions. 100% represents
the total amount of full-length receptor in the translation mix and is
the sum of the 8- and 5-nm forms. Only these two forms were detected in
our assay. Stokes radius measurements were performed as described. B,
Ligand binding activity of GR translated in vitro in the
presence of a control or the hsp90 peptide before and after activation.
The specific glucocorticoid-binding activity of the programmed
reticulocyte lysate was assayed when GR was translated in the presence
of peptides. When submitted to activating conditions, regardless of the
presence of peptides, no ligand-binding activity was detectable. 100%
represents the specific ligand-binding activity of the programmed
lysate stabilized by 20 mM sodium molybdate, which thus
reflects the binding activity of the nonactivated receptor. Ligand
binding assays were performed using the dextran-charcoal method. ,
Nonactivated GR and control peptide; , nonactivated receptor plus
hsp90peptide; , activated receptor, with control or hsp90 peptide.
C, Quantification of the association of GR to hsp90. Reticulocyte
lysate was programmed as described above to generate
[35S]methionine-labeled GR in the presence of 200
µM control peptide and submitted (lane A) or not (lane
NA) to activating conditions. The hsp90 peptide was added either before
initiating the translation reaction (PAPT) or after completion of the
reaction (PAAT). Hsp90 was then immunoprecipitated with the anti-hsp90
monoclonal IgM 3G3, and immune pellets (after extensive washing) as
well as supernatants were analyzed for their content in labeled
receptors by 8% SDS-PAGE and autoradiography. Control precipitations
were performed using protein A alone (Prot A) and protein A coupled to
an anti-IgM IgG to assess the amount of nonspecific adsorption to the
matrix; this was previously reported to be nonnegligible (47). Results
are expressed as the percentage of total receptor (pellet plus
supernatant) immunoprecipitated specifically in the presence of the 3G3
antibody. D, Quantification of the association of PR to hsp90. Assays
were performed as described in C using a PR cDNA (form A).
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The hsp90-Free GR Occurs in a Non-DNA-Binding State
Receptors that do not associate with hsp90 during translation can
potentially generate two forms of polypeptides: an activated receptor,
which binds to DNA with a high affinity, or a misfolded, nonfunctional
polypeptide. To distinguish between these two possibilities, we assayed
GR and PR DNA-binding activities in the presence or absence of the
hsp90 peptide (Fig. 3
). Preliminary experiments revealed
that electrophoretic mobility shift assays were not appropriate to
assess the activation rate of GR. Indeed, this assay yielded high rates
of spontaneous activation and displayed nonspecific complexes migrating
with an electrophoretic mobility similar to that of GR-GRE complexes.
The DNA-cellulose binding assay was thus used to evaluate GR and PR
DNA-binding activities. Results are expressed as a percentage of the
amount of receptor binding to DNA after activation. Typically,
submitting GR (Fig. 3
, A and C) and PR (Fig. 3
, B and D) to activating
conditions led to the conversion of 6070% of the total receptor into
the DNA-binding form. This conversion was completely inhibited in the
presence of 20 mM Na2MoO4, a potent
inhibitor of the activation process (Fig. 3
, A and B). Despite its
ability to prevent hsp90/GR association, the hsp90 peptide did not
induce true activation of GR, as no significant increase in the
DNA-binding activity was observed (Fig. 3A
). PR was also tested for its
sensitivity to peptide interference in the DNA binding assay. No
increase in the DNA-binding activity of PR was detected in the presence
of the hsp90 peptide.

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Figure 3. Inhibition of GR Association to hsp90 from GR Does
not Promote GR Binding to DNA
A, The DNA-binding activity of GR translated in vitro in
the presence of increasing amounts of control or hsp90 peptides was
monitored by the DNA-cellulose binding assay before or after activation
(Fig. 3A ). Results are expressed as relative binding to DNA-cellulose,
with 100% representing the binding activity of native receptors
exposed to activating conditions [50 nM dexamethasone (GR)
or R5020 (PR) and moderate heating]. Results are the average of at
least five independent experiments. , GR, activation, and hsp90
peptide; , GR, activation, and control peptides; , GR, hsp90
peptide, and 20 mM molybdate; , GR, activation, control
peptide, and 20 mM molybdate. B, The DNA-binding activity
of PR translated in vitro in the presence of increasing
amounts of control or hsp90 peptides and activated or not under
conditions analogous to those described in A was quantified similarly.
, PR, activation, and hsp90 peptide; , PR, activation, and
control peptide; , activated receptor, hsp90 peptide, and 20
mM molybdate; , activated receptor, control peptide, and
20 mM molybdate. C and D, The hsp90 peptide specifically
inhibits the DNA-binding activity of activated GR. In
vitro translated GR (C) or PR (D) were submitted to activating
conditions as described in Fig. 2 , C and D, and incubated in the
presence of increasing amounts of control or hsp90 peptide. Control
reactions (nonactivated receptors) were run under similar conditions,
except that 20 mM MoO4 was added before
activation. , Activated receptor plus hsp90 peptide; , activated
receptor plus control peptide; , nonactivated receptor plus hsp90
peptide; , nonactivated receptor plus control peptide.
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We also noted that exposure of this peculiar form of GR to activating
conditions yielded, surprisingly, a receptor able to bind DNA. In
addition, restoration of the DNA-binding activity was strictly
dependent on both agonist binding and exposure to heat. Reagents known
to inhibit the activation process, and thus able to stabilize the
nonactivated, hsp90-associated form of GR [MoO4 (Fig. 3A
)
and antiglucocorticoids (Dao-Phan, H.-P., and P. Lefebvre, unpublished
data)] blocked this process. As we observed that GR is found in the
presence of peptide during the translation process in a hsp90-free form
(see Fig. 2
), this suggests that the peptide-GR interaction is
reversible and that under these conditions, receptor assembly with the
chaperone complex in reticulocyte lysate can occur.
We then asked whether the peptide could prevent the binding of
activated GR and PR to DNA. This would indicate a direct interaction of
the hsp90 segment with or in close proximity to the DBD of both
receptors, as previously speculated (16). The DNA-binding activity of
activated GR and PR was tested in the presence of increasing
concentrations of control or hsp90 peptides (Fig. 3
, C and D). At
peptide concentrations of 100200 µM, no significant
inhibition was noticed for either receptor. However, GR displayed a
decreased affinity for DNA at peptide concentrations above 300
µM, with an I50 around 500 µM
(Fig. 3C
). On the contrary, activated PR bound DNA efficiently, even at
a peptide concentration of 1 mM (Fig. 3D
). Thus, the hsp90
peptide showed again in this assay a clear selectivity for GR despite a
high homology (84%) between the GR and PR DBDs.
The hsp90 Peptide Is a Specific Antiglucocorticoid in Cultured
Cells
The experiments described above demonstrate that the hsp90 peptide
prevents the assembly of the heteromeric GR in vitro,
whereas the hsp90-free GR does not bind DNA. We next tested whether
such an inhibition could occur in live cells and what consequences it
might have on the transcriptional activity of the receptor. COS cells
were transfected with GR or PR complementary DNA expression vectors, a
luciferase (luc) reporter gene containing a
glucocorticoid/progesterone response element (MMTV promoter), and
increasing amounts of an expression vector coding for the sense or the
antisense hsp90 peptide cDNA. In the absence of peptide, we observed a
10-fold increase in luc activity in response to GR
stimulation by glucocorticoids (1 µM dexamethasone; Fig. 4
, A and C). This increase was about 5-fold when PR was
transfected and stimulated with 1 µM R5020, a synthetic
progestin. A dose-dependent decrease in the glucocorticoid-induced
luc response was observed in response to increasing amounts
of transfected cytomegalovirus (CMV) hsp90 plasmid. This inhibition was
almost complete at a ratio of CMVhsp90 plasmid to GR expression vector
of 5 (Fig. 4A
). No effect was noticed when GR was substituted for PR
(Fig. 4C
) despite similar levels of receptor expression, and
overexpression of the antisense peptide under strictly similar
conditions did not affect either the glucocorticoid-dependent or the
progestin-dependent activation of the MMTV promoter (Fig. 4
, B and D,
respectively).

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Figure 4. Antiglucocorticoid Activity of the hsp90 Peptide in
Cultured Cells
Triplicate 35-mm dishes of COS-7 cells were transfected as described in
Materials and Methods and treated with vehicle (0.1%
ethanol) or 1 µM dexamethason (Dex; GR) or R5020 (PR) for
16 h. Luciferase activity was then measured and is expressed as a
percentage of the luc activity detected in the presence of
ligand, without transfected pCMV5hsp90 or pCMV09psh plasmids. A,
Analysis of GR content of transfected COS cells. Cells (2 x
106) were transfected with 10 µg of the GR expression
vector, and a whole cell extract was prepared 48 h after
transfection. Proteins were analyzed by Western blotting using an
anti-DBD monoclonal antibody, directed against the five amino acids
between the two N-terminal cysteinyl residues of the second zinc finger
or D box. The recognized epitope is AGRND and is conserved in both rat
GR and rabbit PR (28). NT, Nontransfected cells; TT, cells transfected
with the rGR expression vector. B, Basal and glucocorticoid-induced
transcriptional activity of GR in the presence of the plasmid
(CMVhsp90) encoding for the hsp90 peptide. C, Basal and
glucocorticoid-induced transcriptional activity of GR in the presence
of the plasmid (pCMV09psh) encoding for the antisense version of the
hsp90 peptide. D, The PR content of COS cells transfected with the PR
expression vector was estimated as described in A. NT, Nontransfected
cells; TT, cells transfected with the rPR expression vector. E, Basal
and progestin-induced transcriptional activity of PR in the presence of
the plasmid (CMVhsp90) encoding for the hsp90 peptide. F, Basal and
progestin-induced transcriptional activity of PR in the presence of the
plasmid (pCMV09psh) encoding for the antisense version of the hsp90
peptide. , Glucocorticoid- or R5020-induced luc activity;
, basal luciferase activity. Results are expressed as a percentage
of the activity of the reporter gene under standard conditions and are
averaged from eight independent experiments.
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To explore further the mechanism of GR inactivation, we analyzed
another property of GR. GR and other nuclear receptors are able to
repress the activity of AP-1-regulated promoters. This transrepressing
activity does not require DNA binding and is observed in the presence
of agonists or antagonists (17). An AP-1-dependent reporter plasmid was
thus transfected into COS cells in the presence of expression vectors
coding for GR and the hsp90 peptide (CMVhsp90). AP-1 activity was
stimulated by the phorbol ester 12-O-tetradecanoyl phorbol
13-acetate (TPA), and inhibition occurred in a GR- and
dexamethasone-dependent manner (Fig. 5
). The anti-AP-1
activity of GR was not, however, observed in the presence of
overexpressed peptide, showing that both trans-activating
and trans-repressing activities of GR are lost under such
conditions. Finally, we wished to characterize the intracellular
localization of GR in response to peptide overexpression in the same
system. COS cells transfected with GR and hsp90 peptide expression
vectors were used in indirect immunofluorescence experiments (Fig. 6
). Unstimulated cells showed a clear cytoplasmic
localization of GR, which was found in the nucleus after dexamethasone
addition to the culture medium (Fig. 6
, panels 1 and 2). The nuclear
translocation was only partial in our system, as mentioned previously
(reviewed in 18 and is very likely due to the high level of
expression of GR in transfected cells. Peptide overexpression was found
to block GR nuclear translocation in response to agonist treatment and
caused a large accumulation of immunoreactive material at the periphery
of the nucleus, suggesting an inhibition of GR nuclear import (Fig. 6
, panels 4 and 5).

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Figure 5. Anti-AP-1 Activity of GR in the Presence of hsp90
Peptide in Cultured Cells
Transrepression assays by GR were carried out in transient transfection
experiments in COS-7 cells. Cells (1.5 x 105) were
transfected with the reporter gene pTPA-RE luc (500 ng),
containing two repeats of a consensus AP-1-binding site, alone or with
a GR expression vector (50 ng) and/or the hsp90 expression vector
CMVhsp90 (250 ng). The 5-fold excess of the CMVhsp90 vector over GR
plasmid yields a complete repression of the GR
trans-activation potential (see Fig. 4 ). Cells were treated
24 h after transfection by 100 nM TPA and/or 1
µM dexamethasone, and reporter gene activity was assayed
18 h later. The luciferase activity detected in the presence of
100 nM TPA was assigned the nominal value (100%), and all
other activities are expressed relative to this value. Data represent
the average of five independent experiments.
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Figure 6. Subcellular Localization of GR in Transfected COS
Cells
Immunofluorescent labeling of GR was carried out in methanol-fixed
cells using the BuGR2 monoclonal antibody. Similar results were
obtained using the monoclonal antibody Mab7. Cells were transfected
with pRSV-rGR alone or with pCMVhsp90. Twenty-fours hours after
transfection, cells were treated with 10-6 M
dexamethasone for 18 h and further processed for immunocytological
detection of the receptor. Photomicrographs of fluorescein
isothiocyanate-labeled cells show typical results. 1) Detection of GR
in control cells. 2) Detection of GR after dexamethasone treatment. 3)
Detection of GR in the presence of overexpressed hsp90 peptide. 4 and
5) Detection of GR in the presence of overexpressed peptide after
dexamethasone treatment. Panel 5 is a higher contrast exposure of panel
4, emphasizing the perinuclear accumulation of GR under these
conditions, which was not observed in other conditions.
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DISCUSSION
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Hsp90 homologs are constitutively expressed stress proteins
located mostly in the cytosol and endoplasmic reticulum of eukaryotic
cells. Interaction with hsp90 has been shown to be required for proper
folding of steroid receptors, and it turned out to be a major component
of a multiprotein complex comprising hsp56 or Cyp-40, hsp70, and p23
(reviewed in Refs. 19 and 20). This "foldosome" assembles
dynamically, in an ATP-dependent manner, and most if not all
polypeptides bind to hsp90 via tetratricopeptide repeats and are
thought to play a major role in the cellular localization of receptors
(21). The regions of GR and PR required for interaction with hsp90 have
been defined by deletion mutagenesis or peptide competition experiments
(10, 11, 12, 22), as the regions of hsp90 required for binding to PR (14),
GR, estrogen receptor, and mineralocorticoid receptor (13, 23, 24).
However, little is known about the quaternary structure of the
hsp90-receptor complexes and about their dimerization interfaces.
Global alignment of hsp90 sequences shows a high degree of similarity
for several regions of these proteins (25). The highly acidic region A
is found in all eukaryotic hsp90, but is lacking in the
Escherichia coli homolog HptG. We have demonstrated that a
35-mer sequence drawn from this region has the propensity to fold into
an
-helical structure and is able to specifically inhibit the
association of hsp90 to GR translated in vitro. Under such
conditions, synthesized receptors are unable to bind DNA, supporting
the idea that this hsp90 fragment is a specific contact region with GR.
This observation is reminiscent of those reporting functionally inert
GRs or dioxin receptors at low levels of hsp84 in yeast (26, 27), a
dysfunction that can be attributed to misfolding of hsp90 target
proteins. However, structure alterations should be minimal because
molecular sizing chromatography of hsp90-free GR and PR (data not
shown) did not reveal any discrepancy between Stokes radii of the these
particular forms of receptor and those of the activated forms. We also
observed that high concentrations of hsp90 peptide could inhibit DNA
binding of activated GR, suggesting that region A of hsp90 interacts
with the DBD of GR. Furthermore, the hsp90 peptide blocked the
interaction of a monoclonal antibody recognizing the second zinc finger
of GR (28), but not that of other antibodies recognizing the N-terminus
of GR (H.-P. Dao-Phan and P. Lefebvre, unpublished data). This could
add another level of inhibition of GR function by blocking homodimer
formation on DNA, as the second zinc finger of nuclear receptors is a
major dimerization interface (29). Additional experiments are in
progress to test this model.
Disruption of oligomeric complexes containing hsp90 by
benzoquinones (ansamycin and geldanamycin) has proven an efficient way
of inhibiting kinases (Raf-1 and
pp60v-src) (30), GR (31), and
PR (32). Similarly, the ability of the hsp90 peptide to selectively
disrupt the GR-hsp90 association in vitro leads to the
prediction that this peptide would act as an antagonist, by blocking GR
hormone-binding capacity. This is a most interesting aspect of our
findings, showing that the antiglucocorticoid activity of our peptide
is highly specific and is not related to a general inhibition of
steroid receptor-mediated transcription, as PR, a closely related
receptor, was not sensitive to hsp90 peptide overexpression. In
addition, the anti-AP-1 activity of the receptor was also abolished,
demonstrating a complete inactivation of GR transcriptional activities.
This can be explained by the subcellular localization of the receptor,
which remained in the cytoplasmic compartment even in the presence of
saturating concentrations of a potent agonist. A related strategy has
been mentioned for the modulation of estrogen receptor activity using
peptides mimicking a phosphorylable homodimerization interface. In this
case, however, no demonstration of the antiestradiol peptide activity
in intact cells has been provided (33).
The association of GR with the chaperoning complex in intact cells,
which is stabilized by antiglucocorticoids (15), is thus a process
requiring region A of hsp90. Moreover, the specific inhibition of GR
DNA-binding activity in vitro suggests that the hsp90
peptide may act by generating misfolded receptors unresponsive to
ligand and unable to translocate into the nucleus. Thus, our results
demonstrate that hsp90/GR association in vivo is a critical
step in the regulation of GR activity. More importantly, they show for
the first time that a nonsteroidal, peptidic antiglucocorticoid can be
generated by mimicking protein-protein interaction interfaces. This may
constitute a new strategy, allowing modification of cellular responses
to nuclear receptors signaling pathways without targeting the
ligand-binding sites of these proteins.
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MATERIALS AND METHODS
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Materials
\[6,7-N-3H\]Dexamethasone (45 Ci/mmol) was purchased
from DuPont-New England Nuclear (Boston, MA).
L-[35S]Methionine (1000 Ci/mmol) and Amplify
were obtained from Amersham (Arlington Heights, IL). TnT SP6- and TnT
T7-coupled transcription-translation kits were obtained from Promega
(Madison, WI). DNA-cellulose, protein A-Sepharose, and all reagents
were purchased from Sigma Chemical Co. (St. Louis, MO). R5020 was a
gift from Dr. J.-M. Renoir (INSERM U-33, Le Kremlin-Bicetre, France).
The anti-hsp90 monoclonal antibody 3G3 was purchased from Affinity
BioReagents (Neshanic Station, NJ), and PR22 was a gift from D. O. Toft
(Mayo Clinic, Rochester, MN). Acrylamide solutions were obtained from
National Diagnostics (Atlanta, GA). The various peptides used for this
study were synthesized and purified as previously described (34).
In Vitro Transcription and Translation
Coupled transcription and translation reactions were carried out
with the TnT kit as suggested by the manufacturer (Promega). Generally,
reaction mixtures contained 12.5 µl rabbit reticulocyte lysate, 1.0
µl TnT buffer, 15 U T7 or SP6 RNA polymerase, 0.5 µl of a 1
mM methionine-depleted amino acid solution, 2.0 µl (20
µCi) labeled methionine, and 1.0 µg plasmid DNA in a final volume
of 25.0 µl. Incubations were performed for 90 min (T7) or 120 min
(SP6) at 30 C for PR
15Fx and pT3.1118, respectively.
High Performance Size-Exclusion Chromatography
A TSK G3000SW column was equilibrated in buffer A (10
mM HEPES, pH 7.4; 10 mM
Na2MoO4; 130 mM NaCl; and 20
mM ß-mercaptoethanol). A flow rate of 0.5 ml/min was
maintained by an LKB 2150 pump (LKB, Rockville, MD), and 0.25-ml
fractions were collected. The column was calibrated as previously
described (35); the void volume was determined by the elution volume of
blue dextran, and the included volume was determined by the peak of
nonincorporated methionine. In all experiments, elution of hemoglobin
from reticulocyte lysate was used as an internal control. One hundred
microliters of each fraction were mixed with 3 ml Aqualyte (J. T.
Baker, Philipsburg, NJ) scintillation fluid and assayed for their
radioactivity content in an LKB 2124 Rack ß-scintillation
counter.
Coimmunoprecipitation of in Vitro Translated
Receptors with the Anti-hsp90 Antibody 3G3
Immunoprecipitations with the IgM 3G3 antibody (36) were
conducted as follows. Protein A-Sepharose was first incubated with an
anti-IgM IgG (50 µg) for 23 h at 4 C, then washed three times with
RIPA buffer (10 mM Tris-HCl, pH 7.4; 150 mM
NaCl; 5 mM ß-mercaptoethanol; and 0.25% Nonidet P-40).
Protein A-anti-IgM complexes were then incubated with 10 µl
reconstituted 3G3 ascite fluid for 2 h at 4 C and washed once with
RIPA buffer and twice with HEPES buffer (10 mM HEPES, pH
7.4; 20 mM Na2MoO4; and 10
mM ß-mercaptoethanol). The programmed lysate was diluted
100-fold in HEPES buffer, and hsp90 from 50-µl samples was
immunoadsorbed on the 3G3-protein A matrix for 2 h at 4 C. This
dilution was necessary to obtain complete immunoadsorption of the
synthesized receptors. Pellets were washed three times with 400 µl
HEPES buffer and analyzed by SDS-PAGE as described below.
Gel Electrophoresis and Autoradiography
SDS-PAGE was performed on 8% slab gels as previously described
(15). Gels were stained with Coomassie blue, destained, soaked in
Amplify (Amersham), and dried. Gels were autoradiographed for 24 h at
-80 C.
Hormone Binding and DNA Binding Assays
In vitro translated GRs were diluted 1:1 in buffer A
and incubated with 100 nM tritiated dexamethasone in the
presence or absence of a 500-fold excess of radioinert dexamethasone.
Steroid binding was assayed by the charcoal adsorption method (37).
Activation of GR or PR to the DNA-binding state was induced by
incubating the lysate at 30 C for 30 min. The DNA-binding activity of
each receptor preparation was assayed as follows. Twenty-five
microliters of DNA-cellulose (12.5%, wt/vol) equilibrated in buffer A
without NaCl were added to 10 µl lysate, stirred at 4 C for 45 min,
and washed three times in the same buffer. The receptor content of each
pellet was then estimated by SDS-PAGE analysis and quantification of
the full-length 35S-labeled receptor. Data are the average
of five independent experiments.
Circular Dichroism Spectra
The 35-mer peptide was solubilized in 0.1 M sodium
phosphate buffer, pH 7.4, at a concentration of 1 mg/ml. The solution
was placed in a 0.1-mm path length cuvette, and spectra were recorded
from 290 to 190 nm by a Jobin-Yvon Dicrographe III (Jobin-Yvon, Paris,
France).
Transient Transfection Experiments
COS cells were transfected by the calcium phosphate
precipitation method as follows. Cells (104/35-mm dish)
were plated in DMEM supplemented with 10% FCS. The following day,
cells were fed 1 ml fresh medium, and calcium-DNA coprecipitate was
added 4 h later. Cells were transfected with a constant amount of
the reporter gene pLTR-Luc (2 µg/35-mm well), receptor expression
vector (50 ng of either RSV-rGR or pSV-rPR, encoding, respectively, for
rat GR or rabbit PR), and increasing amounts of pCMVhsp90 or pCMV09psh.
The DNA concentration (10 µg/well) was kept constant using the empty
pCMV5 vector. Cells were treated for 16 h with 10-6
M dexamethasone (GR) or R5020 (PR). Results are averaged
from eight independent experiments. ß-Galactosidase and luciferase
assays were performed as previously reported (38, 39). Indirect
immunofluorescence detection of GR was performed as previously
described (38), using Mab7 or BuGR2 monoclonal antibodies. Both
antibodies showed a similar cellular localization of GR (data not
shown).
Synthesis and Sequences of Peptides
Peptides were synthesized by the Merrifield solid phase method
as described previously (34). The lyophilized crude peptide was
purified by high performance reverse phase chromatography using a Delta
Pak C4300A (7.8 mm x 30-cm; Millipore, Paris, France) column.
Full-length peptides were characterized by mass spectrometry, and
purity was judged to be more than 85% in all cases.
Sequences were as follows: hsp90 peptide, 232-EEKGE KEEED KEDEE KPKIE
DVGSD EEDDS GKDKK-266; and control peptides, 435-GGLAP PPGSC SPSLS
PSSNR SSPAT HSP-462 and 154-SKESV RNDRN KKKKE VPKPE CSES-177. Sequences
are from the human retinoic acid receptor-
(40).
Plasmids
Plasmids encoding the rat GR (pT3.1118) and the A form of the
chicken PR (PR
15Fx) were provided by Drs. K. R. Yamamoto
(University of California-San Francisco) and H. Gronemeyer (INSERM
U-184, Illkirch, France). The pSV-rPR was obtained from Prof. E.
Milgrom (INSERM U-135, Le Kremlin-Bicetre, France), and pCMV5 was a
gift from Dr. D. W. Russell (University of Texas, Dallas, TX). The
pCMVhsp90 and reverse pCMVhsp90 were constructed as follows. A 151-bp
oligonucleotide encoding for the desired hsp90 sequence was inserted
into pCMV5 as an EcoRI fragment, containing the 5'- and
3'-EcoRI sites, a Kozak consensus sequence (GCCACC) (41)
upstream of the ATG codon, and an additional TTA codon (leucine) after
the initial methionine so as to generate a stop codon when the sequence
was inserted in the antisense orientation. Consequently, the following
peptide was translated from the pCMVhsp90 plasmid: 1-MLEEK GEKEE EDKED
EEKPK IEDDV GSDEE DDSGK DKKK-39, whereas the peptide 1-MLLLL ILTGI
IFLIG SYIFD LWLFL ILLIF LLLFT LFL-38 was synthesized from pCMV09psh.
The pT3.1118 (42), pRSV-rGR (42), PR
15Fx (43), pLTR-Luc (39), pCMV5
(44), and pSV-rPR (45) were described previously. The pGL3-basic
(Promega) is the backbone of the AP-1-inducible reporter gene pTPA-RE
Luc. The TATA box of the adenovirus major late promoter was cloned as a
HindIII-BglII fragment into pGL3-basic, yielding
pTATA-Luc. Two copies of the consensus AP-1 response element ATGAGTCAG
were inserted 30 bp upstream of the TATA box as a
KpnI-EcoRI fragment, separated by a
PvuII site.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. K. R. Yamamoto, D. W. Russell, and H. Gronemeyer
and Profs. P. Chambon and E. Milgrom for the gift of plasmids. We are
grateful to Dr. D. O. Toft and A. C. Wikström for the gift of
antibodies, and to Dr. E. H. Bresnick for thoughtful comments on the
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Phillippe Lefebvre, INSERM U-459, Laboratoire de Biochimie Structurale, Faculté de Médecine Henri Warembourg, 1 place de Verdun, 59045 Lille, France.
This work was supported by grants from INSERM, Association pour
Recherche sur le Cancer, Fédération Nationale des Centres
de Lutte contre le Cancer, and Université de Lille II and an ARC
fellowship (to H.-P.D.-P.).
Received for publication August 16, 1996.
Revision received December 23, 1996.
Accepted for publication February 26, 1997.
 |
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