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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 574–659 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 221–290) and B (region 530–581), whereas the third is a putative leucine zipper called region Z (region 392–419). 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, {alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sequence and Structure of Region 232–266 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. 1AGo). The main structural features of these sequences are their acidic character and their putative organization into a DNA-like, {alpha}-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 208–210 and 220 nm. These values underscore the propensity of the hsp90 peptide to fold into an {alpha}-helical structure under these conditions (Fig. 1BGo).



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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 {alpha}-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 190–290 nm by a Jobin-Yvon Dicrograph III. They are plotted as a function of the molar ellipticity.

 
Inhibition of the Association of hsp90 with GR by the hsp90 Peptide
To assess directly whether this {alpha}-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. 2AGo). 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. 2BGo), 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. 2Go, the anti-hsp90 antibody coprecipitated a significant amount of labeled GR (Fig. 2CGo) and PR (Fig. 2DGo), 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. 2CGo, lanes PAPT). In contrast, binding of hsp90 to PR was not affected in the presence of the hsp90 peptide (Fig. 2DGo). Adding the hsp90 peptide after completion of the translation reaction did not modulate the amount of coimmunoprecipitated GR and PR (Fig. 2Go, 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 {alpha}-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; {blacktriangleup}, nonactivated receptor plus hsp90peptide; {square}, 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).

 
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. 3Go). 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. 3Go, A and C) and PR (Fig. 3Go, B and D) to activating conditions led to the conversion of 60–70% 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. 3Go, 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. 3AGo). 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. 3AGo). 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; {blacktriangleup}, GR, activation, and control peptides; {square}, GR, hsp90 peptide, and 20 mM molybdate; {diamond}, 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; {blacktriangleup}, PR, activation, and control peptide; {square}, activated receptor, hsp90 peptide, and 20 mM molybdate; {diamond}, 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. 2Go, 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; {blacktriangleup}, activated receptor plus control peptide; {square}, nonactivated receptor plus hsp90 peptide; {diamond}, nonactivated receptor plus control peptide.

 
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. 3AGo) 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. 2Go), 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. 3Go, C and D). At peptide concentrations of 100–200 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. 3CGo). On the contrary, activated PR bound DNA efficiently, even at a peptide concentration of 1 mM (Fig. 3DGo). 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. 4Go, 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. 4AGo). No effect was noticed when GR was substituted for PR (Fig. 4CGo) 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. 4Go, 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; {square}, 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.

 
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. 5Go). 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. 6Go). Unstimulated cells showed a clear cytoplasmic localization of GR, which was found in the nucleus after dexamethasone addition to the culture medium (Fig. 6Go, 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. 6Go, 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. 4Go). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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, 1–5 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{Delta} 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 2–3 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 2–4 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 C4–300A (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-{alpha} (40).

Plasmids
Plasmids encoding the rat GR (pT3.1118) and the A form of the chicken PR (PR{Delta} 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{Delta}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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Hutchison KA, Stancato LF, Owens-Grillo JK, Johnson JL, Krishna P, Toft DO, Pratt WB 1995 The 23-kDa acidic protein in reticulocyte lysate is the weakly bound component of the hsp foldosome that is required for assembly of the glucocorticoid receptor into a functional heterocomplex with hsp90. J Biol Chem 270:18841–18847[Abstract/Free Full Text]
  2. Owens-Grillo JK, Hoffmann K, Hutchison KA, Yem AW, Deibel MR, Handschumacher RE, Pratt WB 1995 The cyclosporin A-binding immunophilin CyP-40 and the FK506-binding immunophilin hsp56 bind to a common site on hsp90 and exist in independent cytosolic heterocomplexes with the untransformed glucocorticoid receptor. J Biol Chem 270:20479–20484[Abstract/Free Full Text]
  3. Czar MJ,Owens-Grillo JK, Dittmar KD, Hutchison KA, Zacharek AM, Leach KL, Deibel MR, Pratt WB 1994 Characterization of the protein-protein interactions determining the heat shock protein (hsp90.hsp70.hsp56) heterocomplex. J Biol Chem 269:11155–11161[Abstract/Free Full Text]
  4. Scherrer LC, Picard D, Massa E, Harmon JM, Simons SS, Yamamoto KR, Pratt WB 1993 Evidence that the hormone binding domain of steroid receptors confers hormonal control on chimeric proteins by determining their hormone-regulated binding to heat-shock protein-90. Biochemistry 32:5381–5386[Medline]
  5. Kang KI, Devin J, Cadepond F, Jibard N, Guiochon-Mantel A, Baulieu EE, Catelli MG 1994 In vivo functional protein protein interaction–nuclear targeted hsp90 shifts cytoplasmic steroid receptor mutants into the nucleus. Proc Natl Acad Sci USA 91:340–344[Abstract]
  6. Bresnick EH, Dalman FC, Sanchez ER, Pratt WB 1989 Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the L cell glucocorticoid receptor. J Biol Chem 264:4992–4997[Abstract/Free Full Text]
  7. Chandler VL, Maler BA, Yamamoto KR 1983 DNA sequences bound specifically by glucocorticoid receptor in vitro render a heterologous promoter hormone responsive in vivo. Cell 33:489–499[Medline]
  8. Payvar F, DeFranco DB, Firestone GL, Edgar BA, Wrange Ö, Okret S, Gustafsson JÅ, Yamamoto KR 1983 Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region. Cell 35:381–392[Medline]
  9. Bohen SP, Kralli A, Yamamoto KR 1995 Hold’em and fold’em: chaperones and signal transduction. Science 268:1303–1304[Medline]
  10. Pratt WB, Jolly DJ, Pratt DV, Hollenberg SM, Giguere V, Cadepond FM, Schweizer-Groyer G, Catelli MG, Evans RM, Baulieu EE 1988 A region in the steroid binding domain determines formation of the non-DNA-binding, 9S glucocorticoid receptor complex. J Biol Chem 263:267–273[Abstract/Free Full Text]
  11. Housley PR, Sanchez ER, Danielsen M, Ringold GM, Pratt WB 1990 Evidence that the conserved region in the steroid binding domain of the glucocorticoid receptor is required for both optimal binding of hsp90 and protection from proteolytic cleavage. A two-site model for hsp90 binding to the steroid binding domain. J Biol Chem 265:12778–12781[Abstract/Free Full Text]
  12. Dalman FC, Scherrer LC, Taylor LP, Akil H, Pratt WB 1991 Localization of the 90-kDa heat shock protein-binding site within the hormone-binding domain of the glucocorticoid receptor by peptide competition. J Biol Chem 266:3482–3490[Abstract/Free Full Text]
  13. Cadepond F, Binart N, Chambraud B, Jibard N, Schweizer-Groyer G, Segard-Maurel I, Baulieu EE 1993 Interaction of glucocorticosteroid receptor and wild-type or mutated 90-kDa heat shock protein coexpressed in baculovirus-infected Sf9 cells. Proc Natl Acad Sci USA 90:10434–10438[Abstract]
  14. Sullivan WP, Toft DO 1993 Mutational analysis of hsp90 binding to the progesterone receptor. J Biol Chem 268:20373–20379[Abstract/Free Full Text]
  15. Lefebvre P, Danze PM, Sablonnière B, Richard C, Formstecher P, Dautrevaux M 1988 Association of the glucocorticoid receptor binding subunit with the 90K nonsteroid-binding component is stabilized by both steroidal and nonsteroidal antiglucocorticoids in intact cells. Biochemistry 27:9186–9194[Medline]
  16. Binart N, Chambraud B, Dumas B, Rowlands DA, Bigogne C, Levin JM, Garnier J, Baulieu EE, Catelli MT 1989 The cDNA-derived amino acid sequence of chick heat shock protein Mr 90,000 (hsp90) reveals a DNA-like structure: potential site of interaction with steroid receptors. Biochem Biophys Res Commun 159:140–147[Medline]
  17. Heck S, Kullman M, Gast A, Ponta H, Rahmsdorf HJ, Herrlich P, Cato ACB 1994 A distinct modulating domain in glucocorticoid receptor monomers in the repression of activity of the transcription factor AP-1. EMBO J 13:4087–4095[Abstract]
  18. Akner G, Wikström AC, Gustafson JA 1995 Subcellular distribution of the glucocorticoid receptor and evidence for its association with microtubules. J Steroid Biochem Mol Biol 52:1–16[CrossRef][Medline]
  19. Pratt WB 1993 The role of heat shock proteins in regulating the function, folding, and trafficking of the glucocorticoid receptor. J Biol Chem 268:21455–21458[Free Full Text]
  20. Smith DF, Toft DO 1993 Minireview–steroid receptors and their associated proteins. Mol Endocrinol 7:4–11[Medline]
  21. Owens-Grillo JK, Czar MJ, Hutchison KA, Hoffman K, Perdew GH, Pratt WB 1996 A model of protein targeting mediated by immunophilins and other proteins that bind th hsp90 via tetratricopeptide repeat domains. J Biol Chem 271:13468–13475[Abstract/Free Full Text]
  22. Schowalter DB, Sullivan WP, Maihle NJ, Dobson ADW, Conneely OM, O’Malley BW, Toft DO 1991 Characterization of progesterone receptor binding to the 90- and 70- kDa heat shock proteins. J Biol Chem 266:21165–21173[Abstract/Free Full Text]
  23. Cadepond F, Jibard N, Binart N, Schweizer-Groyer G, Segard-Maurel I, Baulieu EE 1994 Selective deletions in the 90 kDa heat shock protein (hsp90) impede hetero-oligomeric complex formation with the glucocorticosteroid receptor (GR) or hormone binding by GR. J Steroid Biochem Mol Biol 48:361–367[CrossRef][Medline]
  24. Binart N, Lombes M, Baulieu EE 1995 Distinct functions of the 90 kDa heat-shock protein (hsp90) in oestrogen and mineralocorticosteroid receptor activity: effects of hsp90 deletion mutants. Biochem J 311:797–804[Medline]
  25. Gupta RS 1995 Phylogenetic analysis of the 90 kD heat shock family of protein sequences and an examination of the relationship among animals, plants, and fungi species. Mol Biol Evol 12:1063–1073[Abstract]
  26. Picard D, Khursheed B, Garabedian MJ, Fortin MG, Lindquist S, Yamamoto KR 1990 Reduced levels of hsp90 compromise steroid receptor action in vivo. Nature 348:166–168[CrossRef][Medline]
  27. Whitelaw ML, Mcguire J, Picard D, Gustafsson JA, Poellinger L 1995 Heat shock protein hsp90 regulates dioxin receptor function in vivo. Proc Natl Acad Sci USA 92:4437–4441[Abstract]
  28. Dahlman-Wright K, Grandien K, Nilsson S, Gustafsson J, Carlstedt-Duke J 1993 Protein-protein interactions between the DNA-binding domains of nuclear receptors-influence on DNA-binding. J Steroid Biochem Mol Biol 45:239–250[CrossRef][Medline]
  29. Freedman LP, Luisi BF 1993 On the mechanism of DNA binding by nuclear hormone receptors–a structural and functional perspective. J Cell Biochem 51:140–150[Medline]
  30. Schulte TW, Blagosklonny MV, Ingui C, Neckers L 1995 Disruption of the Raf-1-Hsp90 molecular complex results in destabilization of Raf-1 and loss of Raf-1-ras association. J Biol Chem 270:24585–24588[Abstract/Free Full Text]
  31. Whitesell, L, Cook, P 1996 Stable and specific binding of heat shock protein 90 by geldanamycin disrupts glucocorticoid receptor function in intact cells. Mol Endocrinol 10:705–712[Abstract]
  32. Smith DF, Whitesell L, Nair SC, Chen SY, Prapapanich V, Rimerman RA 1995 Progesterone receptor structure and function altered by geldanamycin, an hsp90-binding agent. Mol Cell Biol 15:6804–6812[Abstract]
  33. Arnold SF, Notides AC 1995 An antiestrogen: a phosphotyrosyl peptide that blocks dimerization of the human estrogen receptor. Proc Natl Acad Sci USA 92:7475–7479[Abstract]
  34. Tbarka N, Richard-Mereau C, Formstecher P, Dautrevaux M 1993 Biochemical and immunological evidence that an acidic domain of hsp-90 is involved in the stabilization of untransformed glucocorticoid receptor complexes. FEBS Lett 322:125–128[CrossRef][Medline]
  35. Sablonnière B, Lefebvre P, Formstecher P, Dautrevaux M 1987 Improved Stokes radius measurement of the glucocorticoid receptor using TSK G4000SW and TSK G3000SW high-performance size-exclusion columns. Analytical and preparative applications. J Chromatogr 403:183–196[CrossRef][Medline]
  36. Perdew GH, Whitelaw ML 1991 Evidence that the 90-kDa heat shock protein (hsp90) exists in cytosol in heteromeric complexes containing hsp70 and three other proteins of Mr 63,000, 56,000, and 50,000. J Biol Chem 266:6708–6713[Abstract/Free Full Text]
  37. Rousseau GG, Kirchkoff J, Formstecher P, Lustenberger P 1979 17-beta carboxamide steroids are a new class of glucocorticoid antagonists. Nature 279:158–160[Medline]
  38. Tahayato A, Lefebvre P, Formstecher P, Dautrevaux M 1993 A protein kinase C-dependent activity modulates retinoic acid-induced transcription. Mol Endocrinol 7:1642–1653[Abstract]
  39. Lefebvre P, Berard DS, Cordingley MG, Hager GL 1991 Two regions of the mouse mammary tumor virus long terminal repeat regulate the activity of its promoter in mammary cell lines. Mol Cell Biol 11:2529–2537[Medline]
  40. Giguere V, Ong ES, Segui P, Evans RM 1987 Identification of a receptor for the morphogen retinoic acid. Nature 330:624–629[CrossRef][Medline]
  41. Kozak M 1991 Structural features in eukaryotic messenger RNAs that modulate the initiation of translation. J Biol Chem 266:19867–19870[Free Full Text]
  42. Rusconi S, Yamamoto KR 1987 Functional dissection of the hormone and DNA binding activities of the glucocorticoid receptor. EMBO J 6:1309–1315[Abstract]
  43. Gronemeyer H, Turcotte B, Quirin-Stricker C, Bocquel MT, Meyer ME, Krozowski Z, Jeltsch JM, Lerouge T, Garnier JM, Chambon P 1987 The chicken progesterone receptor: sequence, expression and functional analysis. EMBO J 6:3985–3994[Abstract]
  44. Andersson S, Davis DL, Dahlback H, Jornvall, H Russell DW 1989 Cloning, structure, and expression of the mitochondrial cytochromeP-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem 264:8222–8229[Abstract/Free Full Text]
  45. Guiochon-Mantel A, Loosfelt H, Ragot T, Bailly A, Atger M, Misrahi M, Perricaudet M, Milgrom E 1988 Receptors bound to antiprogestin form abortive complexes with hormone responsive elements. Nature 336:695–698[CrossRef][Medline]
  46. Thompson JD, Higgins DG, Gibson TJ 1994 CLUSTAL W: improving the sensitivity of progressive multiple sequencealignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680[Abstract]
  47. Hutchison KA, Dittmar KD, Pratt WB 1994 All of the factors required for assembly of the glucocorticoid receptor into a functional heterocomplex with heat shock protein 90 are preassociated in a self-sufficient protein folding structure, a "foldosome." J Biol Chem 269:27894–27899[Abstract/Free Full Text]