Molecular Origins for the Dominant Negative Function of Human Glucocorticoid Receptor Beta

This study molecularly elucidates the basis for the dominantnegative mechanism of the glucocorticoid receptor (GR) isoformhGRß, whose overexpression is associated with humanglucocorticoid resistance. Using a series of truncated hGR ${alpha}$ mutantsand sequential mutagenesis to generate a series of hGR ${alpha}$ /ßhybrids, we find that the absence of helix 12 is neither necessarynor sufficient for the GR dominant negative phenotype. Moreover,we have localized the dominant negative activity of hGRßto two residues and found that nuclear localization, in additionto heterodimerization, is a critical feature of the dominantnegative activity. Molecular modeling of wild-type and mutanthGR ${alpha}$ and hGRß provides structural insight and a potentialphysical explanation for the lack of hormone binding and thedominant negative actions of hGRß.

INTRODUCTION

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Introduction

Glucocorticoids are among the most widely used classes of drugsin the world. The anti-inflammatory effects of glucocorticoidsare routinely exploited in treatment of many pathological conditionsranging from asthma, rheumatoid arthritis, ulcerative colitis,and common eczema. Likewise, the immunosuppressive benefitsof glucocorticoids afford them a role in most chemotherapeuticregimes as well as for management of autoimmunity. However,prolonged glucocorticoid treatment, as well as rare geneticdispositions, can result in glucocorticoid resistance (7). Anincreasing number of studies in the last several years haveimplicated alternative splicing of the glucocorticoid receptor(GR) gene and subsequent expression of the hGRß proteinisoform as a contributing factor to glucocorticoid resistancein a variety of pathological conditions (17, 21, 28, 38, 39,41). Since hGRß does not undergo ligand-induced downregulation and has an increased half-life, the expression ofhGRß becomes more significant (31). Expression ofhGRß is also enhanced by proinflammatory cytokines,such as tumor necrosis factor alpha and interleukin 1; however,a precise physiological role for hGRß remains elusive(43).

The GR, a member of the nuclear hormone receptor superfamilyof ligand-activated transcription factors, participates in numeroussignaling pathways leading to altered gene expression in targetcells and tissues and is essential for life (8). The GR canmodulate gene expression either positively or negatively bydirectly binding as a homodimer to glucocorticoid response elements(GRE) located in the promoter regions of target genes. Alternatively,the ligand-activated GR is known to repress or antagonize othernuclear factors involved in regulating gene expression, suchas NF- ${kappa}$ B and AP-1, through direct protein interactions (29).Both of these functions of GR signaling and transactivationand transrepression, appear to involve distinct and separableregions of the receptor and are major components of the physiologicalresponse to both natural and synthetic glucocorticoids.

In humans, alternative splicing of the ninth and final GR exongives rise to hGR ${alpha}$ and hGRß proteins divergent at onlythe extreme carboxy termini (11, 20). Although the two proteinsare 94% identical, the hGRß isoform fails to bindhormone or activate gene expression and functions as a dominantnegative inhibitor of hGR ${alpha}$ (3, 31). In addition, hGRßdoes not undergo ligand-dependent down regulation and consequentlyhas a half-life longer than that of hGR ${alpha}$ (43). As observed amongall nuclear receptor superfamily members, the GR carboxy-terminalregion encodes the ligand-binding domain (LBD). Extensive structuralanalysis of numerous nuclear receptor LBDs has revealed a commonfold and structural mechanism for hormone action (40, 42). Essentiallya 12-helix bundle, nuclear receptor LBDs contain a transactivationfunction (AF-2) created in large part by ligand-induced conformationalchanges involving the 12th and final helix (helix 12 [H12])of the domain. The H12/AF-2 core region is absent in hGRßand is replaced by a unique carboxy-terminal tail distinct fromthat of hGR ${alpha}$ . It is unclear if the absence of H12 or the presenceof unique carboxy-terminal residues gives rise to the dominantnegative phenotype of hGRß.

Our interest lies in understanding the molecular mechanism ofthe hGRß dominant negative activity and its potentialrole in human pathology. Previous studies have proposed thata likely mechanism for the dominant negative activity of hGRßis the formation of inactive heterodimers with hGR ${alpha}$ ; however,the precise mechanism and structural basis is unknown. The purposeof the present study was to functionally discriminate betweenthe absence of H12 and the presence of the unique carboxy-terminalend of hGRß and to determine which residues, if any,were critical for the dominant negative effect. Using carboxy-terminalhGR ${alpha}$ truncation mutants and site-directed mutagenesis, we havemapped the dominant negative activity to two amino acids withinthe unique hGRß region. Molecular modeling and secondarystructure alignments provide further insight and suggest a mechanisticmodel for the dominant negative action of hGRß.

MATERIALS AND METHODS

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Materials and Methods

Reagents and plasmids. Dexamethasone (Dex) was purchased from Steraloids (Wilton, N.H.).trans-³⁵S label (1,108 Ci/mmol) was purchased from ICN Radiochemicals(Irvine Calif.). [¹⁴C]Chloramphenicol (40 to 60 mCi/mmol) wasobtained from NEN Life Science Products. Acetyl-coenzyme A (CoA)and protease inhibitors were purchased from Roche MolecularBiochemicals (Indianapolis, Ind.). Oligonucleotide primers formutagenesis and PCR were synthesized by Oligo's Etc. (Bethel,Maine). For all mutagenesis, the QuickChange site-directed mutagenesiskit from Stratagene was utilized. The peroxidase-labeled anti-rabbitimmunoglobulin G secondary antibody and the enhanced chemiluminescencedetection kit (ECL) were purchased from Amersham Pharmacia Biotech.The TNT coupled reticulocyte lysate translation system was purchasedfrom Promega (Madison, Wis.). Reagents for immunocytochemistrywere purchased from BioGenex (San Ramon, Calif.).

The use and construction of the GR expression vectors pCMVhGR ${alpha}$ ,pCMVhGRß, pCMVhGR ${alpha}$ (1-727), pT7/T3-hGR ${alpha}$ , and pT7/T3-hGRß,were previously described (reference 30 and references therein).Note that pCMVhGR ${alpha}$ (1-727) was originally named pCMVhGR728T. Forsynthesis of pCMVhGR ${alpha}$ (1-742) and pCMVhGR ${alpha}$ (1-706), stop codons(TAG) were introduced at positions 743 and 707, respectively,of pCMVhGR ${alpha}$ . Analogous mutagenesis of pT7/T3 was carried outto create the pT7/T3 hGR ${alpha}$ truncation mutants (1-706, 1-727, and1-742) used for in vitro synthesis with [³⁵S]methionine. Generationof the hGR ${alpha}$ /ß hybrids was carried out by sequentialmutagenesis of pCMVhGR ${alpha}$ , substituting the corresponding residuesof hGRß(728-742) for hGR ${alpha}$ . All mutants were verifiedby DNA sequencing at the National Institutes of EnvironmentalHealth Sciences core sequencing facility. Other plasmids usedin these studies, pGMCS (mouse mammary tumor virus-chloramphenicolacetyltransferase [MMTV-CAT]), the empty pCMV5 vector, and theglutathione S-transferase (GST) fusion protein vectors, pGEX4Tand pGEX4T-hGR ${alpha}$ (523-777), have been previously described (30).

Cell culture, transfections, and CAT assays. COS-1 cells were maintained in Dulbecco's minimum essentialmedium with high glucose containing 2 mM glutamine and a 10%(vol/vol) mixture of heat-inactivated fetal calf-calf serum(1:1). For transactivation assays, cells were incubated for1 to 2 days in medium containing dextran-coated charcoal-strippedserum to remove endogenous steroids. All cell culture mediacontained 100 IU of penicillin/ml and 100 mg of streptomycin/ml.Cell cultures were maintained in a 5%-CO₂ humidified incubatorat 37°C and passaged every 3 to 4 days. The COS-1 cellswere transfected with plasmid DNA using the DMRIE-C reagent(Gibco-BRL) or Effectene transfection reagent (Qiagen).

For CAT assays, COS-1 cells were transfected with pGMCS (MMTV-CAT)and hGR ${alpha}$ /hGRß mutants and treated as described above.Cells were harvested in phosphate-buffered saline (PBS), andcell pellets were resuspended in 0.25 M Tris-HCl, pH 8, and5 mM EDTA. The cells were sonicated for 30 s and then pelletedat 20,800 x g at 4°C for 5 min. The supernatant was removedand heat inactivated for 10 min at 68°C. Protein concentrationswere determined using a Bradford protein assay (Bio-Rad). Tenmicrograms of cell extract was assayed for CAT activity andquantitated as previously described (1, 30).

For luciferase assays, cells were transfected with pHHLuc (MMTV-Luc)and hGR plasmids as described above. Cells were harvested in1x cell lysis buffer for reporter assays following the manufacturer'sinstructions (Roche Applied Science). Duplicate samples of 50µl were assayed using a MLX-3 luminometer (Dynex Technologies),and numbers of relative light units were adjusted for proteinconcentration.

GST pull-down assays. GST pull-down assays were carried out essentially as describedpreviously (22). Briefly, GST or GST-hGR ${alpha}$ (523-777) fusion proteinswere induced in Escherichia coli DH5 ${alpha}$ by addition of 0.1 mM isopropyl-ß-D-thiogalactopyranosideto log-phase cells. Following a 4 h incubation, the cells wereharvested and lysed by sonication in a solution containing 10mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl, 10% glycerol,0.2-mg/ml lysozyme, 75 µM PeFa block (Boehringer Mannheim),and 0.2-µg/ml leupeptin. Following centrifugation to removeinsoluble protein, the soluble protein extract was incubatedovernight with PBS-washed glutathione agarose (Amersham Pharmacia)in the presence of 1% Triton X-100 and 1 mM dithiothreitol.Following extensive washing, the agarose with the immobilizedfusion proteins was then incubated with full-length [³⁵S]methionine-labeledhGR ${alpha}$ , hGRß, or hGR ${alpha}$ truncation mutants overnight at4°C with constant rotation in a binding buffer containingTris-EDTA (pH 8.0) with 100 mM NaCl, 10% glycerol, 1 mM dithiothreitol,0.01% NP-40, and the protease inhibitors PeFa block and leupeptin.After extensive washing of the agarose beads with the above-describedbuffer, bound proteins were resolved on sodium dodecyl sulfate-polyacrylamidegel electrophoresis and visualized by autoradiography. Typically,more than 25% of total bacterial expressed hGR(523-777) wasrecovered in the soluble fraction; however, only 5 to 10% ofthis fraction bound the glutathione-agarose under the conditionsdescribed.

Western blotting and immunocytochemistry. For Western blotting, cell extracts prepared for CAT assayswere separated on precast 8% Tris-glycine gels (Novex, San Diego,Calif.) and transferred to nitrocellulose membranes (0.2 µm).The membranes were washed in Tris-buffered saline with 0.1%Tween 20 (TBST) and blocked in TBST containing 10% nonfat milkfor a minimum of 2 h at room temperature. Blots were next incubatedin TBST supplemented with affinity-purified primary anti-GRantibody, Ab57 (1:1,000) overnight at 4°C. Following extensiverinsing and washing in TBST (3x, 10 to 15 min), the blots wereprobed with peroxidase-conjugated goat anti-rabbit secondaryantibody (1:10,000) for 2 h at room temperature. Bands werevisualized using ECL reagents (Amersham).

Immunocytochemistry was carried out essentially as previouslydescribed (31). The day following transfection, cells were spliton two-chamber glass slides. Approximately 48 h posttransfection,the cells were treated with 100 nM Dex or vehicle for 2 h. Thecells were fixed in 2% paraformaldehyde, washed in PBS, andpermeabilized with 0.2% Triton X-100. Cells were again washedin PBS, treated with 2% normal goat serum, washed in PBS, andincubated overnight with a 1:8,000 dilution of Ab57. The followingday, the slides were washed and incubated with a secondary antibodyat 1:7,500 (Texas Red conjugated antibody from Molecular Probes)for 20 h at 4°C. After washing, fluorescence was measuredusing a Zeiss LSM 510 laser scanning confocal microscope witha Plan-Apochromat 100x oil immersion objective and the recommendedexcitation and emission wavelengths for Texas Red (MolecularProbes). Digital images were optimized in Photoshop to revealboth intensity and distribution of immunoreactive GR. The cellsstained with peroxidase were processed as described above usingthe Super sensitive detection kit for mouse and rat (Biogenex)followed by a 6-min reaction with diaminobenzidine-peroxidasesolution. Cells were counterstained with hematoxylin.

Structure predictions and molecular modeling. The following programs were used for secondary structure prediction:DPM (10); DSC (26); GOR1 (13); GOR2 (15); HNN (hierarchal neuralnetwork method) and MLRC (16); PHD (35); Predator (12); SOPM(self-optimized prediction method) (14); PSIpred (position-specificiterated prediction) (25); and Sspro (2). The majority of thesealgorithms can be found and run on the Internet or are availableat no charge from the authors.

LBD models were developed for hGR ${alpha}$ , hGRß, the hGR ${alpha}$ (1-742)truncation mutant, and the hGR ${alpha}$ (ß6-7) double mutantusing the Molecular Simulations Homology module and associatedprograms. Models were based on the coordinates for the solved,ligand-bound progesterone receptor LBD structure (Protein DataBank entry 1A28). The alignment and models developed were checkedto ensure that they were in agreement with a variety of secondarystructure predictions (19; Wisconsin Package version 10.3 [Accelrys,Inc., San Diego, Calif.]). The models developed were also checkedusing the programs PROCHECK and WhatIf to insure that psi andphi angles occurred in the expected regions of the Ramachandranmap and that there were no steric bumps or overlaps in the structure.Energy minimization and dynamics were performed as part of thehomology modeling using the Discover CVFF force field in theMolecular Simulations software. For comparison of our modelwith the solved structure (5), root mean square deviation wascalculated using the program MVP (27).

RESULTS

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Results

The domain organization of the full-length wild type (wt) hGR ${alpha}$ is shown in Fig. 1A. Nuclear receptor LBDs exhibit a highlyconserved three-dimensional architecture (6, 42, 46). The recentsolution of the GR LBD further demonstrates the structural homologyamong nuclear receptors (5). However, the GR LBD coordinatesare not yet publicly available. In order to understand the potentialstructural differences between hGR ${alpha}$ and hGRß, we havemodeled the hGR ${alpha}$ LBD using the coordinates from the progesteronereceptor (hPR) (Protein Data Bank entry 1A28), the most closelyrelated superfamily member whose LBD crystal structure has beensolved (Fig. 1B) (45). The tertiary structure of hGR ${alpha}$ , (LBD,residues 525 to 777) was predicted using the Molecular SimulationsHomology module and contains the structure of Dex bound to thereceptor in place of progesterone. Our model was compared withthe solved structure of hGR ${alpha}$ , and the overall root mean squaredeviation on all backbone atoms between the reported experimentalstructure (residues 531 to 776) and the model developed is 1.78Å (M. Lambert, personal communication).

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FIG. 1. (A) Linear representation of the hGR ${alpha}$ , indicating the boundaries of the DBD and LBD. (B) Homology model of the hGR ${alpha}$ LBD (residues 525 to 777) based on the crystal structure coordinates of the progesterone receptor (PR) LBD (45). The three amino acids labeled, S706, E727, and D742, represent the boundaries of the truncation mutants described below. The residues, which are unique for hGRß (amino acids 728 through 742), are shown in yellow, while the entire H11-H12 loop and H12 are shown in green (residues 743 to 777). (C) Illustration of hGR ${alpha}$ C-terminal deletion constructs used in the experiments described. hGR ${alpha}$ (1-742) represents an hGR ${alpha}$ truncation that is the same length as hGRß. hGR ${alpha}$ (1-727), previously described by Oakley et al., is truncated at the hGR ${alpha}$ and hGRß divergence point. Finally, in the hGR ${alpha}$ (1-706) truncation, the entirety of H10/11 and H12 is removed.

To functionally distinguish between the absence of H12 and therole of the 15 unique residues at the carboxy terminus of hGRß,we created several hGR ${alpha}$ truncation mutants (Fig. 1C). It is noteworthythat the boundaries of the hGRß unique sequence lieat structurally sensible points: the end of H10 (that is theboundary of two distinct helices in several nuclear receptors)and the end of H11. The hGR ${alpha}$ (1-742) construct represents thehGR ${alpha}$ homologue to hGRß in that both proteins are thesame length but are composed of 15 distinct amino acids at thecarboxy terminus. Our second hGR truncation, hGR ${alpha}$ (1-727), isuseful for understanding the role of residues 728 to 742 inhGR function (30). Finally, we created a third strategic truncation,hGR ${alpha}$ (1-706), which lacks the entire putative H10/11 region andshould be severely compromised in both structure and function.

Functional analysis of hGR ${alpha}$ , hGRß, and truncation mutants. The truncation mutants described in Fig. 1 were evaluated fortransactivation and dominant negative activity. Of the fivehGR constructs shown in Fig. 1C, only full-length hGR ${alpha}$ is transcriptionallyactive from the glucocorticoid-responsive promoter MMTV-CAT(Fig. 2A). This result is consistent with reports in which lesssevere carboxy-terminal truncations of the mouse GR result incomplete loss of binding and transcriptional activity (48).Western blots shown below the graph indicate that the lack oftransactivation is not a result of different protein expressionlevels. The three hGR ${alpha}$ truncations were next compared to hGRßfor dominant negative activity (Fig. 2B). Only hGR ${alpha}$ (1-742) exhibitedan inhibitory effect on full-length hGR ${alpha}$ activity, and that wasonly partially as strong as that observed for hGRß[40% inhibition for hGR ${alpha}$ (1-742) compared to 62% for hGRß].Thus, the lack of H12 alone cannot entirely account for thedominant negative activity of hGRß. Western blotsshown beneath the graph indicate that protein expression levelsare equivalent and do not account for the differences in dominantnegative activity observed. The Western blots also show thatonly hGR ${alpha}$ undergoes ligand-dependent down regulation; the truncationmutants and hGRß do not.

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FIG. 2. A) Transactivation of hGR ${alpha}$ , hGRß, and hGR ${alpha}$ truncation mutants. The hGR ${alpha}$ proteins described in Fig. 1C were analyzed for transactivation potential following transient transfection in GR-deficient COS-1 cells. Cells were cotransfected with the indicated GR expression vector and a glucocorticoid-responsive MMTV-CAT reporter gene (pGMCS). Hormone response was measured as the fold induction of CAT activity in response to 100 nM Dex treatment (DEX, hatched bars) over that of untreated cells (CON, solid bars). Data are an average from two experiments with the indicated standard error of the mean. Protein expression levels, determined by Western blotting for each condition, are shown beneath the graph. (B) Dominant negative activities of hGRß and the three-hGR ${alpha}$ truncation mutants. Dominant negative activity is measured as the decrease in hormone response (fold induction) upon coexpression of hGR ${alpha}$ with 10-fold more of the empty vector (pCMV5 [CMV]), hGRß, or the other hGR ${alpha}$ truncation mutants. The percentage of transactivation is shown in parentheses above each bar, where hGR ${alpha}$ plus the empty pCMV5 vector is 100%. The relative amounts of transfected expression vectors are indicated below the graph. Transfection and CAT assays were carried out as for panel A. Data shown are an average for four to seven experiments with the indicated standard error of the mean.

The formation of inactive heterodimers is the likely mechanismfor dominant negative activity (30). A possible explanationfor the observed difference between hGRß and hGR ${alpha}$ (1-742)and the other truncation mutants is an altered dimerizationsurface. To ascertain how the hGR ${alpha}$ LBD interacts with the otherGR constructs, GST fusion protein pull-down assays were developed.The entire molecular surface involved in dimerization of theGR is not completely understood, although regions within boththe DNA binding domain (DBD) and the LBD are involved (5, 36).Since all of the constructs used in Fig. 2 contain the DBD,bacterially expressed hGR ${alpha}$ LBD (residues 525 to 777) was preparedas a GST fusion protein in order to assess the contributionof only the LBD in dimerization. Glutathione agarose-immobilizedhGR ${alpha}$ LBD was incubated with ³⁵S-labeled, in vitro-transcribedand -translated hGR ${alpha}$ , hGRß, and hGR ${alpha}$ truncations. Theradiolabeled hGR ${alpha}$ , hGRß, and hGR ${alpha}$ (1-742) interact equivalentlywith the hGR ${alpha}$ LBD in a ligand-independent manner, whereas thehGR ${alpha}$ (1-727) truncation exhibits weak interaction while the hGR ${alpha}$ (1-706)interaction approaches background levels (Fig. 3A). None ofthe labeled proteins interacts with GST alone (data not shown).A representative experiment is shown indicating the bound andinput protein for each construct (Fig. 3B). Since hGR ${alpha}$ (1-742)and hGRß heterodimerize with equal affinity to hGR ${alpha}$ LBD, this suggests that the weaker dominant negative activityof hGR ${alpha}$ (1-742) (compared to that of hGRß) is not aresult of a weaker dimerization interface in this region ofthe LBD.

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FIG. 3. In vitro interaction of hGR ${alpha}$ LBD with hGR ${alpha}$ , hGRß, and hGR ${alpha}$ C-terminal truncation mutants. A) Summary of GST pull-down assays. The E. coli-expressed hGR ${alpha}$ LBD (amino acids 525 to 777) was immobilized on glutathione agarose and incubated with the indicated ³⁵S-labeled, in vitro-translated hGR proteins described in Fig. 1C. The percent of ³⁵S-protein bound by GST-hGR ${alpha}$ LBD averaged from three independent experiments was determined by densitometry of autoradiographs. (B) A representative autoradiograph of the data summarized in panel A. I indicates input protein, and B is the bound fraction. Positions of molecular size markers are shown to the left.

To further investigate the biochemical and functional differencesbetween hGR ${alpha}$ , hGRß, and the truncation mutants, wecompared their subcellular distribution by immunocytochemistry.Surprisingly, unlike the constitutively nuclear hGRß,the hGR ${alpha}$ (1-742) truncation remains predominantly cytosolic inthe absence or presence of Dex (Fig. 4). Interestingly, thehGR ${alpha}$ (1-727) truncation is nuclearly localized, similar to thecase for hGRß. The hGR ${alpha}$ (1-706) truncation, which ismissing the entire H10-H12 region and putative dimerizationinterface, exhibits diffuse cellular staining relative to theother proteins studied. Only hGR ${alpha}$ changes cellular distributionin response to the agonist Dex, which is consistent with ligandbinding and transactivation data. It thus appears that bothsubcellular localization and physical interaction with hGR ${alpha}$ areimportant prerequisites for hGRß imparting a dominantnegative effect. Although the truncation hGR ${alpha}$ (1-727) is mostlynuclear, as is hGRß, its inability to interact withhGR ${alpha}$ likely explains its lack of dominant negative activity.

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FIG. 4. Immunocytochemical analysis of hGRs. The subcellular distribution of hGR ${alpha}$ , hGRß, and the indicated hGR ${alpha}$ truncation mutants was measured in the absence (CON) and presence (DEX) of Dex. A representative image for each condition is shown. The data were quantified by ranking cell staining according to their predominantly cytosolic (C > N), predominantly nuclear (N > C), or equivalent (C = N) distribution. The numbers represent the percentages of cells counted which fell into the indicated rank. Approximately 150 cells were counted from two or three individual experiments.

Generation of hGR ${alpha}$ /ß hybrids. We next asked whether the dominant negative activity of hGRßcould be attributed to any particular amino acid or was dependenton the entire 15-residue sequence of hGRß. To addressthis, sequential cumulative mutagenesis of hGR ${alpha}$ residues 728to 742 to the corresponding amino acids in hGRß wasperformed. These ${alpha}$ /ß hybrids were first tested forhormone-induced transactivation. No significant changes in transactivationwere observed when each of the first three unique residues (728,730, and 731) of hGRß were swapped into hGR ${alpha}$ . However,the next amino acid replacement, leucine (L) 733 to lysine (K),totally abolished ligand-dependent transactivation. This lossof activity was maintained for each successive mutation in the ${alpha}$ /ß hybrids (Fig. 5, top panel). This abrupt all-or-nothingeffect on transactivation prompted us to generate single anddouble point mutations surrounding residues 733 and 734, replacinghGR ${alpha}$ residues with the corresponding beta sequence. Althoughdiminished transactivation was observed with several mutants,only one double-mutant hybrid, hGR ${alpha}$ (ß6-7), in whichleucine 733 and asparagine (N) 734 were replaced with lysineand proline (P), as in hGRß, was completely inactive(Fig. 5, bottom panel).

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FIG. 5. Transactivation and dominant negative activity of hGR ${alpha}$ /ß hybrids. (A) The amino acids 728 to 742 of hGR ${alpha}$ were replaced with the corresponding amino acids of hGRß in a sequential fashion. The residues from 728 through 742 were numbered 1 to 15, shown in the center. Note that three residues, Val729 (position 2), Leu732 (position 5), and Leu741 (position 14), are conserved in both hGR ${alpha}$ and hGRß (_*). Beginning with the mutation of Val728 to Asp, hGR ${alpha}$ (ß1), each additional mutant included the one before it, until a complete hGR ${alpha}$ /ß hybrid was generated. - indicates that no change from wt hGR ${alpha}$ was made. Only the first 12 sequential mutants are shown. Dex-induced transactivation was measured as for Fig. 2A with the data expressed as percent conversion of [¹⁴C]chloramphenicol to the acetylated form. The top panel shows the sequential mutants, where each residue in hGR ${alpha}$ was changed with a corresponding hGRß residue in succession. The bottom panel shows data from single and double point mutants described in the text. For each data set the wt hGR ${alpha}$ is shown as the top sequence and the hGRß sequence is on the bottom. Note that each hybrid contains the H12 carboxy-terminal extension ( $->$ ), except hGRß, which terminates following Ile742. The shaded region through positions 6 and 7 (amino acids 733 and 734) indicates the two amino acid mutations sufficient to completely block transactivation. As in Fig. 2, Western blots indicated similar expression levels for each receptor tested (data not shown). (B) The hybrid receptors from panel A that were completely deficient in Dex-induced transactivation were tested for dominant negative activity following the experimental protocol described for Fig. 2A. The top two bars, hGR ${alpha}$ (+ pCMV5) and hGR ${alpha}$ + hGRß, are the controls. The dashed line indicates the level of dominant negative activity observed with hGRß. All receptors tested for dominant negative activity were transfected at 10-fold-higher levels relative to levels for hGR ${alpha}$ . The middle bars are the sequential mutants that lack transactivation, and the double mutant, hGR ${alpha}$ (ß6-7), is shown at the bottom.

We next evaluated the dominant negative activity of the transactivation-deficientreceptors described in Fig. 5. The ${alpha}$ /ß hybrid, hGR ${alpha}$ (ß1-6),although completely defective in transactivation, is a poordominant negative inhibitor of hGR ${alpha}$ . However, the next sequentialhybrid, hGR ${alpha}$ (ß1-7), in which the first seven uniqueresidues in hGRß are swapped into the correspondinghGR ${alpha}$ sequence, is a potent dominant negative inhibitor. The remainingsequential hybrids, hGR ${alpha}$ (ß1-8) to hGR ${alpha}$ (ß1-12),are also dominant negative proteins. Interestingly, the doublemutant, hGR ${alpha}$ (ß6-7), is also a strong dominant negativeinhibitor of hGR ${alpha}$ function, illustrating that only two residuesfrom hGRß, Lys733 and Pro734, are necessary for dominantnegative activity (Fig. 5B).

The hGR ${alpha}$ (ß6-7) mutant. The hGR ${alpha}$ (ß6-7) mutant was further characterized andcompared with hGRß. Immunocytochemistry of the doublemutants hGR ${alpha}$ (ß6-7) and hGR ${alpha}$ (ß7-8) supportsthe hypothesis that constitutive nuclear localization is animportant feature in dominant negative activity. The dominantnegative hGR ${alpha}$ (ß6-7) is essentially nuclear in the absenceand presence of hormone. However, the hybrid hGR ${alpha}$ (ß7-8),which transactivates the reporter gene and is nuclearly localizedin response to Dex, similar to wt hGR ${alpha}$ , is found mainly in thecytosol in the absence of Dex (Fig. 6A). Interestingly, manyof the other hGR ${alpha}$ /ß hybrids exhibit a diffuse cellularGR staining pattern, suggesting that H12 may have an effecton cellular localization and that the complex functions associatedwith this region of the receptor are not freely dissociated(data not shown).

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FIG. 6. Analysis of hGR ${alpha}$ (ß6-7). (A) Nuclear localization of double mutants hGR ${alpha}$ (ß6-7) and hGR ${alpha}$ (ß7-8). The dominant negative double mutant hGR ${alpha}$ (ß6-7) was compared with the double mutant hGR ${alpha}$ (ß7-8), which appears normal in transactivation (see Fig. 5). Immunocytochemistry was carried out as for Fig. 4. (B) Repression of p65 by hGR ${alpha}$ but not hGRß or hGR ${alpha}$ (ß6-7). COS1 cells were transfected with the 3X MHC-luc reporter plus pCMV5, hGR ${alpha}$ , hGRß, or hGR ${alpha}$ (ß6-7) and treated with 100 nM Dex or left untreated. Overexpression of hGRß or hGR ${alpha}$ (ß6-7) does not repress transactivation of an NF- ${kappa}$ B reporter gene in the presence of hormone.

A potentially important distinction between hGR ${alpha}$ and hGRßis the repressive effect on NF- ${kappa}$ B-mediated transactivation. wthGR ${alpha}$ will transrepress p65-mediated reporter in response to hormone,whereas hGRß shows no hormone-dependent repression(30). When tested in this system, the hGR ${alpha}$ (ß6-7) mutantalso failed to further transrepress p65 reporter gene activityin the presence of hormone (Fig. 6B). These data further indicatethat these two residues are sufficient for creating GRß-likeactivity.

To test whether the biochemical properties of the hGR ${alpha}$ (ß6-7)mutant were dependent on the particular amino acid side chainsor simply a result of changing the existing residues in hGR ${alpha}$ ,additional mutagenesis of hGR ${alpha}$ residues 733 and 734 was carriedout. Both amino acids were mutated to alanine, thereby generatingthe hGR ${alpha}$ L733A/N734A double mutant. Functional analysis of thismutant showed transactivation ability similar to that of wthGR ${alpha}$ as well as hormone-induced down regulation and nuclear translocation(Fig. 7). Therefore, it appears that residues K733 and P734are necessary and sufficient for generation of a dominant negativeinhibitor of hGR ${alpha}$ that is functionally homologous to hGRßdespite the presence of H12 and the associated AF-2 function.

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FIG. 7. Transactivation and nuclear localization of an hGR ${alpha}$ alanine mutant. The amino acids at positions 6 and 7 of hGR ${alpha}$ previously described (L733 and N734) were mutated to alanine [hGR ${alpha}$ (6-7AA]. (A) Transactivation of MMTV-Luc and corresponding protein expression. (B) Nuclear localization of hGR ${alpha}$ , hGRß, and hGR ${alpha}$ (6-7AA) in the absence and presence of Dex. Localization of hGR ${alpha}$ (6-7AA) is the same as that of wt hGR ${alpha}$ .

Molecular modeling. Several strategies were employed to elucidate the structuralbasis for the dominant negative activity of hGRß andto evaluate the contribution of the side chains of residues733 and 734. Twelve unique secondary structure prediction algorithmswere employed in concert with homology modeling to develop three-dimensionalstructures and alignments for hGR ${alpha}$ , hGRß, hGR ${alpha}$ (ß6-7),and the truncation mutants. The secondary structure alignmentsare shown in Fig. 8A. Not surprisingly, these algorithms clearlypredict that the hGR ${alpha}$ (728-742) sequence, VVENLLNYCFQTFLD, formsan alpha helix in agreement with its homology to solved LBDstructures. However, the unique hGRß sequence, NVMWLKPESTSHTLI,is predicted to be largely disordered. The same secondary structurepredictions carried out with the truncation mutant hGR ${alpha}$ (1-742)indicate that the random coil predicted for the unique hGRßregion is not simply a result of truncation. Furthermore, predictionsfor the hGR ${alpha}$ (ß6-7) double mutant indicate a kink, break,or distortion at that point in H11 (Fig. 8A).

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FIG. 8. Structure predictions and molecular modeling. (A) A total of 12 secondary structure algorithms were run on the entire sequences of hGR ${alpha}$ , hGRß, hGR ${alpha}$ (ß6-7), and hGR ${alpha}$ (1-742). The single-letter amino acid abbreviation for each receptor from residue 701 until the end is shown at the top of each sequence prediction block. Below the wt hGR ${alpha}$ sequence is a comparison with the hPR. Below the hPR is the actual secondary structure found for the crystallographic solution of the hPR structure. The solid lines between hGR and hPR indicate a conserved amino acid. (B) Multiple sequence alignment of hGR ${alpha}$ , hGRß, and several other related receptors (hPR, hAR, hER ${alpha}$ , and RXR ${gamma}$ ). Alignment of hGRß was done using the program Clustal W. Symbols underneath alignment indicate conserved residues. C) Three-dimensional models of hGR ${alpha}$ , hGRß, and hGR ${alpha}$ (ß6-7). Modeling was done using Molecular Simulations software. The common helices of each model (residues 525 to 727) are shown in red, the nonhomologous region from amino acid 728 through 742 is colored yellow, and the H12 region (residues 743 to 777), present in hGR ${alpha}$ and hGR ${alpha}$ (ß6,7), is colored green. The hGR ${alpha}$ model is shown with ligand occupying the binding pocket. The images also show the side chain orientations of residues 733 and 734 in each molecule.

The solved crystal structure for the ligand-bound progesteronereceptor was used as the target structure for the developmentof a structural homology model of hGR ${alpha}$ and hGRß (45).The basis for the hGRß structural model is a multiplesequence alignment of several members of the nuclear receptorsuperfamily ligand binding domains using the Clustal W alignmentalgorithm (Fig. 8B) (19). An alignment was also performed usingthe GCG Pileup program (Wisconsin Package version 10.3). Publishedalignments of the nuclear receptor ligand binding domains (46)and the Pfam protein domain databases were consulted as well.As in Fig. 1B, Dex replaces progesterone in the ligand-bindingsite of the hGR ${alpha}$ model (45). The three-dimensional structuremodels of hGR ${alpha}$ , hGRß, and hGR ${alpha}$ (ß6-7) are shown(Fig. 8C). Whereas H10/11 in the truncated hGR ${alpha}$ is relativelyundisturbed, the unique hGRß sequence adopts a significantlydifferent structure with the unwinding of H11 beginning nearresidue 733. This gross unwinding likely reflects the constraintsapplied from the alignment in Fig. 8B. Nevertheless, these models,along with that of hGR ${alpha}$ (ß6-7), predict that the disruptionof H10/11 in hGRß is caused by replacing leucine andasparagine in hGR ${alpha}$ with lysine and proline, respectively, therebygenerating side chain interactions that are both electrostaticallyand sterically unfavorable.

DISCUSSION

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Discussion

The dominant negative activity of hGRß results fromseveral cooperating factors associated with the extreme carboxy-terminalends of hGR ${alpha}$ and hGRß. Hormone binding, transactivation(AF-2), dimerization, and surprisingly, nuclear localizationall depend upon sequences within the last 50 or so residuesof hGR ${alpha}$ . In hGRß, alternative splicing replaces thefinal 50 amino acids of hGR ${alpha}$ with 15 unique residues. It remainsunclear which of the properties listed above are affected bythis change and how they contribute to the dominant negativephenotype of hGRß. We have determined that hGRß'sunique sequence and the missing H12 both contribute to dominantnegative activity. First, the absence of H12 renders the receptorunable to bind ligand or transactivate reporter genes. Second,the 15 unique residues function to keep the receptor constitutivelynuclear localized. This feature, in addition to physical interaction,is critical for maximum dominant negative activity. Surprisingly,the dominant negative function of hGRß can be localizedto just two amino acids within the unique hGRß sequence.These data, along with secondary structure predictions and molecularmodeling, support an atomic-scale hypothesis describing howthe different secondary and tertiary structures of hGRßand hGR ${alpha}$ result in the dominant negative phenotype.

Other dominant negative nuclear receptor mechanisms. Many nuclear receptors have naturally occurring dominant negativeisoforms in which the mutant form blocks the activity of thewt protein when the two are coexpressed in the same cell. Dominantnegative forms of the thyroid hormone receptor and the peroxisomeproliferator activated receptor gamma are important factorsin disease pathology associated with these receptors (4, 9).Similar reports with ER dominant negative mutants (23, 37),as well as other nuclear receptors (24), suggest that H12 andthe associated AF-2 contribute to the dominant negative effectand that most dominant negative receptors are generated viaalterations in the length, sequence, conformation, or stabilityof the carboxy-terminal end of the LBD.

The hGRß dominant negative mechanism contains distinguishingfeatures in comparison to most other nuclear receptors. First,it appears that the GR ligand binding is more sensitive to perturbationsof H10/11 and H12 than other receptors, such as ER or RXR, wheredisruption of H12 does not prevent agonist binding (37, 48).Second, hGR ${alpha}$ is a cytosolic protein in the absence of hormone,while most nuclear receptors are constitutively nuclear. Althoughnone of the dominant negative GRs generated bind Dex, whetherdisruption of binding is necessary for dominant negative activityis unclear. For example, the hGR ${alpha}$ (ß1-6) receptor istranscriptionally inactive but lacks full dominant negativeactivity, suggesting that these two features are separable.

Subcellular localization, DNA binding, and dimerization. The classical view of GR action involves ligand binding-inducedconformational changes with concurrent release of cytoplasmicaccessory proteins and unmasking of a nuclear localization signallocated immediately carboxy-terminal to the DBD (32). However,the immunocytochemistry experiments presented here suggest arole of the carboxy-terminal end of the GR LBD in nuclear orcytoplasmic retention. That hGRß and hGR ${alpha}$ (1-727) areprimarily nuclear while hGR ${alpha}$ (1-742) is cytoplasmic suggests thatthe region from amino acid 728 to 742 is somehow involved incytoplasmic retention and/or nuclear export. Whether this isdirectly coded within this region of the receptor or relieson other parts of the receptor is not clear. However, Zelkoet al. recently reported that the homologous H10/11 region inthe nuclear receptor CAR is involved in nuclear localizationof that receptor (47). Two conserved regions within H10/11 ofhGR somewhat resemble the leucine-rich regions of well-definednuclear export signals found in human immunodeficiency virusRev and I ${kappa}$ B (18). Whether this region of the GR harbors a functionalsignaling or localization motif remains to be established.

One possible mechanism for dominant negative activity is theblocking of DNA binding sites by the transcriptionally silenthGRß. However, several lines of evidence do not supportthis mechanism. First, overexpression of hGRß doesnot significantly affect progesterone or androgen receptor transactivation(30). Since the recognition sites are identical for all threereceptors, the lack of an effect of hGRß suggestsa mechanism other than squelching of response elements. In addition,preliminary analysis of mutations in the DNA binding domainshows no effect on the ability of hGRß to functionas a dominant negative inhibitor of hGR ${alpha}$ (M. R. Yudt and J. A.Cidlowski, unpublished observations).

Our model for dominant negative action centers on the formationof inactive, or weakly active, heterodimers between hGR ${alpha}$ andhGRß. The recently published crystal structure ofthe hGR ${alpha}$ LBD suggests that the dimerization interface is distinctfrom other solved nuclear receptor LBD structures. Before thestructure of the hGR ${alpha}$ was solved, the interface of all nuclearreceptor LBD dimers involved H10 and/or H12 (6). However, thehGR ${alpha}$ LBD dimer interface involves H4 and beta strands 3 and 4,located on the opposite face of the folded domain from H10 toH12. This suggests that the gross structural changes in H11and H12 associated with hGRß or hGR ${alpha}$ (ß6-7)would not be expected to perturb the dimer interface found inhGR ${alpha}$ . The crystal structure also provides a potential explanationfor our observations that the hGR ${alpha}$ LBD can dimerize with thefull-length in vitro-translated receptor in a ligand-independentfashion, since the conformational changes believed to occurupon hormone binding are far away from the published dimer interface(5).

Molecular modeling and the structure of hGRß. Despite being the first receptor cloned (44) and its obviouspharmacological interest, the GR LBD structure has only recentlybeen determined, and the structural information is not yet publiclyaccessible (5). To date, well over 20 different nuclear receptorLBD structures have been published, including apo-, agonist-bound,antagonist-bound, and ternary complexes with coactivator orcorepressor peptides (for reviews, see reference 42). Nuclearreceptor LBDs conform to a canonical folding architecture (46).The model of the hGR ${alpha}$ presented here is similar to previouslypublished homology models of the GR LBD also generated fromthe structure of the progesterone receptor LBD (33, 34). Inaddition, comparison of our model with the solved structure(M. Lambert, personal communication) supports the claim thatthe hGR ${alpha}$ model presented here is a valid model and is sufficientlysimilar to the experimental structure to support our conclusions.

The purpose of our modeling experiments is to identify the structuralconsequences of alternative splicing in relation to the functionof hGR ${alpha}$ and hGRß and to better understand the molecularenvironment of residues 733 and 734 in particular. The resultsfrom 12 structure prediction algorithms strongly indicate anonhelical or random conformation of the 15 unique residuesin hGRß. The same predictions on hGR ${alpha}$ (1-742) show thatthe disorder of these residues is not merely a result of truncationand the absence of H12. In fact, secondary structure predictionsfor both full-length hGR ${alpha}$ and the truncated hGR ${alpha}$ (1-742) concurwith homologous solved structures, such as that of progesteronereceptor (45), which predict helicity up to residue 741 to 743.Inspection of side chain conformations in hGR ${alpha}$ and hGRßas well as in the hGR ${alpha}$ (ß6-7) double mutant supportsthe experimental evidence that disruption of H11 at or nearresidue 733 is a critical feature for the dominant negativeactivity of hGRß. In conclusion, the 15 unique residuesof hGRß, by keeping the receptor nuclearly localized,play an important role in dominant negative activity of thishGR isoform. We have shown that the absence of H12 itself isneither necessary nor sufficient for a full dominant negativephenotype.

ACKNOWLEDGMENTS

We thank Mill Lambert for comparing our model with the solvedhGR X-ray structure.

FOOTNOTES

* Corresponding author. Mailing address: Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, 111 Alexander Dr., Research Triangle Park, NC 27709. Phone (919) 541-1564. Fax (919) 541-1367. E-mail: cidlowski{at}niehs.nih.gov.

Present address: Wyeth Research, Philadelphia, PA 19101.

REFERENCES

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