The Distinct Agonistic Properties of the Phenylpyrazolosteroid Cortivazol Reveal Interdomain Communication within the Glucocorticoid Receptor

Noritada Yoshikawa, Keiko Yamamoto, Noriaki Shimizu, Sachiko Yamada, Chikao Morimoto and Hirotoshi Tanaka

Division of the Clinical Immunology, the Advanced Clinical Research Center, the Institute of Medical Science, the University of Tokyo, Tokyo 108-8639; and the Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo 101-0062, Japan

Address all correspondence and requests for reprints to: Hirotoshi Tanaka, M.D., Ph.D., the Division of the Clinical Immunology, the Advanced Clinical Research Center, the Institute of Medical Science, the University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail: hirotnk{at}ims.u-tokyo.ac.jp.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recent structural analyses of the nuclear receptors establish a paradigm of receptor activation, in which agonist binding induces the ligand binding domain (LBD)/activation function-2 helix to form a charge clamp for coactivator recruitment. However, these analyses have not sufficiently addressed the mechanisms for differential actions of various synthetic steroids in terms of fine tuning of multiple functions of whole receptor molecules. In the present study, we used the glucocorticoid receptor (GR)-specific agonist cortivazol (CVZ) to probe the plasticity and functional modularity of the GR. Structural docking analysis revealed that although CVZ is more bulky than other agonists, it can be accommodated in the ligand binding pocket of the GR by reorientation of several amino acid side chains but without major alterations in the active conformation of the LBD. In this induced fit model, the phenylpyrazole A-ring of CVZ establishes additional contacts with helices 3 and 5 of the LBD that may contribute to a more stable LBD configuration. Structural and functional analysis revealed that CVZ is able to compensate for the deleterious effects of a C-terminal deletion of the LBD in a manner that mimics the stabilizing influence of the F602S point mutation. CVZ-mediated productive recruitment of transcriptional intermediary factor 2 to the C-terminally deleted LBD requires the receptor’s own DNA binding domain and is positively influenced by the N-terminal regions of GR or progesterone receptor. These results support a model where ligand-dependent conformational changes in the LBD play a role in GR-mediated gene regulation via modular interaction with the DBD and activation function-1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GLUCOCORTICOIDS ARE PRODUCED in the adrenal cortex under the strict control of the hypothalamus-pituitary-adrenal axis and exert a variety of biological actions including the regulation of glucose and lipid metabolism, electrolyte balance, and modulation of the immune, cardiovascular, and central nervous system (1, 2). Multiple compounds with glucocorticoid activity including dexamethasone (DEX), prednisolone, and cortivazol (CVZ) are widely used as an antiinflammatory and/or immunosuppressive agents (3). At pharmacological doses, however, patients often suffer from side effects of glucocorticoids, the molecular basis for which have not been fully clarified. Indeed, dissociation of their therapeutic effects and adverse reactions is still one of the most challenging clinical issues to be solved (4, 5).

Glucocorticoids act by the binding to their cognate receptor, the glucocorticoid receptor (GR), the prototypic member of the nuclear receptor superfamily, which also includes the receptors for the mineralocorticoids (MR), estrogens, progestins (PR), and androgens (AR), as well as those for peroxisome proliferators, vitamin D, and thyroid hormones (6, 7). Phylogenetic and sequence analysis indicate that the GR, MR, PR, and AR form a subfamily of oxosteroid receptors (6, 7). Like most nuclear receptors, the GR is a modular protein that is organized into three major domains: an N-terminal regulatory domain harboring a strong transcriptional activation function (AF)-1, a central DNA binding domain (DBD), and a C-terminal ligand binding domain (LBD) (6, 8). The LBD harbors a second AF-2 directly regulated by ligands. Agonist binding to the LBD induces the reorientation of a critical {alpha}-helix (AF-2 helix) and the formation of a binding pocket for a family of coactivator proteins that play essential roles in transactivation (9, 10). Among nuclear receptors, the AF-2 pocket is highly conserved, whereas the molecular size and amino acid composition of AF-1 is much more diverse (6, 9). In the absence of ligand, the GR is retained in the cytoplasm in association with chaperone proteins such as heat shock protein 90 (hsp90) (11, 12). Hormone binding initiates the release of the chaperone proteins and translocation of the receptor into the nucleus where GR binds to DNA promoter elements termed glucocorticoid response element (GRE) from which it can either activate or repress transcription depending on the context of the target promoter (6, 8). In addition, the GR also interacts with other transcription factors such as nuclear factor-{kappa}B (NF-{kappa}B) to repress their transcriptional activities. This GR-mediated repression has been postulated to be one of the major mechanisms for the therapeutic antiinflammatory and immunosuppressive activities of glucocorticoids (13, 14).

Recent crystallographic analyses of the nuclear receptors have clarified the relationship between receptor structure and function and established a paradigm of receptor activation. The GR LBD, similar to other nuclear receptor LBDs, is composed of {alpha}-helices and ß-strands folded into a three-layer helical sandwich. The ligand binding pocket is composed of residues from helices 3, 4, 5, 6, 7, 10, and the AF-2 helix as well as residues from ß-strands between helices 5 and 6. Following AF-2 helix is an extended strand that forms a conserved ß-sheet with a ß-strand between helices 8 and 9. This C-terminal ß-strand also appears to play an important role in receptor activation by stabilizing AF-2 helix in an active conformation (15, 16). Many AF-2 coactivators for the GR have been identified to date, including steroid receptor coactivator-1, transcriptional intermediary factor (TIF) 2/GR-interacting protein-1 and cAMP response element binding protein-binding protein/p300 (17, 18, 19, 20, 21). These coactivators directly associate with the GR LBD via their LXXLL motif. For example, the LLRYLL sequence in the TIF2 forms a two-turn {alpha}-helix that orients the hydrophobic leucine side chains into a groove formed in part by the AF-2 helix and residues from helices 3, 3', 4, and 5. The N- and C-terminal ends of the coactivator helix are clamped by Glu-755 from the AF-2 helix and Lys-579 in helix 3, respectively (15). Mutations that disrupt either the first (Glu-755) or the second (Arg-585 and Asp-590) charge clamp dramatically reduce activation mediated by the GR LBD, demonstrating that they are critical for transactivation function of the GR (15). On the other hand, AF-1 coactivators have only recently been described. For example, basal transcription factors including TBP and TFIID have been shown to associate with the AF-1 of GR (22, 23). TSG101 and DRIP150 have also been reported to interact with GR AF-1 and regulate GR function in a reciprocal manner; GR transcriptional activities are repressed by TSG101 but enhanced by DRIP150 (24). These cofactors are shown to interact with distinct regions of AF-1 (22, 23, 24). Although we now have at hand a large number of regulatory proteins that interact directly or indirectly with the various modular domains of nuclear receptors, how ligands differentially regulate the functional interplay between them remains poorly understood.

The phenylpyrazologlucocorticoid CVZ is a unique synthetic glucocorticoid agonist with complex binding properties and is more potent than DEX (25). We previously demonstrated that CVZ selectively binds to the GR but not to the MR and, based on two criteria, we proposed that the functional interaction of CVZ with the GR LBD is different from that of DEX. Firstly, deletion of the last 12 amino acids of GR severely compromises DEX but not CVZ binding and secondly, the point mutant L753F, in which Leu-753 in AF-2 is substituted to Phe, can efficiently recruit TIF2 to the LBD when bound to CVZ but not when bound to DEX (26). These results prompted us to propose that occupancy of the GR LBD by CVZ might lead to a more stable active conformation that can tolerate the disrupting effects of LBD mutations and may have unique effects on the structure and function of the whole GR molecule. In the present study, we explore the distinct properties of CVZ-bound GR by modeling its structure, analyzing the influence of both destabilizing and stabilizing LBD mutations, and probing the role played by other receptor domains. We provide evidence that the CVZ-specific LBD conformation allows efficient TIF2 recruitment to the receptor at least in part through intrareceptor communication between the LBD and DBD, as well as through collaboration with N-terminal sequences.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Docking Model of CVZ-Bound GR LBD
Because we and others showed that CVZ-bound GR has distinct functional characteristics when compared with DEX-bound GR (see introductory section), we first examined whether CVZ could be accommodated into the classical ligand binding pocket of the GR LBD or whether a distinct binding mode must be invoked. For this purpose, we modeled the three-dimensional structure of CVZ-bound GR using the coordinates of the crystal structure between the F602S mutant LBD and DEX (15). In brief, we docked CVZ in silico into the ligand binding pocket of the GR LBD by superimposing its steroid backbone with that of DEX. The calculated volume of DEX and CVZ is 386 Å3 and 541 Å3, respectively, reflecting that CVZ has a bulky phenylpyrazole ring attached to the A-ring of steroid backbone as well as a C21-acetoxyl group at the D-ring (Fig. 1AGo). The estimated volume of the ligand binding pocket of the GR LBD is 600 Å3, which appears to be large enough for binding either ligand. Energy minimization of the CVZ/GR LBD complex suggested that favorable configurations between GR and CVZ could be reached by the induced fit mechanism. The resultant model for CVZ-GR LBD is shown in Fig. 1BGo. CVZ, as well as DEX, is completely enclosed within the bottom half of the GR LBD and spatial position of the {alpha}-helices and ß-strands of the CVZ-bound GR LBD is almost identical with that of DEX-bound one, including the orientation of helix 12. One or more hydrophobic residues within the GR protein contact nearly every atom of the steroid core of DEX and CVZ (Fig. 1CGo). In addition, the model allows for all of the hydrophilic groups of CVZ to form hydrogen bonds with the protein, which is what is observed in the DEX structure (Fig. 1CGo). A similar and extensive hydrogen bond network between GR and either DEX or CVZ is likely to contribute to their high affinity binding. Moreover, both ligands make direct contacts with AF-2 helix at Leu-753 and the loop preceding AF-2 helix at Ile-747 and Phe-749 (Fig. 1CGo). These interactions are likely to stabilize the AF-2 helix in the active conformation in CVZ-bound LBD and are consistent with the strong agonistic activities of CVZ.



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Fig. 1. Putative Three-Dimensional Structure of the GR LBD Docked with DEX and CVZ

A, Structure of DEX and CVZ. B, Overall arrangement of DEX- and CVZ-bound GR LBD. Ribbons represent {alpha}-helices and ß-strands, and tubes represent loops of the protein. The GR-LBD docked with DEX or CVZ is depicted with space fill model. C, Ligand binding pocket of GR-DEX complex superimposed with that of GR-CVZ one (StereoView 20/20, 3D Experience, Hertfordshire, UK). Structures are drawn with stick model. Amino acid residues of GR-DEX and GR-CVZ complex are drawn by green and orange sticks, respectively. DEX and CVZ are depicted with blue and red, respectively. D, Amino acid sequence and secondary structure of the GR LBD. Amino acid sequence of the GR LBD and the location of {alpha}-helices (H1 to H12, bold bars) and ß-strands (ß1 to ß4, arrows) are schematically illustrated. Number depicts the position of amino acids. Open and filled circles indicate the GR residues closer than 4.5 Å to DEX and CVZ, respectively.

 
Given the extra volume of CVZ, several differences between the DEX-bound GR LBD complex and our model are also evident. To accommodate the bulky phenylpyrazole ring and 21-acetoxyl group of CVZ, the position of the side chain of Arg-611 is shifted outward of the ligand binding pocket and side chain conformations of Asn-564, Gln-570, Met-604, Leu-608, Met-646, and Phe-749 are altered, resulting in distinct hydrogen bond network (Fig. 1Go, C and D). Notably, all of these amino acids except for Phe-749 originate from helices 3 and 5. Taken together, the modeling suggests that CVZ might stably bind with the GR and elicit its distinct effects on GR function via minor local alteration in the conformation of these helices.

Discrimination of Glucocorticoid Ligands by GR Variants with Destabilizing and Stabilizing Mutations
We have previously shown that deletion of the last 12 amino acids severely compromises DEX binding and DEX-dependent activity but CVZ binding activity is preserved (26). In contrast to this deleterious deletion, the Phe-602 to Ser substitution in the GR LBD has been shown to stabilize the binding of various ligands and was instrumental in the successful crystallization of the GR LBD (15, 16, 27). We therefore hypothesized that this substitution might counteract the deleterious effects of C-terminal deletion and restore DEX function. We thus introduced this mutation in the context of the C-terminally deleted GR-(1–765) (resultant mutant is GR-(1–765)/F602S, Fig. 2AGo). These mutants should be useful tools to contrast the characteristics of the interaction between CVZ and DEX with the GR.



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Fig. 2. Association of hsp90 with the GR LBD Is Not Affected by Either C-Terminal Truncation or F602S Substitution in the GR

A, Schematic illustration of the wild-type and C-terminally truncated human GR. The number depicts the position of amino acid. GR-(1–765) lacks 12 amino acids from the C-terminal end of the LBD. GR-(1–765)/F602S contains the additional amino acid substitution from Phe-602 to Ser (F602S, arrowhead) in the context of GR-(1–765). B, Association of hsp90 with the GR and its C-terminally truncated mutants. After transfection of pCMX, pCMX-GR, pCMX-GR-(1–765), or pCMX-GR-(1–765)/F602S into COS7 cells, the cells were cultured in the absence of ligand for 24 h and whole cell extracts were prepared and coimmunoprecipitated with hsp90-specific antibodies or control mouse Ig. Whole cell extracts or immunoprecipitated proteins were run on 6% SDS-polyacrylamide gels. Western immunoblotting was performed using anti-GR or anti-hsp90 antibodies as described in Materials and Methods. IP, Antibodies for immunoprecipitation. Blot, antibodies for Western immunoblotting. C, Subcellular localization of the GFP-tagged wild-type and C-terminal truncated GR after heat shock. COS7 cells expressing either GFP-tagged protein GFP-GR (wild type), GFP-GR-(1–765), or GFP-GR-(1–765)/F602S were cultured in a humidified 5% CO2 atmosphere at 43 C, and subcellular localization of the proteins was analyzed as described in Materials and Methods. Results represent the percentage of nuclear-dominant fluorescent cells (%N > C). Experiments were repeated three times with almost identical results, and representative graph is shown.

 
Initially, we characterized these mutants biochemically. Because hsp90 is essential for ligand binding, we addressed whether these mutant GR could bind hsp90 in situ (Fig. 2BGo). To this end, COS7 cells were transfected with expression plasmids for the wild-type and mutant GR forms, and cellular lysates were prepared in the absence of ligand. Protein expression of the wild-type GR and its mutants was comparable (Fig. 2BGo). As shown in Fig. 2BGo, the wild-type GR and its mutant forms can be immunoprecipitated with anti-hsp90 antibodies but not control Ig. The functional significance of this association was confirmed in heat-shock experiment. As seen in Fig. 2CGo, treatment of transfected cells at 43 C for 2 h promoted nuclear translocation of green fluorescent protein (GFP)-fused GR and its mutants. We concluded that neither the C-terminal deletion nor F602S substitution within GR-(1–765) grossly affected the association of hsp90 with the GR LBD in the absence of ligand.

Next, we performed protease digestion experiments because this assay, albeit indirectly, can simultaneously assess ligand binding and the subsequent conformational alteration of the receptor (28, 29). For this purpose, GR proteins were translated in vitro in the presence of [35S]Met and incubated in the presence or absence of DEX or CVZ, followed by digestion with various concentrations of trypsin. As shown in Fig. 3AGo, in the absence of ligand, wild-type and mutant GRs were completely processed at a trypsin concentration of 5 µg/ml. In the presence of 10 µM DEX or CVZ, however, the wild-type GR was protected from the enzymatic digestion and stable 30- and 27-kDa fragments were produced, indicating that ligand binding altered and stabilized the LBD conformation into trypsin-resistant form. In contrast to the wild-type GR, the C-terminally deleted receptor discriminated between the two ligands because CVZ was able to protect the LBD from digestion with 50 and 100 µg/ml of trypsin (generating 25- and 28-kDa fragments), whereas DEX was substantially less effective. Consistent with our hypothesis, addition of the F602S mutation within the context of the C-terminal deletion partially restored the ability of DEX to protect against 50 and 100 µg/ml of trypsin. This suggests that this amino acid substitution enabled DEX to more efficiently bind and stabilize the deleted LBD just as CVZ can do in the absence of the point mutation (Fig. 3AGo).



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Fig. 3. Effects of DEX and CVZ on Proteolytic Digestion of in Vitro-Translated Wild-Type and C-Terminal Truncated GR

Schematic illustration of the wild-type GR, GR-(1–765), and GR-(1–765)/F602S (A), and NLS-fused GR LBD, GRLBD-(499–765), and GRLBD-(499–765)/F602S (B) are shown in the left. The predicted trypsin-resistant fragments are depicted as bold lines with their calculated molecular weights (45 61 ). Representative results from trypsin digestion assays are presented in the right. In brief, in vitro-synthesized [35S]GR and its mutants (A), or [35S]GRLBD and its mutants (B) was incubated with either vehicle or 10 µM of DEX or CVZ for 30 min at 20 C; then the indicated concentrations of trypsin were added and digestion was preceded for 10 min at 20 C as described in Materials and Methods. Samples were denatured and separated on 12% SDS-polyacrylamide gels. Gels were processed as described in Materials and Methods for visualization and representative results are shown.

 
These results were further supported in separate experiments using constructs expressing a fusion protein between the simian virus 40 nuclear localization signal (NLS) and the GR LBD alone (Fig. 3BGo). In the absence of ligands, in vitro-translated wild-type and mutant LBDs were completely digested (Fig. 3BGo). In the presence of DEX or CVZ, however, trypsin treatment of the wild-type LBD generated stable 30- and 27-kDa fragments (Fig. 3BGo). As in the case of the full-length GR, the C-terminally deleted LBD [LBD-(499–765)] was resistant to trypsin in the presence of CVZ but not DEX. Moreover, the F602S substitution enabled DEX to protect this LBD. Taken together, these results indicate that CVZ-bound GR mimics the more stable conformation achieved by DEX in the presence of the F602S point mutation.

Distinct Effects of DEX and CVZ on the Function of C-Terminally Truncated GR
We next studied the effect of treatment with DEX and CVZ on nuclear translocation of GR and its mutants using GFP-tagged forms. The mutations did not alter the behavior of the receptor in the absence of ligands because GFP-tagged GR and its mutants are all localized in the cytoplasm. In the case of wild-type GFP-GR, both DEX and CVZ promoted efficient nuclear translocation (Fig. 4AGo). In contrast, whereas CVZ was able to induce nuclear translocation of GFP-GR-(1–765), DEX failed to do so (Fig. 4AGo). Notably, introduction of the F602S mutation restored the ability of DEX to induce the translocation of the C-terminally deleted receptor and eliminated the difference between the ligands (Fig. 4AGo). These results again suggested that the F602S substitution appears to stabilize DEX-binding to GR-(1–765).



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Fig. 4. C-Terminal Truncation of the LBD with F602S Substitution Unveils Differential Effects of DEX and CVZ on Transactivation Function of the GR

A, Effects of ligands on subcellular localization of the GR and its C-terminally truncated mutants. GFP-tagged wild-type GR, GR-(1–765), and GR-(1–765)/F602S were transiently expressed in COS7 cells and the cells were cultured in the presence or absence of 1 µM of DEX or CVZ for 2 h, then digital images were taken as described in Materials and Methods and representative results are shown. B, Differential effects of DEX and CVZ on GRE-driven reporter gene expression. COS7 cells were cotransfected with 2 µg of GRE-driven reporter plasmid pGRE-LUC and 100 ng of expression plasmids for either wild-type GR, GR-(1–765), or GR-(1–765)/F602S, and cultured in the absence or presence of the indicated concentrations of DEX or CVZ for 24 h. Cellular luciferase activities were measured as described in Materials and Methods. Experiments were performed in triplicate and results are expressed as relative light units (RLU) per microgram of protein in the extract, and the means ± SD are shown. C, Effects of DEX and CVZ on receptor protein levels. COS7 cells were transiently transfected with expression plasmids for GFP-tagged wild-type GR, GR-(1–765), or GR-(1–765)/F602S. The cells were further cultured and treated with vehicle or 1 µM of DEX or CVZ for 0, 12, or 24 h. Whole cell extracts were prepared and 10 µg of protein was separated by SDS-PAGE. Expression levels of each protein were analyzed by Western immunoblotting using anti-GFP antibodies as described in Materials and Methods. Data were quantitated and expressed as percentage of density, which was given relative to the density obtained from the cells treated with each ligand for 0 h as described in Materials and Methods. Experiments were repeated three times with almost identical results, and representative results are shown.

 
To assess the effect of DEX and CVZ on the transactivation function of wild-type and mutant GRs, we used a GRE-driven luciferase reporter plasmid in COS7 cells. As previously reported, DEX and CVZ induced GRE-dependent transactivation by the wild-type GR, whereas reporter gene activation by GR-(1–765) was only observed for CVZ (Fig. 4BGo). Surprisingly, although DEX and CVZ were able to induce nuclear translocation and DNA binding activities of GR-(1–765)/F602S (Fig. 4AGo and data not shown), DEX scarcely induced transactivation even at high concentrations, whereas CVZ elicited gene activation in a concentration-dependent manner (Fig. 4BGo). Because it has been shown that ligand binding affects stability of the GR (30), we examined GR protein levels after treatment with DEX or CVZ. As previously reported, treatment with DEX or CVZ decreased the protein levels of the GFP-fused full-length GR (Fig. 4CGo). In clear contrast, DEX treatment did not lead to reduced protein levels of either GFP-GR-(1–765) or GFP-GR-(1–765)/F602S (Fig. 4CGo). Moreover, CVZ showed only marginal decrease in the protein levels of these mutant GRs (Fig. 4CGo). These results reveal a role for the C-terminal 12 amino acids in down-regulation of the receptor but indicate that the differential effects of DEX and CVZ on GR-mediated reporter gene activation is not simply due to ligand-induced decrease of GR protein stability. The striking difference in transactivation between DEX- and CVZ-bound GR mutants coupled with our structural prediction that CVZ-bound LBD might lead to only minor conformational alteration in the LBD when compared with DEX suggests that such subtle conformational changes might affect ligand-dependent cofactor recruitment to the receptor, especially because it has already been reported that in addition to AF-2, N-terminal segments of the LBD are important for stable interaction with coactivators (15, 31). We therefore examined the effect of exogenous expression of TIF2 on the ability of the various GR mutants to induce transcription in response to DEX and CVZ. Overexpression of TIF2-enhanced CVZ-induced transactivation by the wild-type GR as well as GR-(1–765) and GR-(1–765)/F602S (Fig. 5BGo). On the other hand, TIF2 only marginally rescued DEX-dependent transactivation by either mutant (Fig. 5BGo).



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Fig. 5. Role of Ligands and AF-1 in Functional Interaction between the LBD of C-Terminally Truncated GR and TIF2

A, Schematic illustration of AF-1 deleted GR and AF-1-chimeric receptors. GR{Delta}AF-1 lacks N-terminal domain containing AF-1 of the GR. MR AF-1 and PR AF-1, the N-terminal domain containing AF-1 of the human MR and PR B form, were fused to GR{Delta}AF-1, and resultant chimeric receptors are designated as M/GG and P/GG, respectively. M/GG/765 and P/GG/765 are C-terminally 12 amino acid-truncated form of the M/GG and P/GG, respectively. M/GG/765/F602S and P/GG/765/F602S contain additional F602S amino acid substitution (arrowheads) in M/GG/765 and P/GG/765, respectively. Ligand-dependent nuclear translocation of these chimeric proteins was examined with indirect immunofluorescence assay as described in Materials and Methods. B, Effects of exogenous expression of TIF2 on DEX- and CVZ-dependent GRE-driven reporter gene expression by C-terminally truncated, AF-1 truncated, or AF-1-chimeric GR. COS7 cells were cotransfected with 2 µg of pGRE-LUC and 100 ng of either empty vector pCMX or expression plasmids for wild-type GR, GR-(1–765), GR-(1–765)/F602S, GR{Delta}AF-1, or their AF-1-chimeric receptors with or without 600 ng of expression plasmid for TIF2 as indicated, and cultured in the presence or absence of 1 µM of DEX (D) or CVZ (C) for 24 h. Experiments were performed in triplicate and results are expressed as relative light units (RLU) per microgram of protein in the extract, and the means ± SD are shown. C, Effects of DEX and CVZ on the subnuclear colocalization of the C-terminal truncated GR with TIF2. GFP-tagged wild-type GR, GR-(1–765), or GR-(1–765)/F602S was transiently expressed with TIF2 in COS7 cells and the cells were cultured in the presence or absence of 1 µM of DEX or CVZ for 2 h. Then digital images were taken as described in Materials and Methods and representative results are shown.

 
To test the possibility that TIF2 recruitment is influenced by sequences within the N-terminal domain of the receptor, we exchanged this region of the GR and its mutants with those of the MR and PR (Fig. 5AGo). Deletion of AF-1 (GR{Delta}AF-1) resulted in a 60% decrease in activity, which was still enhanced by TIF2. As previously reported (32), replacement of the N-terminal region of the GR with that of the MR did not significantly enhance activity compared with GR{Delta}AF-1. Enhancement of CVZ-mediated transactivation by TIF2 was observed in M/GG/765 and M/GG/765/F602S as seen in GR{Delta}AF-1 and M/GG. The N-terminal region of PR functionally substituted for that of GR and TIF2 enhancement is observed for both P/GG/765 and P/GG/765/F602S (Fig. 5BGo). These results argue that although AF-1 cooperates with AF-2, it does not play a critical role in CVZ-mediated recruitment of TIF2 to the receptor.

To confirm the effects of these ligands on the interaction between the GR and TIF2 in situ, we performed immunofluorescence colocalization assays in which GFP-GR and TIF2 were coexpressed. As previously reported (26), wild-type GR displace a speckled localization in the nucleus in the presence of TIF2 (Fig. 5CGo and data not shown). Consistent with the transactivation data, CVZ induced this speckled pattern for both GR-(1–765) and GR-(1–765)/F602S, whereas DEX treatment failed to translocate GR-(1–765) and resulted in a diffuse nuclear localization of GR-(1–765)/F602S (Fig. 5CGo). Thus, although DEX-bound GR-(1–765)/F602S accumulates in the nucleus, it does not appear to form transcriptionally productive complex with TIF2.

Ligand-Dependent Scenario for Communication between DBD and the LBD
To further delineate the receptor domains involved in the differential properties of DEX- vs. CVZ-bound GR, we next examined the ability of CVZ to counteract the deleterious effects of the C-terminal deletion and support the recruitment of TIF2 in the context of the GR LBD alone. To test this, we constructed plasmids for the expression of GAL4 DBD-GR LBD fusion proteins [GAL4-GRLBD, GAL4-GRLBD-(489–765), and GAL4-GRLBD-(489–765)/F602S; see Fig. 6AGo], and tested them in a mammalian one-hybrid assay using GAL4-responsive reporter plasmid in COS7 cells. Nuclear translocation assays paralleled the observations using the intact GR and confirmed that CVZ could achieve nuclear entry of all the chimeric proteins, whereas DEX induced the translocation of only GAL4-GRLBD and GAL4-GRLBD-(489–765)/F602S (Fig. 6AGo). The GAL4-GRLBD chimera activated the reporter gene in response to both DEX and CVZ and heterologous expression of TIF2 enhanced this response, indicating that binding of either ligand facilitated the recruitment of TIF2 to the LBD alone. Deletion of the last 12 amino acids of the LBD severely compromised CVZ as well as DEX induction even in the presence of TIF2 or after combining it with the F602S substitution (Fig. 6BGo). Both ligands induced substantial down-regulation of the wild-type LBD fusion chimera, whereas the levels of GAL4-GRLBD-(489–765) and GAL4-GRLBD-(489–765)/F602S were mildly affected or unchanged (Fig. 6CGo). These results indicate that the deleterious effects of the C-terminal truncation cannot be counteracted by CVZ binding or the F602S substitution in the context of the LBD alone and suggest that additional domains are required.



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Fig. 6. Cooperation of the DBD in Ligand-Dependent Coactivator Recruitment of the LBD

A, Schematic illustration and subcellular localization of the GR LBD fused to the DBD of the GAL4 or GR and their C-terminally truncated mutants. GAL4-GRLBD (left), GR{Delta}AF-1 (right), and their C-terminally truncated mutants are schematically illustrated. Ligand-dependent nuclear translocation of these chimeric proteins was examined with indirect immunofluorescence assay as described in Materials and Methods. B, Role of the DBD on the ligand-dependent interaction between the LBD and TIF2 in the GR. For modified one-hybrid assay, COS7 cells were cotransfected with 2 µg of the GAL4-driven reporter plasmid tk-GALpx3-LUC (left) or GRE-driven reporter plasmid pGRE-LUC (right) and 100 ng of either empty vector pCMX or expression plasmids for GAL4-GRLBD (left), GR{Delta}AF-1 (right), or their mutants with or without 600 ng of expression plasmid for TIF2 as indicated. After further 24 h of culture in the presence or absence of 1 µM of DEX (D) or CVZ (C), the cells were harvested and luciferase activities were measured as described in Materials and Methods. Experiments were performed in triplicate and results are expressed as relative light units (RLU) per microgram of protein in the extract and the means ± SD are shown. C, Expression levels of GAL4-GRLBD, GR{Delta}AF-1 and their C-terminally truncated mutants. COS7 cells were transiently transfected with expression plasmid for GAL4-GRLBD (left), GR{Delta}AF-1 (right), or their C-terminally truncated mutants as indicated. The cells were further cultured and treated with vehicle or 1 µM of DEX or CVZ for 0, 12, or 24 h. Whole cell extracts were prepared and 10 µg of protein was separated by SDS-PAGE. Expression levels of each protein were analyzed by Western immunoblotting using anti-GAL4 (DBD) or anti-GFP antibodies as described in Materials and Methods. Data were quantitated as described in Materials and Methods and expressed as percentage of density, which is given relative to the density obtained from the cells before addition of ligands (0 h). Experiments were repeated three times with almost identical results, and representative results are shown.

 
To examine the involvement of the GR DBD, we carried out parallel one-hybrid assays in the context of N-terminally deleted GR{Delta}AF-1 construct where the LBD is recruited to the promoter through the GR’s own DBD. As in the case of the GAL4 fusions, CVZ could achieve nuclear entry of all three mutant GR forms, whereas DEX could do so only for GR{Delta}AF-1 and GR{Delta}AF-1/765/F602S (Fig. 6AGo). In clear contrast to the GAL4 fusion chimeras, CVZ was able to support transactivation after deletion of the last 12 amino acids and TIF2-mediated enhancement was preserved (Fig. 6BGo). Because the behavior of these N-terminally deleted forms resembled that of the full-length receptor (including the ligand-induced changes in protein level; see Fig. 6CGo), the results indicate that the DBD of GR is sufficient to support the ability of CVZ to counteract the deleterious effects of the C-terminal deletion and that productive recruitment of TIF2 in this context likely involves a ligand-based functional communication between the LBD and the DBD.

The Functional Differences between CVZ and DEX Are Also Evident during GR Transrepression of NF-{kappa}B
To examine whether the induced structural change of the LBD upon CVZ binding affects other receptor functions, we tested the ability of both DEX and CVZ to support GR mediated transrepression of NF-{kappa}B-stimulated transcription. Although the mechanism of NF-{kappa}B repression by the GR remains to be fully defined, it is known that the p65 subunit of NF-{kappa}B physically interact with the GR DBD (33, 34, 35, 36, 37). Wild-type GR suppressed NF-{kappa}B-dependent transcription in response to both DEX and CVZ (Fig. 7Go). Deletion of the last 12 amino acids again revealed a marked difference between ligands because CVZ-mediated repression was preserved, whereas the DEX response was essentially abrogated. As in the case of activation, inclusion of the F602S substitution did not restore DEX-mediated inhibition (Fig. 7Go). These results suggest that CVZ differentially modulates receptor function not only in transactivation but also in transrepression, likely through the establishment of a distinct LBD conformation and functional interaction with the DBD.



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Fig. 7. CVZ Elicits Transrepression Function of the GR Despite the Lack of the C-Terminal End

HeLa cells were cotransfected with 2 µg of pNF{kappa}BHL reporter plasmid and 1 µg of either empty vector pCMX or expression plasmids for the wild-type GR, GR-(1–765), or GR-(1–765)/F602S. The cells were cultured and treated with or without 10 nM phorbol 12-myristate acetate (PMA) in the presence or absence of 1 µM of DEX or CVZ for 24 h. Assays were performed in triplicate and results are expressed as percentage of induction, which is given relative to the luciferase activity obtained from the cells treated with PMA alone, and the means ± SD are shown.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study, we have explored the mode of binding of the unique glucocorticoid agonist CVZ and have taken advantage of C-terminally deleted GR variants to unveil otherwise hidden differences between CVZ and the prototypic agonist DEX. Ligand binding is believed to give the receptor a cue for activation by the adoption of a conformation that is conducive to interaction with coactivator proteins (38). Data from multiple members of this family indicate that the chemical structure of individual agonists can produce distinct conformations of the LBD with unique regulatory properties (9, 39). Given bulky phenylpyrazole substituent at the A-ring of CVZ, it is thus reasonable to hypothesize that CVZ-bound GR is likely to have a distinct structure when compared with the DEX-bound one. According to our modeling data, CVZ could fit in the ligand binding pocket through an induced fit mechanism (Fig. 1Go). The additional volume contributed by the phenylpyrazole ring of CVZ provides additional contacts with the protein and can be accommodated through minor alterations in the receptor structure involving the reorientation of amino acid side chains emerging mainly from helices 3 and 5. These alterations may result in the generation of a more stable bound conformation and distinct functional surfaces. Although it is obvious that the direct determination of the structure of CVZ-bound LBD is required to determine the actual mode of binding, data from existing structures are compatible with the model. In the case of the liver X receptor ß, the structure of the LBD is flexible and can adapt to different sized ligands. When compared with the small agonist T0901317, the larger agonist GW3965 shifts many side-chains and enlarges the volume of the ligand binding pocket. This results in a different local conformation in a sector of the LBD while preserving the interaction between the ligand and helix 12 intact (40). In our model, the bulky A-ring of CVZ intimately contacts with and modulates the conformation of helices 3 and 5 (Fig. 1Go). Along this line, Ali et al. (41) have reported on compounds that have a phenylpyrazole group at the A-ring. Replacement of the C- and D-ring of the steroid backbone by alkyl, alkenyl, or benzyl groups yields compounds that selectively bind to the GR and induce transactivation (41). As in the case of CVZ and DEX, when the phenylpyrazole group in such compounds is replaced by a C3-ketone, their GR binding ability is completely abolished (41). This suggests that the mode of binding between these compounds is similar and is consistent with the idea that contacts between the phenylpyrazole A-ring and helices 3 and 5 are compatible with a stable active conformation. Interestingly, a similar modeling and functional approach in the MR argues that the Ser-810 to Leu substitution in helix 5 of the MR, which renders progesterone and the antagonist spironolactone into an agonist, is due to the creation of new interactions between these ligands and helices 3 and 5 (42). Concerning the GR, the conformation of helix 3 is likely to be important for agonism because Lys-579 within this helix is involved in clamping the C-terminal end of the coactivator helix and mutation of this residue reduces GR transactivation without affecting ligand binding (15, 31).

The C-terminal end of the GR LBD forms an extended ß-sheet that is important for binding of certain agonists and stable interaction with coactivators (15, 16, 38, 43). Deletion of the C-terminal 12 amino acids from GR severely compromises the ability of DEX to bind, to induce a trypsin-resistant conformation of the LBD, and to support transactivation. Because this deletion does not extend into the AF-2 helix and preserves the essential constituents of the AF-2 pocket, the observations suggest that the role of these residues is to stabilize an active conformation of the receptor. Our current results indicate that the deleterious effects of this deletion can be surmounted when CVZ occupies the binding pocket. This indicates that the deleted receptor possesses all the necessary features for an active conformation as long as an appropriate ligand is bound. In this view, it is likely that the additional contacts provided by the bulky phenylpyrazole substituent yield a stable active conformation without the contributions provided by the C-terminal residues. The fact that the stabilizing F602S substitution can restore the ability of DEX to bind and induce the nuclear translocation of the C-terminally deleted GR is also consistent with CVZ stabilizing an active conformation. The fact that DEX-dependent transcriptional activity of the deletion mutants is not restored by the F602S mutation suggests that, in this context, the stabilizing influence of the F602S is not sufficient and that conformations other than an active one are favored. A similar behavior is also observed in the case of the antagonist RU486 because it is capable of binding and inducing the nuclear translocation of GR even after deletion of the last 28 C-terminal amino acids (44, 45). Our results, thus, clearly show that the C-terminal 12 amino acids are not absolutely essential for agonistic activity and highlight the critical role played by the ligand in sculpting the functional surfaces of the receptor.

The unique ability of CVZ to support transactivation by the C-terminally deleted receptor allowed us to examine the role played by domains other than the LBD. Our results indicate that, at least in terms of ligand-dependent transactivation, the N-terminal region of the receptor is not essential but contributes to overall activity, most likely through cooperation between AF-1 and AF-2 (46). More importantly, direct targeting of the C-terminally deleted LBD to the DNA via the GAL4 DBD does not support CVZ-driven activity and TIF-2 recruitment. In contrast, when the deleted LBD is brought to the promoter by the GR’s own DBD, CVZ is able to support transactivation and TIF-2 recruitment. Together with the protease digestion studies, these results indicate that, although CVZ can promote a trypsin-resistant conformation of the C-terminally deleted LBD alone, the transcriptional effects of CVZ require the DBD of GR. The unique role of the GR DBD might involve an appropriate orientation of the two LBDs upon DNA binding-induced dimerization or more likely, reflects a direct contribution of the DBD to the active conformation of the CVZ-bound LBD. Several laboratories have shown that the DBD stabilizes the LBD and enhances ligand-dependent nuclear translocation (47, 48, 49, 50). Moreover, ligand-driven conformations of the LBD can influence both DNA binding (51) and anti-NF-{kappa}B activities (52). Kumar et al. (53) have suggested that the DBD plays an important role in the structural stabilization of the GR. Importantly, interdomain communication is not exclusive to GR and has been observed in other receptors as well, especially in AR (54). Steroid pharmacology is increasingly focused in the development of ligands with selective modulatory activities. Because the mode of interdomain communication may be distinct for each receptor and may be modulated in a ligand-, tissue-, and promoter-context-dependent manner, ligands such as CVZ and other phenylpyrazole analogs that manipulate this regulatory avenue will not only provide a better understanding of the mechanisms of interdomain communication but also provide novel leads in the development of selective GR modulators.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents and Antibodies
DEX was purchased from Sigma (St. Louis, MO). CVZ was a kind gift from Aventis Pharma (Strasbourg, France). Other chemicals were obtained from Wako Pure Chemical (Osaka, Japan) unless otherwise specified. Monoclonal anti-hsp90 antibodies were obtained from Affinity Bioreagents, Inc. (Golden, CO). Goat antimouse IgM and control mouse IgM TEPC183 were obtained from Sigma. Monoclonal anti-GFP antibodies were obtained from CLONTECH (Palo Alto, CA). Polyclonal anti-GAL4 (DBD) and antipolyhistidine antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA).

Plasmids
The expression plasmids for the wild-type and C-terminal 12 amino acid-truncated human GR, pCMX-GR, and pCMX-GR-(1–765) have been previously described (26). Construction of pCMX-GR-(1–765)/F602S was carried out using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using pCMX-GR-(1–765) as a template. The expression plasmids for the fusion between the simian virus 40 NLS and the human GR LBD (amino acids 499–777) and for the fusion of GFP and wild-type human GR, pCMX-NLS-GRLBD and pCMX-GFP-GR have been previously described (55, 56). The expression plasmid for the chimera between the GAL4 DBD and the human GR LBD (amino acids 489–777), pCMX-GAL4-GRLBD, was a kind gift of Dr. K. Umesono (Kyoto University, Kyoto, Japan). To construct the expression plasmid for GFP- or polyhistidine-tagged AF-1-deleted human GR (amino acids 417–777), pCMX-GFP-GR{Delta}AF-1 or pCMX-GR{Delta}AF-1, the DNA fragments encoding human GR DBD and LBD were inserted into the parent pCMX-GFP or pCMX-6xHis vectors, respectively. To construct the expression plasmids for chimeric proteins of the AF-1 of either the human MR (amino acids 1–598) or PR B form (amino acids 1–562) and GR{Delta}AF-1 (resultant plasmids are pCMX-MRAF-1/GR{Delta}AF-1 and pCMX-PRBAF-1/GR{Delta}AF-1, respectively), the N-terminal domain of each receptor was amplified by PCR using pRShMR or pEGFP-PRB as templates with appropriate flanking sequences [these template plasmids were a kind gift of Dr. R. M. Evans (Salk Institute, La Jolla, CA) and Dr. G. L. Hager (National Cancer Institute, Bethesda, MD), respectively], and inserted into the parent pCMX-GR{Delta}AF-1. To exchange the mutations within the LBD, PstI-BamHI fragments encoding a part of the human GR LBD (amino acids 596–765) from pCMX-GR-(1–765) or pCMX-GR-(1–765)/F602S were inserted into the same sites of the recipient expression plasmids. The expression plasmid for TIF2 pSG5-TIF2 was kindly provided by Dr. P. Chambon (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France). The GRE-driven reporter plasmid pGRE-LUC, GAL4-responsive reporter plasmid tk-GALpx3-LUC, and NF-{kappa}B-responsive reporter plasmid pNF{kappa}BHL have been described previously (57).

Cell Culture and Heat Shock Treatment
COS7 and HeLa cells were obtained from the RIKEN Cell Bank (Tsukuba, Japan) and maintained in DMEM (Sigma) supplemented with 10% fetal calf serum and antibiotics. In all experiments, serum steroids were stripped with dextran-coated charcoal, and cells were cultured in a humidified atmosphere at 37 C with 5% CO2. Heat shock treatment for COS7 cells was achieved by shifting flasks to another 5% CO2 incubator set at 43 C.

Graphical Manipulations and Ligand Docking
Graphical manipulations were performed using SYBYL 6.9 (Tripos, St. Louis, MO). The atomic coordinates of the crystal structure of human GR LBD (amino acids 523–777) were retrieved from Protein Data Bank (entry 1M2Z) (15). We docked CVZ into the ligand binding pocket manually by superimposing its steroid backbone with that of DEX (58, 59, 60). Energy minimization of CVZ/GR-LBD complex was performed until the energy gradient was lower than 0.1 kcal/(mol)(Å) on Tripos force field by using subset minimization command.

Immunoprecipitation and Western Immunoblot Assay
For analysis of the interaction between the GR and hsp90, we transiently transfected expression plasmids for wild-type GR or its mutants in COS7 cells and the assays were performed as described previously (56). In brief, whole cell extracts were prepared by lysing cells and immunoprecipitating with either the anti-hsp90 IgM antibody 3G3 or control mouse IgM antibody TEPC 183 as follows. We first prepared goat antimouse IgM coupled to CNBr-activated Sepharose 4B (Amersham Biosciences, Piscataway, NJ) as described previously (56). Seventy micrograms of cellular protein was added to the goat antimouse IgM-coupled Sepharose. The reaction mixtures were incubated on ice for 90 min, after which Sepharose beads were pelleted by centrifugation and washed three times with MENG buffer [25 mM Mops (pH 7.5), 1 mM EDTA, 0.02% NaN3, 10% glycerol] containing 20 mM sodium molybdate and 2 mM dithiothreitol. Immunoprecipitated proteins were eluted by boiling in sample buffer and analyzed by SDS-PAGE and electrophoretically transferred to an Immobilon-NC Pure nitrocellulose membrane (Millipore, Bedford, MA). Subsequently, Western immunoblot analysis was performed with polyclonal anti-GR antibodies diluted at 1:1000, followed by horseradish peroxidase-conjugated antirabbit Ig (Amersham Biosciences) diluted at 1:2000. After stripping off the immune complexes, the same membrane was probed for detection of hsp90, using monoclonal mouse anti-hsp90 IgG antibodies 3B6 (1:500), followed by horseradish peroxidase-conjugated antimouse Ig diluted at 1:1000. In parallel, 20 µg of whole cell extracts were independently used for immunodetection of wild-type and mutant GR or hsp90. Antibody-protein complexes were visualized using the enhanced chemiluminescence method according to the manufacturer’s protocol (Amersham Biosciences).

Visualization of GFP Fusion Proteins
For analysis of subcellular localization of the human GR and its mutants, we transiently expressed GFP-tagged receptors in COS7 cells and assays were performed as described previously (26). Briefly, after 6 h of transient transfection of the expression plasmids for GFP-fusion proteins, the medium was replaced with phenol red-free DMEM supplemented with 2% dextran-coated charcoal-treated fetal calf serum, and the cells were cultured at 37 C for at least 24 h. After various treatments, cells were examined using an IX70 microscope (Olympus, Tokyo, Japan) enclosed by an incubator and equipped with a heating-stage and a fluorescein isothiocyanate filter set. Digital images were randomly taken in eight views and analyzed on FLUOVIEW FV 500 systems (Olympus).

Limited Proteolysis Assay
The expression plasmids for the GR and its mutants, which contain the coding sequences under control of the T7 promoter, were transcribed and translated with the TNTT7-coupled reticulocyte lysate system (Promega Corp., Madison, WI) in the presence of [35S]Met (1000 Ci/mmol, Amersham Biosciences) according to the manufacturer’s instruction. Three microliters of [35S]Met-labeled translation mixtures including in vitro-translated GR were incubated for 30 min at 20 C with 1 µl of vehicle (0.4% ethanol) or 10 µM of DEX or CVZ. Limited proteolysis was performed by the addition of 1 µl of trypsin solution to the translation mixtures (final trypsin concentrations were 5–100 µg/ml). Digestion was conducted for 10 min at 20 C and stopped by cooling in ice, followed by the addition of 5 µl of sodium dodecyl sulfate (SDS) sample buffer and boiling for 5 min. The proteolysis products were separated on a 1.5-mm thick 12% SDS-polyacrylamide gels. After electrophoresis, the gels were vacuum-dried for 60 min at 80 C and autoradiographed.

Transfection and Reporter Gene Assay
Cells were plated on 6-cm diameter culture dishes (Iwaki Glass, Chiba, Japan) to 30–50% confluence and cell culture medium was replaced with Opti-MEM lacking phenol red (Invitrogen, Carlsbad, CA) before transfection. Plasmid cocktail was mixed with TransIT-LT1 transfection reagent (Panvera Corp., Madison, WI) and added to the culture. Total amount of the plasmids was kept constant by adding an irrelevant plasmid (pGEM3Z was used unless otherwise specified). After 6 h of incubation, the medium was replaced with fresh DMEM supplemented with 2% dextran-coated charcoal-treated fetal calf serum, and the cells were cultured in the presence or absence of various ligands for 24 h at 37 C. Luciferase enzyme activity was determined using a luminometer (Promega) essentially as described (26).

Indirect Immunofluorescence Assay
For assessment of subcellular localization of chimeric GR proteins, indirect immunofluorescence assay was performed as described previously (26). After transfection of expression plasmids for various GR mutants into COS7 cells, the cells were grown on eight-chambered sterile glass slides (Nippon Becton Dickinson, Tokyo, Japan) for 24 h and were treated without or with l µM of DEX or CVZ for 2 h. The cells were fixed in ice-cold acetone for 2 min and air-dried. After fixation, the cells were washed with PBS and incubated with anti-GR polyclonal rabbit antibody at a dilution of 1:100 in PBS containing 0.1% Triton X-100 for 1 h at 37 C. Then, the cells were washed three times with PBS and incubated with fluorescein isothiocyanate-conjugated antirabbit IgG (Santa Cruz Biotechnology) at a dilution of 1:200 in PBS containing 0.1% Triton X-100 for 1 h at 37 C. The cells were finally washed three times with PBS and mounted with GEL/MOUNT (Biomeda Corp., Foster City, CA) for examination on a confocal laser-scanning microscope IX70. Digital images were randomly taken in eight views and analyzed on FLUOVIEW FV 500 systems.

Quantitative Analysis of Chimeric Proteins
For determination of expression levels of GFP-, GAL4-, and polyhistidine-tagged proteins, we transiently expressed each chimeric protein in COS7 cells and the cells were cultured in the presence or absence of ligands for 0, 12, or 24 h. After various treatments, whole cell extracts were prepared and 10 µg of proteins ware separated by SDS-PAGE and transferred to nitrocellulose membranes. Subsequently, Western immunoblot analysis was performed with anti-GFP, -GAL4 (DBD), and -polyhistidine antibodies followed by appropriate secondary horseradish peroxidase-conjugated antibodies. Antibody-protein complexes were visualized using the enhanced chemiluminescence method. Expression levels of each chimeric protein were quantified by scanning the blot and using image analysis software from the National Institutes of Health (NIH Image 1.62).


    ACKNOWLEDGMENTS
 
We thank Drs. H. Ogawa, P. Chambon, R. M. Evans, G. L. Hager, and K. Umesono for plasmids and the members of Morimoto laboratory for fruitful discussions and help.


    FOOTNOTES
 
This work was supported in part by the grants from the Ministry of Education, Science, Technology, Sports, and Culture, the Ministry of Health, Labour, and Welfare, the Takeda Science Foundation, the Uehara Memorial Foundation, the Vehicle Racing Commemorative Foundation, and Japan Society for the Promotion of Science.

First Published Online January 27, 2005

Abbreviations: AF, Activation function; AR, androgen receptor; CVZ, cortivazol; DBD, DNA binding domain; DEX, dexamethasone; GFP, green fluorescent protein; GR, glucocorticoid receptor; GRE, glucocorticoid response element; hsp90, heat shock protein 90; LBD, ligand binding domain; MR, mineralocorticoid receptor; NF-{kappa}B, nuclear factor-{kappa} B; NLS, nuclear localization signal; PR, progesterone receptor; SDS, sodium dodecyl sulfate; TIF, transcriptional intermediary factor.

Received for publication June 30, 2004. Accepted for publication January 20, 2005.


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