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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-1893
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 4 1535-1543
Copyright © 2006 by The Endocrine Society

Functional Characterization of the Natural Human Glucocorticoid Receptor (hGR) Mutants hGR{alpha}R477H and hGR{alpha}G679S Associated with Generalized Glucocorticoid Resistance

Evangelia Charmandari, Tomoshige Kino, Takamasa Ichijo, Keith Zachman, Anton Alatsatianos and George P. Chrousos

Pediatric Endocrinology Section, Reproductive Biology and Medicine Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Evangelia Charmandari, M.D., Department of Pediatric Endocrinology, Great Ormond Street Hospital for Children, 9th Floor, Southwood Building, Great Ormond Street, London, WC1N 3JH, United Kingdom. E-mail: charmane{at}mail.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Glucocorticoid resistance is often a result of mutations in the human glucocorticoid receptor {alpha} (hGR{alpha}) gene, which impair one or more of hGR{alpha}’s functions. We investigated the molecular mechanisms through which two previously described mutant receptors, hGR{alpha}R477H and hGR{alpha}G679S, with amino acid substitutions in the DNA- and ligand-binding domains, respectively, affect glucocorticoid signal transduction.

Methods and Results: In transient transfection assays, hGR{alpha}R477H displayed no transcriptional activity, whereas hGR{alpha}G679S showed a 55% reduction in its ability to stimulate the transcription of the glucocorticoid-responsive mouse mammary tumor virus promoter in response to dexamethasone compared with the wild-type hGR{alpha}. Neither hGR{alpha}R477H nor hGR{alpha}G679S exerted a dominant negative effect upon the wild-type receptor. Dexamethasone binding assays showed that hGR{alpha}R477H preserved normal affinity for the ligand, whereas hGR{alpha}G679S displayed a 2-fold reduction compared with hGR{alpha}. Nuclear translocation studies confirmed predominantly cytoplasmic localization of the mutant receptors in the absence of ligand. Exposure to dexamethasone resulted in slower translocation of hGR{alpha}R477H (25 min) and hGR{alpha}G679S (30 min) into the nucleus than the wild-type hGR{alpha} (12 min). In chromatin immunoprecipitation assays in cells stably transfected with the mouse mammary tumor virus promoter, hGR{alpha}R477H did not bind to glucocorticoid-response elements, whereas hGR{alpha}G679S preserved its ability to bind to glucocorticoid-response elements. Finally, in glutathione-S-transferase pull-down assays, hGR{alpha}G679S interacted with the glucocorticoid receptor-interacting protein 1 coactivator in vitro only through its activation function (AF)-1, unlike the hGR{alpha}R477H and hGR{alpha}, which interacted with the glucocorticoid receptor-interacting protein 1 through both their AF-1 and AF-2.

Conclusions: The natural mutants hGR{alpha}R477H and hGR{alpha}G679S cause generalized glucocorticoid resistance by affecting different functions of the glucocorticoid receptor, which span the cascade of the hGR signaling system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOIDS REGULATE A VARIETY of biological processes and play a pivotal role in the maintenance of basal and stress-related homeostasis (1). At the cellular level, their actions are mediated by a 94-kDa intracellular receptor protein, the glucocorticoid receptor (GR), which belongs to the superfamily of steroid/thyroid/retinoic acid receptor proteins (2, 3) (Fig. 1Go, A and B). Alternative splicing of the human GR (hGR) gene in exon 9 generates two highly homologous receptor isoforms, termed {alpha} and ß. hGR{alpha} is ubiquitously expressed in almost all human tissues and cells and represents the classic hGR that functions as a ligand-dependent transcription factor. In the absence of ligand, hGR{alpha} resides mostly in the cytoplasm of cells as part of a large multiprotein complex (4). Upon ligand-induced activation, the receptor dissociates from this protein complex and translocates into the nucleus, where it binds as a homodimer to glucocorticoid-response elements (GREs) located in the promoter regions of target genes. hGR{alpha} regulates the expression of glucocorticoid-responsive genes positively or negatively, depending on the GRE sequence and promoter context (5, 6) (Fig. 1CGo). The receptor can also modulate gene expression as a monomer independently of GRE binding, by physically interacting with other transcription factors, such as activator protein-1 and nuclear factor-{kappa}B (7, 8, 9).


Figure 1
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  		FIG. 1. Schematic representation of the structure and function of the hGR{alpha}. A, Linear representation of the 777-amino-acid receptor showing its principal functional domains. B, Enlargement of part of the DBD showing the amino acid sequence (single-letter codes) of the two zinc fingers and the dimerization loop (in bold). The R to H substitution at amino acid position 477 is indicated in bold. In addition, the A to T mutation at position 458 that could produce a dimerization defective receptor is shown. C, Nucleocytoplasmic shuttling of hGR{alpha}. Upon binding to the ligand, the activated receptor dissociates from heat-shock proteins (HSPs) and translocates into the nucleus, where it homodimerizes and binds to GREs in the promoter region of target genes. TF, Transcription factor; TFRE, transcription factor response element. D, Schematic representation of the interaction of AF-1 and AF-2 of hGR{alpha} with coactivators. DRIP/TRAP, Vitamin D receptor-interacting protein/thyroid hormone receptor-associated protein; SWI/SNF, switching/sucrose non fermenting. E, Location of the known mutations of the hGR{alpha} gene (top) and protein (bottom).

 
Two regions of hGR{alpha} possess intrinsic transcriptional activation function (AF): AF-1, which is located at the amino-terminal domain and is glucocorticoid independent, and AF-2, which is located at the ligand-binding domain (LBD) and is glucocorticoid dependent (5, 6). To initiate transcription, hGR{alpha} uses its transcriptional activation domains as surfaces to recruit chromatin-remodeling factors and to interact with general transcription factors or adaptor proteins, termed coactivators or cofactors, which serve to link enhancer-bound transcription factors to general transcription factors (10, 11, 12, 13). Several families of nuclear receptor coactivators, including the p160 coactivators, such as the steroid receptor coactivator 1 and the GR-interacting protein 1 (GRIP1), have been described (10, 11, 12, 13, 14). The p160 coactivators play a critical role in the transcriptional activation of glucocorticoid-responsive genes. They interact directly with both the AF-1 of hGR{alpha} through their carboxyl-terminal domain and the AF-2 through multiple amphipathic LXXLL signature motifs located in their nuclear receptor-binding (NRB) domain (15). They also have histone acetyltransferase activity, which promotes chromatin decondensation, thus allowing the transcriptional initiation complex of the RNA polymerase II and its ancillary components to initiate and promote transcription (10, 12, 13) (Fig. 1DGo).

Glucocorticoid resistance is a rare, familial or sporadic condition characterized by generalized, partial, end-organ insensitivity to glucocorticoids (16, 17, 18, 19). The molecular basis of generalized glucocorticoid resistance has been ascribed to mutations in the hGR{alpha} gene, which impair one or more of the molecular mechanisms of GR action, thus altering tissue sensitivity to glucocorticoids. Inactivating mutations within the LBD and DNA-binding domain (DBD), and a 4-bp deletion at the 3' boundary of exon 6 of the hGR{alpha} gene have been described in five kindreds and four sporadic cases (20, 21, 22, 23, 24, 25, 26, 27, 28, 29) (Fig. 1EGo). Two recently described novel mutations of hGR{alpha} causing generalized glucocorticoid resistance, R477H and G679S, are located in the DBD and LBD of the receptor, respectively (24). Molecular studies demonstrated that the R477H mutation abolishes the transactivational activity of the receptor completely but preserves normal affinity for the ligand, whereas the G679S mutation results in decreased transcriptional activity and a 50% decline in the affinity for ligand compared with the wild-type hGR{alpha} (24). In this study, we extended the functional characterization of the above mutant receptors and systematically investigated the molecular mechanisms through which the R477H and G679S mutations of hGR{alpha} affect glucocorticoid signal transduction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids

The pRShGR{alpha} plasmid expresses hGR{alpha} under the control of the Rous sarcoma virus (RSV) promoter, and was a gift from Dr. R. M. Evans (Salk Institute, La Jolla, CA). The plasmids pRShGR{alpha}R477H and pRShGR{alpha}G679S were constructed by introducing the respective mutations into the pRShGR{alpha} plasmid using PCR-assisted site-directed mutagenesis (Stratagene, La Jolla, CA) (29). The plasmids pF25-hGR{alpha} and pBK/CMV-hGR{alpha}, which express, respectively, the green fluorescent protein (GFP)-fused hGR{alpha} and wild-type hGR{alpha} have been reported previously (25, 27, 29). The plasmids pF25-hGR{alpha}R477H and -hGR{alpha}G679S and pBK/CMV-hGR{alpha}R477H and -hGR{alpha}G679S were constructed by introducing the respective mutations into the pF25GFP-hGR{alpha} and pBK/CMV-hGR{alpha}, respectively, using PCR-assisted site-directed mutagenesis (29).

The plasmids pGEX4T3-GRIP1(1-1462), pGEX4T3-GRIP1(596-774) and pGEX4T3-GRIP1(740-1217), which express glutathione-S-transferase (GST)-fused full-length GRIP1, NRB fragment of GRIP1, and carboxyl-terminal fragment of GRIP1, respectively, were constructed as previously described (29, 30). The pRSV-erbA–1 plasmid was used as a negative control in parallel experiments. The pMMTV-luc plasmid, which expresses luciferase under the control of the glucocorticoid-inducible mouse mammary tumor virus (MMTV) promoter, was a gift from Dr. G. L. Hager (National Cancer Institue, National Institutes of Health, Bethesda, MD). The pSV40-ß-gal plasmid encodes the ß-galactosidase gene under the control of the simian virus 40 promoter (Promega, Madison, WI).

Cell cultures and transient transfection assays

CV-1 and COS-7 embryonic African green monkey kidney cells and HeLa human cervical uterine carcinoma cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics. HCT-116 human colon carcinoma cells stably integrated with the MMTV promoter were grown in McCoy’s 5A medium supplemented with 10% FBS, G418 (0.4 mg/ml), and antibiotics. Cells were incubated in a humidified atmosphere of 5% CO2 at 37 C and passaged every 3–4 d. Twenty-four hours before transfection, subconfluent cells were removed from their flasks and plated in appropriate plates, flasks, or dishes for additional transfection studies.

CV-1, COS-7, and HCT-116 cells were transfected using lipofectin (Life Technologies, Gaithersburg, MD) (31), whereas HeLa cells were transfected using FuGENE 6 (Roche Diagnostics Corp., Indianapolis, IN) according to the instructions of the manufacturer. The FuGENE 6 to transfected DNA ratio was 2.5:1.

Transactivation assays

CV-1 cells were seeded in six-well plates at a concentration of 1.5 x 105 cells per well. Twenty-four hours later, cells were cotransfected with pRShGR{alpha}, pRShGR{alpha}R477H, pRShGR{alpha}G679S, or a control plasmid (pRSV-erbA–1) (0.05 µg/well), pMMTV-luc (0.5 µg/well), and pSV40-ß-gal (0.1 µg/well). Forty-eight hours after transfection, dexamethasone (Sigma Chemical Co., St. Louis, MO) was added to the medium at concentrations ranging from 10–10 to 10–6 M.

For experiments designed to determine whether the mutant receptors exert a dominant negative effect upon the wild-type receptor, CV-1 cells were cotransfected with pMMTV-luc (0.5 µg/well), pSV40-ß-gal (0.1 µg/well), a constant amount of pRShGR{alpha} (0.05 µg/well), and five progressively increasing concentrations of pRShGR{alpha}R477H or pRShGR{alpha}G679S, so that the ratio between the wild-type and mutant receptors would range from 1:0 to 1:10 (1:0, 1:1, 1:3, 1:6, and 1:10). pRSV-erbA–1 was added in appropriate quantities to maintain a constant amount of DNA in each well. Twenty-four hours later, the transfection medium was replaced with supplemented DMEM. Forty-eight hours after transfection, dexamethasone or vehicle (100% ethanol) was added to the medium at a concentration of 10–6 M.

Luciferase and ß-galactosidase assays

Seventy-two hours after transfection, cells were washed with PBS twice and lysed at 4 C using a reporter lysis buffer (Promega). Luciferase and ß-galactosidase activity in the cell lysates was determined as previously described (22, 29, 33). All experiments were performed in triplicate and repeated at least three times.

Western blot analyses

CV-1 and COS-7 cells were seeded in 75-cm2 flasks at a concentration of 1 x 106 cells per flask and grown in supplemented DMEM. Subconfluent cells were transfected with pRShGR{alpha}, pRShGR{alpha}R477H, or pRShGR{alpha}G679S (15 µg/flask) using lipofectin (31). Twenty-four hours (CV-1) or 6 h (COS-7) after transfection, the transfection medium was replaced with supplemented DMEM. After additional incubation for 48 h, the cells were harvested, whole homogenates were produced, and Western blot analyses were performed as previously described (29).

Whole-cell dexamethasone binding assays

COS-7 cells were seeded in six-well plates (1.5 x 105 cells per well) and transfected with pRShGR{alpha}, pRShGR{alpha}R477H, or pRShGR{alpha}G679S (1.5 µg/well) using lipofectin (31). Six hours later, the transfection medium was replaced with supplemented DMEM. Confluent cells were incubated in plain DMEM with six different concentrations (1.56, 3.125, 6.25, 12.5, 25, and 50 nM) of [1,2,4,6,7-3H]dexamethasone (Amersham Pharmacia Biotech, Little Chalfont, UK) at 37 C in the presence or absence of a 500-fold molar excess of nonradioactive dexamethasone for 1 h. After incubation, cells were harvested and radioactivity was measured using a ß-counter (Beckman LS6000IC counter; Beckman Coulter Inc., Fullerton, CA) as previously described (29). Specific binding was calculated by subtracting nonspecific from total binding, and these data were analyzed using the Scatchard method. Binding capacity was expressed as femtomoles per 106 cells, and the apparent dissociation constant (Kd) was expressed in nanomolar concentrations. All experiments were repeated at least three times. Student’s t test was used to compare the mean apparent dissociation constant of the mutant receptors, hGR{alpha}R477H and hGR{alpha}G679S, and the wild-type hGR{alpha}.

Detection and localization of GFP-fused GRs

HeLa cells were plated on coated 35-mm-diameter dishes (1.5 x 105 cells per well) in supplemented DMEM. Twenty-four hours later, cells were transfected with GFP-fused hGR{alpha}-, GFP-fused hGR{alpha}R477H-, or GFP-fused hGR{alpha}G679S-expressing plasmids (2 µg/dish) using FuGENE 6. In additional experiments, and to determine the effect of the mutant receptors upon the nuclear translocation of the wild-type receptor, HeLa cells were transfected with equal amounts of GFP-fused hGR{alpha} (1 µg/dish) and pRShGR{alpha}R477H or pRShGR{alpha}G679S (1 µg/dish). Forty-eight hours after transfection, the medium was replaced by phenol red-free DMEM supplemented with 10% charcoal-treated FBS and antibiotics. Sixteen hours later, cells were exposed to dexamethasone (10–6 M), and fluorescence was detected sequentially by an inverted fluorescence microscope (Leica DM IRB, Wetzlar, Germany), as previously described (29, 34). All experiments were repeated at least three times, and a population of seven to 10 cells was examined in each experiment. Results are expressed as mean ± SEM.

Chromatin immunoprecipitation assays

HCT-116 human colon carcinoma cells, in which the MMTV promoter was stably integrated within chromatin, were seeded in 150-mm-diameter dishes (2.5 x 106 cells per dish) and grown in supplemented McCoy’s 5A medium. Subconfluent cells were transiently transfected with pRShGR{alpha}, pRShGR{alpha}R477H, or pRShGR{alpha}G679S (10 µg/dish) using lipofectin (31). Chromatin immunoprecipitation assays were performed as previously described (29, 35).

GST pull-down assay

GST-fused GRIP1(1-1462), GRIP1(559-774), and GRIP1(740-1217) were bacterially produced, purified, and immobilized on the GST beads as previously described (29, 36). In vitro transcription/translation reactions (TNT Quick Coupled Transcription/Translation System; Promega) were used to produce 35S-labeled hGR{alpha}, hGR{alpha}R477H, and hGR{alpha}G679S in rabbit reticulocyte lysate by using pBK/CMV-hGR{alpha}, pBK/CMV-hGR{alpha}R477H, and pBK/CMV-hGR{alpha}G679S, respectively, as templates (29). The in vitro interaction between hGR{alpha}-related plasmids and GST-fused GRIP1 proteins was tested as previously described (29).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mutant receptors hGR{alpha}R477H and hGR{alpha}G679S demonstrate decreased transcriptional activity compared with the wild-type hGR{alpha}

In transient transfection assays, the mutant receptor hGR{alpha}R477H displayed no transcriptional activity. Compared with the wild-type hGR{alpha}, hGR{alpha}G679S showed a 55% reduction in its ability to stimulate the transcriptional activity of the MMTV promoter in response to 10–6 M dexamethasone and an 82.5% reduction in its ability to stimulate the transcriptional activity of the MMTV promoter in response to 10–9 M dexamethasone, the latter concentration of dexamethasone being equivalent to physiological serum cortisol concentration. The concentration of dexamethasone required to achieve 50% of transactivation was 10–9 M for the wild type and 10–8 M for the mutant receptor hGR{alpha}G679S, suggesting that hGR{alpha}G679S has lower affinity for dexamethasone than the wild-type hGR{alpha} (Fig. 2AGo).


Figure 2
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  		FIG. 2. A, Transcriptional activity of the wild-type hGR{alpha}, the mutant receptors hGR{alpha}R477H and hGR{alpha}G679S, and a control plasmid. Compared with the wild-type receptor, the mutant receptor hGR{alpha}R477H displayed no transcriptional activity, whereas hGR{alpha}G679S demonstrated a 55% reduction in its ability to transactivate the MMTV promoter in response to dexamethasone. Bars represent mean ± SE of at least three independent experiments. B and C, Absence of a dominant negative effect of the mutant receptors hGR{alpha}R477H (B) and hGR{alpha}G679S (C) upon the wild-type hGR{alpha}. Cotransfection with a constant amount of hGR{alpha} and five progressively increasing concentrations of hGR{alpha}R477H (B) or hGR{alpha}G679S (C) did not reveal a dose-dependent inhibition of hGR{alpha}-mediated transactivation of the MMTV promoter. Bars represent mean ± SE of at least three independent experiments.

 
The mutant receptors hGR{alpha}R477H and hGR{alpha}G679S do not exert a dominant negative effect upon the wild-type hGR{alpha}

In transient transfection assays, cotransfection with a constant amount of the wild-type hGR{alpha} and five progressively increasing concentrations of the mutant receptors hGR{alpha}R477H or hGR{alpha}G679S did not reveal a dose-dependent inhibition of hGR{alpha}-mediated transactivation of the MMTV promoter (Fig. 2Go, B and C). These findings suggest that the mutant receptors do not exert a dominant negative effect upon the wild-type hGR{alpha}.

The mutant receptor hGR{alpha}R477H preserves normal affinity, whereas hGR{alpha}G679S demonstrates decreased affinity for the ligand compared with the wild-type hGR{alpha}

Dexamethasone binding studies showed that the Kd of hGR{alpha}G679S was significantly higher than that of the wild-type receptor (14.8 ± 0.7 vs. 8.8 ± 0.5 nM; P = 0.01), suggesting that the affinity of the mutant receptor hGR{alpha}G679S for the ligand was 2.0-fold lower than that of the wild-type hGR{alpha}. There was no significant difference in the apparent dissociation curve between the hGR{alpha}R477H and hGR{alpha} (7.9 ± 0.3 vs. 8.8 ± 0.5; P = 0.3), suggesting that the mutant receptor hGR{alpha}R477H preserves normal ligand-binding affinity. No difference in the number of dexamethasone-binding sites was noted between the wild-type and mutant receptors.

Western blot analyses demonstrated no differences in the expression of hGR{alpha}, hGR{alpha}R477H, and hGR{alpha}G679S proteins in CV-1 or COS-7 cells, indicating that the above-described findings did not reflect differences at the protein expression level (data not shown).

The mutant receptors hGR{alpha}R477H and hGR{alpha}G679S demonstrate delayed nuclear translocation compared with the wild-type hGR{alpha}

We studied the subcellular localization and nuclear translocation of the wild-type and mutant receptors in HeLa cells by creating GFP-fused constructs of the three receptors. In the absence of dexamethasone, GFP-fused hGR{alpha} was primarily localized in the cytoplasm of cells. Addition of dexamethasone (10–6 M) resulted in translocation of the wild-type receptor into the nucleus within 12 min (mean ± SE, 12.00 ± 0.71 min) (Fig. 3AGo). The pathological mutant receptors GFP-hGR{alpha}R477H and GFP-hGR{alpha}G679S were also observed predominantly in the cytoplasm of cells in the absence of ligand. However, exposure to the same concentration (10–6 M) of dexamethasone induced a slower translocation of these receptors into the nucleus, which took 25 min for the GFP-hGR{alpha}R477H receptor (25.00 ± 1.2 min) (Fig. 3BGo) and 30 min for the GFP-hGR{alpha}G679S receptor (30.00 ± 1.3 min) (Fig. 3CGo). These findings suggest that the mutant receptors GFP-hGR{alpha}R477H and GFP-hGR{alpha}G679S show, respectively, a 2.1- and 2.5-fold delay in nuclear translocation compared with the wild-type receptor. Coexpression of either mutant with the wild-type receptor at a 1:1 ratio had no apparent effect on the nuclear translocation of the wild-type hGR{alpha}.


Figure 3
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  		FIG. 3. Nuclear translocation of GFP-hGR{alpha} (A), GFP-hGR{alpha}R477H (B), and GFP-hGR{alpha}G679S (C) after exposure to dexamethasone. HeLa cells transiently expressing GFP-hGR{alpha}, GFP-hGR{alpha}R477H, or GFP-hGR{alpha}G679S were exposed to the same concentration of dexamethasone (10–6 M). Images of the same cells were obtained at the indicated time points.

 
The mutant receptor hGR{alpha}G679S preserves its ability to bind to DNA, whereas hGR{alpha}R477H does not bind to DNA in vivo

We investigated the ability of the mutant receptors to bind to DNA in chromatin immunoprecipitation assays. Both the wild-type hGR{alpha} and the mutant receptor hGR{alpha}G679S coprecipitated with MMTV GREs similarly, in a ligand-dependent fashion, suggesting that hGR{alpha}G679S preserves its ability to bind to DNA. However, hGR{alpha}R477H did not coprecipitate with MMTV GREs, indicating that the presence of the R477H mutation in the DBD of the receptor abolishes its ability to bind to DNA (Fig. 4Go).


Figure 4
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  		FIG. 4. Chromatin immunoprecipitation assay performed on HCT-116 cells stably transformed with the MMTV promoter. Both hGR{alpha} and hGR{alpha}G679S coprecipitated with MMTV GREs in a ligand-dependent fashion, indicating that the mutant receptor hGR{alpha}G679S preserves its ability to bind to DNA. By contrast, the mutant receptor hGR{alpha}R477H did not coprecipitate with MMTV GREs. MW, Molecular weight.

 
The mutant receptor hGR{alpha}G679S interacts with the GRIP1 coactivator in vitro only through its AF-1

We investigated the in vitro interaction between the mutant receptors hGR{alpha}R477H and hGR{alpha}G679S and the GRIP1 coactivator in a GST pull-down assay. GRIP1 contains two sites that bind to steroid receptors; one site, the NRB site, is located between amino acids 542 and 745 and interacts with the AF-2 of hGR{alpha} in a ligand-dependent fashion, whereas the other site is located at the carboxy terminus of GRIP1, between amino acids 1121 and 1250, and binds to the AF-1 of hGR{alpha} in a ligand-independent fashion (37) (Fig. 5AGo).


Figure 5
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  		FIG. 5. A, Linearized GRIP1 molecule and distribution of its functional domains. AD1, Activation domain 1; AD2, activation domain 2; HLH, helix-loop-helix; NIDaux, auxiliary nuclear receptor interacting domain; PAS, period arylhydrogen receptor and single-minded (adapted from Ref. 30 ). B, GST pull-down assay. In vitro translated and 35S-labeled hGR{alpha}, hGR{alpha}R477H, or hGR{alpha}G679S was incubated with bacterially produced GST-fused GRIP1(1-1462), GRIP1(597-774), and GRIP1(740-1217) in the absence or presence of dexamethasone (10–5 M). There was no interaction between the mutant receptor hGR{alpha}G679S and the NRB fragment of GRIP1 in vitro, indicating that the AF-2 domain of hGR{alpha}G679S is ineffective.

 
Both the wild-type and mutant receptors bound to full-length GRIP1 and the carboxyl-terminal fragment of GRIP1. However, although the wild-type hGR{alpha} and the mutant receptor hGR{alpha}R477H interacted with the NRB fragment of GRIP1 in a ligand-dependent fashion, there was no interaction between hGR{alpha}G679S and this fragment of GRIP1 (Fig. 5BGo). These results suggest that the mutant receptor hGR{alpha}G679S interacts with the GRIP1 coactivator in vitro only through its AF-1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The mutant receptor hGR{alpha}R477H displayed no transcriptional activity and did not bind to MMTV GREs but demonstrated normal affinity for the ligand and preserved its ability to interact with the GRIP1 coactivator in vitro through both its AF-1 and AF-2 domains. On the other hand, hGR{alpha}G679S had decreased transcriptional activity and reduced affinity for dexamethasone, and although it preserved its ability to bind to GREs, it interacted with the GRIP1 coactivator in vitro only through its AF-1 domain. Both receptors showed delayed translocation into the nucleus after exposure to dexamethasone and did not exert a dominant negative effect upon the transcriptional activity of hGR{alpha}. These findings suggest that the mutant receptors hGR{alpha}R477H and hGR{alpha}G679S cause generalized glucocorticoid resistance by affecting multiple steps in the cascade of GR action, which primarily involve inability to bind to target GREs (hGR{alpha}R477H) and abnormal interaction with GRIP1 and possibly other p160 coactivators (hGR{alpha}G679S).

In standard dexamethasone-binding assays, the mutant receptor hGR{alpha}G679S had reduced affinity for ligand binding compared with the wild-type receptor. This may reflect the location of this mutation between helices 8 and 9 of hGR{alpha}, which line the ligand-binding pocket and may, therefore, affect the affinity of the receptor for ligand directly (Fig. 6Go). The structure of the hGR LBD contains 12 {alpha}-helices and four small ß-strands that fold into a three-layer helical domain (38, 39). After helix 12 (H12), there is an extended strand that forms a conserved ß-sheet with a ß-strand between helices 8 and 9. This C-terminal ß-strand appears to play an important role in receptor activation by stabilizing H12 in the active conformation (38). Thus, the presence of the G679S mutation in the LBD of the receptor may induce a conformational change that ultimately affects the affinity of the receptor for the ligand. The normal affinity for ligand of the mutant receptor hGR{alpha}R477H concurs with previous observations (24) and reflects the integrity of its LBD, given that the mutation is located in the DNA-binding domain of the receptor.


Figure 6
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  		FIG. 6. Crystal structure of the LBD of hGR{alpha}. The lower arrow indicates the position of H12 in the agonist-bound form of the receptor. The upper arrow indicates the position of the G679S mutation (in yellow), which is located between helices 8 and 9 of the hGR{alpha} LBD.

 
In the absence of ligand, both mutant receptors hGR{alpha}R477H and hGR{alpha}G679S were observed primarily in the cytoplasm, whereas exposure to dexamethasone induced a delayed translocation into the nucleus compared with the wild-type receptor. After exposure to dexamethasone (10–6 M), GFP-hGR{alpha} was completely transported from the cytoplasm into the nucleus within 12 min, whereas the mutant receptors hGR{alpha}R477H and hGR{alpha}G679S required approximately 25 and 30 min, respectively. These findings indicate that the mutations R477H and G679S affect the nucleocytoplasmic shuttling of hGR{alpha}, probably through impairment of the nuclear localization sequence (NL)1 and/or NL2 functions. Impairment of the NL1 function may be the result of the decreased affinity for ligand, which may prevent a proper ligand-induced allosteric conformation of the receptor and, hence, a normal interaction between NL1 and components of the importin system (40, 41). On the other hand, impairment of the NL2 function might be specifically dependent upon the conformation of the LBD induced by the ligand, which could be altered by the presence of the mutation (40). Differential binding of the mutant and wild-type receptors to heat-shock proteins, which partially inactivate NL1 and NL2, might also explain the delays observed in the nuclear translocation of the mutant receptors (42).

In the chromatin immunoprecipitation assays, hGR{alpha}R477H did not coprecipitate with MMTV GREs, whereas both hGR{alpha}G679S and wild-type hGR{alpha} did bind to MMTV GREs. A major function of the C-terminal zinc finger of the DBD of hGR{alpha} is to contribute to receptor homodimerization, a prerequisite for potent receptor binding to GREs and efficient transactivation of glucocorticoid-responsive genes (43, 44). This function is achieved by a group of five amino acids in the N-terminal knuckle of the C-terminal zinc finger of the receptor, known as the D loop or dimerization domain (Fig. 1BGo). Point mutations in the DBD of the GR may abolish DNA binding, resulting in silencing of transcriptional activation, although they may not affect the ability of the mutant receptors to transrepress activator protein-1-, nuclear factor-{kappa}B-, and/or other target gene-dependent transcription, possibly through protein-protein interactions and/or tethering of other cofactors to the transcriptional machinery (44, 45, 46). The latter concept is further supported by the fact that mice with GRs defective in homodimerization and DNA binding show similar CRH expression but markedly increased proopiomelanocortin and, to a lesser extent, ACTH expression (45, 47), suggesting that the negative glucocorticoid feedback control of CRH expression involves mechanisms independent of DNA binding, whereas that of proopiomelanocortin and ACTH expression is DNA binding dependent. These findings may explain the better suppression by a single overnight dexamethasone dose observed in the patient with the R477H mutation than that of the patient with the G679S mutation, despite the absent GRE-dependent transcriptional activity of the hGR{alpha}R477H receptor (24).

Unlike hGR{alpha} and hGR{alpha}R477H, the mutant receptor hGR{alpha}G679S interacted with the GRIP1 coactivator in vitro through its AF-1 but not its AF-2 domain. The binding of agonist compounds to GR induces a conformational change in H12 of the LBD of the receptor, revealing a recognition site groove for interaction with coactivators (48, 49). GRIP1 binds to AF-2 via LXXLL sequence motifs termed NR-boxes, found in multiple copies within the coactivator protein. It is likely that the presence of the G679S mutation in the LBD of hGR{alpha} influences the orientation of H12, either by preventing contact between this helix and the ligand or by displacing it from its active position (32, 38, 50). These findings indicate that hGR{alpha}G679S may form a defective complex with GRIP1, which is partially or completely ineffective. Furthermore, the mutant receptor may also display an abnormal interaction with other AF-2-associated proteins, such as the p300/cAMP response element-binding protein (CREB)-binding protein (CBP) cointegrators and components of the vitamin D receptor-interacting protein/thyroid hormone receptor-associated protein (DRIP/TRAP) complex (10, 11, 12, 13).

We conclude that the mutant receptors hGR{alpha}R477H and hGR{alpha}G679S cause generalized glucocorticoid resistance by affecting multiple steps in the cascade of the GR signaling pathway, which include, respectively, inability to bind to target GREs and abnormal interaction with GRIP1 and possibly other p160 coactivators. These differential effects upon the glucocorticoid signal transduction pathway may explain the differences observed in the clinical phenotype of the affected subjects.


   Footnotes
 
This work was supported in part by the intramural program of the National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD.

All authors have nothing to declare.

First Published Online January 31, 2006

Abbreviations: AF, Activation function; DBD, DNA-binding domain; FBS, fetal bovine serum; GFP, green fluorescent protein; GR, glucocorticoid receptor; GRIP1, GR-interacting protein 1; GST, glutathione-S-transferase; H12, helix 12; hGR, human GR; LBD, ligand-binding domain; MMTV, mouse mammary tumor virus; NL, nuclear localization sequence; NRB, nuclear receptor-binding; RSV, Rous sarcoma virus.

Received August 23, 2005.

Accepted January 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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