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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1920
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 6 3696-3705
Copyright © 2005 by The Endocrine Society

A Novel Point Mutation in the Ligand-Binding Domain (LBD) of the Human Glucocorticoid Receptor (hGR) Causing Generalized Glucocorticoid Resistance: The Importance of the C Terminus of hGR LBD in Conferring Transactivational Activity

Evangelia Charmandari, Annaswamy Raji, Tomoshige Kino, Takamasa Ichijo, Anatoly Tiulpakov, Keith Zachman and George P. Chrousos

Pediatric and Reproductive Endocrinology Branch (E.C., T.K., T.I., A.T., K.Z., G.P.C.), National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and Department of Endocrinology, Brigham and Women’s Hospital, Harvard Medical School (A.R.), Boston, Massachusetts 02115

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


   Abstract
Top
 Abstract
Introduction
 Case Reports
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoid resistance is a rare, familial or sporadic condition characterized by partial end-organ insensitivity to glucocorticoids. The clinical spectrum of the condition is broad, ranging from completely asymptomatic to severe hyperandrogenism and/or mineralocorticoid excess. The molecular basis of glucocorticoid resistance has been ascribed to mutations in the human glucocorticoid receptor-{alpha} (hGR{alpha}) gene, which impair one or more of the molecular mechanisms of GR action, thus altering tissue sensitivity to glucocorticoids. We identified a new case of generalized glucocorticoid resistance in a young woman who presented with a long-standing history of fatigue, anxiety, hyperandrogenism, and hypertension. The disease was caused by a novel, heterozygous mutation (T->C) at nucleotide position 2318 (exon 9) of the hGR{alpha} gene, which resulted in substitution of leucine by proline at amino acid position 773 in the ligand-binding domain of the receptor. We systematically investigated the molecular mechanisms through which the natural hGR{alpha}L773P mutant impaired glucocorticoid signal transduction. Compared with the wild-type hGR{alpha}, hGR{alpha}L773P demonstrated a 2-fold reduction in the ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter, exerted a dominant negative effect on the wild-type receptor, had a 2.6-fold reduction in the affinity for ligand, showed delayed nuclear translocation (30 vs. 12 min), and, although it preserved its ability to bind to DNA, displayed an abnormal interaction with the GR-interacting protein 1 coactivator in vitro. We conclude that the carboxyl terminus of the ligand-binding domain of hGR{alpha} is extremely important in conferring transactivational activity by altering multiple functions of this composite transcription factor.


   Introduction
Top
 Abstract
 Introduction
Case Reports
 Materials and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOID RESISTANCE IS a rare, familial or sporadic condition characterized by generalized, partial end-organ insensitivity to glucocorticoids (1, 2, 3, 4, 5). Affected subjects have compensatory elevations in circulating cortisol and ACTH concentrations, which maintain circadian rhythmicity and appropriate responsiveness to stressors, and resistance of the hypothalamic-pituitary-adrenal axis to dexamethasone suppression, but no overt clinical evidence of hypo- or hypercortisolism, with the exception of fatigue, which has been the presenting sign in some patients. Although adequate compensation is achieved by the elevated cortisol concentrations in the majority of the patients with the condition, the excess ACTH secretion often results in increased production of adrenal steroids with androgenic and/or mineralocorticoid activities (1, 2, 3, 4, 5). The former accounts for manifestations of androgen excess, such as ambiguous genitalia in girls, precocious puberty, acne, hirsutism, infertility, male-pattern hair loss and menstrual irregularities in women, and adrenal rests in the testes and oligospermia in men. The latter accounts for symptoms and signs of mineralocorticoid excess, such as hypertension and/or hypokalemic alkalosis. The clinical spectrum of the condition is broad, ranging from completely asymptomatic (displaying biochemical alterations only) to severe cases of hyperandrogenism, fatigue, and/or hypertension with or without electrolyte abnormalities (2, 3, 4, 5). The fatigue has been considered a potential manifestation of muscular or central nervous system glucocorticoid deficiency (2).

The molecular basis of generalized glucocorticoid resistance has been ascribed to mutations in the human glucocorticoid receptor-{alpha} (hGR{alpha}) gene that impair one or more of the molecular mechanisms of glucocorticoid receptor (GR) function, thus altering tissue sensitivity to glucocorticoids. Inactivating mutations within the ligand-binding (LBD) and DNA-binding domains as well as a 4-bp deletion at the 3' boundary of exon 6 of the hGR{alpha} gene have been described in five kindreds and three sporadic cases (6, 7, 8, 9, 10, 11, 12, 13, 14).

The hGR{alpha} isoform, which results from alternative splicing of hGR in exon 9, is ubiquitously expressed in almost all tissues and cells and represents the classic GR 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, which consists of the receptor polypeptide, two molecules of 90-kDa heat shock protein, and several other proteins (15). Upon hormone binding, the receptor undergoes an allosteric change that results in dissociation from the cytoplasmic heterooligomer and translocation into the nucleus, where it binds as a homodimer to glucocorticoid response elements (GREs) located in the promoter regions of target genes, and regulates the expression of glucocorticoid-responsive genes positively or negatively, depending on the GRE sequence and promoter context (16). The receptor can also modulate gene expression independently of GRE binding, by physically interacting with other transcription factors, such as activator protein-1 and nuclear factor-{kappa}B (17, 18).

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 carboxyl-terminal domain and is glucocorticoid dependent (19). 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, that link enhancer-bound transcription factors to general transcription factors (20, 21, 22, 23). Several families of nuclear receptor coactivators have been described, including the p160 coactivators, such as steroid receptor coactivator 1 and glucocorticoid receptor-interacting protein 1 (GRIP1), the p300/cAMP response element-binding protein (CREB)-binding protein (CBP) cointegrators, the p300/CBP-associated factor, the switching/sucrose nonfermenting complex, and the vitamin D receptor-interacting protein/thyroid hormone-associated protein complex (20, 21, 22, 23). The p160 coactivators 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. They also play an important role in the initiation of transcription, because they have histone acetyltransferase activity, which is believed to prepare target gene promoters for transactivation by decondensation of the corresponding chromatin (20, 21, 22, 23).

In the present study we describe a new case of generalized glucocorticoid resistance caused by a novel heterozygous mutation of the hGR{alpha} gene, and we present the molecular mechanisms through which the pathological, mutant receptor impairs glucocorticoid signal transduction.


   Case Reports
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 Abstract
 Introduction
 Case Reports
Materials and Methods
 Results
 Discussion
 References
 
A 29 yr-old female presented with a long-standing history of fatigue, profound anxiety, acne, hirsutism, menstrual irregularities, and hypertension. She had been treated with calcium channel blockers and contraceptives with no improvement for 3 yr. The family history revealed anxiety and hypertension in her father and a paternal aunt. On clinical examination, she was noted to have acne, hirsutism, and elevated blood pressure despite adherence to antihypertensive treatment (blood pressure, 140/90 mm Hg), but no clinical signs suggestive of Cushing’s syndrome. Her weight was 78.92 kg, her height was 170.18 cm, and her body mass index was 27.25 kg/m2. Biochemical and endocrinological evaluation at presentation revealed elevated 0800 h serum cortisol concentrations [56.2 µg/dl; normal range (nr), 8–19 µg/dl], increased 24-h urinary free cortisol excretion (187.6 µg/d; nr, 10–34 µg/d), and elevated 0800 h plasma ACTH (80 pg/ml; nr, 10–60 pg/ml) and serum testosterone (93 ng/dl; nr, 10–55 ng/dl), androstenedione (209 ng/dl; nr, 85–275 ng/dl), and dehydroepiandrosterone sulfate (458 ng/dl; nr, 60–255 ng/dl) concentrations. A low dose dexamethasone suppression test (0.5 mg dexamethasone every 6 h for 48 h) revealed resistance of the hypothalamic-pituitary-adrenal axis to dexamethasone suppression (0800 h serum cortisol, 13.9 µg/dl; 0800 h plasma ACTH, 53 pg/ml). Additional investigations excluded Cushing’s disease, hyperaldosteronism, and other causes of hypertension.

The above clinical manifestations, in association with the findings of the endocrinological evaluation, suggested the diagnosis of generalized glucocorticoid resistance. Written informed consent was obtained from the patient, and additional molecular studies were undertaken. A thymidine incorporation assay, a dexamethasone binding assay, and sequencing of the hGR{alpha} gene (all described in Materials and Methods below) confirmed the diagnosis. After treatment with high dose dexamethasone (2 mg at night), the clinical manifestations of the condition, including the profound anxiety, subsided, the antihypertensive treatment was discontinued, the serum cortisol concentration was suppressed (1.5 µg/dl), and the concentrations of plasma ACTH (12 pg/ml) and serum testosterone (42 ng/dl), androstenedione (190 ng/dl), and dehydroepiandrosterone sulfate (80 ng/dl) were normalized.

The patient’s father, aged 59 yr, was also chronically anxious and hypertensive. He had elevated 0800 h serum cortisol concentrations (25.7 µg/dl) and increased 24-h urinary free cortisol excretion (150 µg/d), but normal 0800 plasma ACTH (26 pg/ml; nr, 10–60 pg/ml) and serum testosterone (357 ng/dl; normal range, 241–827 ng/dl) concentrations. However, he did not provide consent for any additional endocrinological or genetic testing despite our advice to the contrary.


   Materials and Methods
Top
 Abstract
 Introduction
 Case Reports
 Materials and Methods
Results
 Discussion
 References
 
Thymidine incorporation assay

Peripheral blood mononuclear leukocytes from the patient and a control subject were isolated using Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, UK) according to the instructions of the manufacturer. Cells were seeded in 24-well plates (105 cells/well), stimulated with phytohemagglutinin (Sigma-Aldrich Corp., St. Louis, MO; 5 µg/well) and exposed to seven different concentrations (0, 2.5, 10, 25, 100, 250, and 1000 nM) of dexamethasone (Sigma-Aldrich Corp.). After incubation at 37 C for 72 h, [3H]thymidine (Amersham Biosciences) was added to each well (0.1 µCi/well), and incubation was continued for an additional 4 h. Subsequently, cells were harvested, centrifuged at 1300 rpm for 15 min, and treated with 10% trichloroacetic acid (MG Scientific, Inc., Pleasant Prairie, WI) twice. Cells were transferred to scintillation vials, and radioactivity was measured using a ß-counter (LS6000IC counter, Beckman Coulter, Inc., Fullerton, CA).

Establishment of permanent cell lines

Epstein-Barr virus (EBV)-transformed peripheral lymphocytes were established from peripheral blood lymphocytes as previously described (24). Cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics, and incubated in a humidified atmosphere of 5% CO2 at 37 C.

Whole cell dexamethasone binding assay

EBV-transformed lymphocytes were seeded in 96-well plates (2 x 106 cells/well) and incubated in plain RPMI 1640 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 Biosciences) at 37 C in the presence or absence of a 500-fold molar excess of nonradioactive dexamethasone for 1 h. Cells were centrifuged at 1300 rpm for 15 min and washed with PBS (200 µl/well) twice. Cells were transferred to scintillation vials, and radioactivity was measured using a ß-counter (LS6000IC counter, Beckman Coulter). 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. Experiments were performed in duplicate and repeated twice.

Amplification and sequencing of hGR{alpha} gene

Genomic DNA was extracted from EBV-transformed peripheral lymphocytes using a rapid extraction protocol (QIAamp DNA Blood Mini Kit, Qiagen, Valencia, CA) according to the instructions of the manufacturer. The entire coding region of the hGR{alpha} gene was amplified by the PCR (GeneAmp PCR System 9700, PerkinElmer Applied Biosystems, Foster City, CA) using Taq DNA polymerase (Invitrogen Life Technologies, Inc., Carlsbad, CA) and sequenced in an automatic sequencer (ABI PRISM 310 Genetic Analyzer, PerkinElmer Applied Biosystems). The primers used for PCR amplification of genomic DNA were designed using the exon/intron junction sequences as previously described (7) (Table 1Go). Initiation was performed at 94 C for 7 min, followed by 30 cycles of denaturation at 94 C for 45 sec, annealing at 55 C (exons 3–7 and 9) or 60 C (exons 2 and 8) for 30 sec, and extension at 72 C for 1 min, and a final period of extension at 72 C for 7 min. PCR-amplified products were electrophoresed on 1.5% agarose gel and visualized by ethidium bromide staining. The expected molecular mass of both hGR{alpha} and hGR{alpha}L773P was 97 kDa.


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  		TABLE 1. Primers used to amplify each exon of the hGR{alpha} gene

 
RT-PCR

Total RNA was isolated from EBV-transformed peripheral lymphocytes using TRIzol (Invitrogen Life Technologies, Inc.) and was further purified using RNeasy maxi kits (Qiagen) according to the manufacturer’s instructions. Briefly, EBV-transformed peripheral lymphocytes (1 x 108) were pelleted and lysed in 5 ml TRIzol reagent, and 1 ml chloroform was added. The samples were shaken vigorously and centrifuged at 12,000 x g for 15 min at 4 C. The colorless upper phase was harvested, 2.5 ml ethanol was added, and samples were applied to RNeasy maxi columns for additional purification. Approximately 500 ng total RNA were used directly in the RT-PCR using AccessQuick RT-PCR system (Promega Corp., Madison, WI) with the following primers corresponding to the flanking regions of the coding sequence of the hGR: forward, 5'-GAGCGGCTCCTCTGCCAGAGTTG-3'; and reverse, 5'-GGCCCTCTATAAACCACATGTAGTG-3'. RT-PCRs were performed according to the instructions of the manufacturer. Initiation was performed at 95 C for 2 min, followed by 30 cycles of denaturation at 95 C for 45 sec, annealing at 62 C for 30 sec, and extension at 72 C for 3 min, and a final period of extension at 72 C for 5 min. PCR products were purified from the gel and directly sequenced in an automatic sequencer (ABI PRISM 310 Genetic Analyzer, PerkinElmer Applied Biosystems). The primers used for sequencing were the two primers described above and an additional internal forward primer (5'-ACACTAAACCCAAAATTAAGG-3').

Plasmids

The pRShGR{alpha} plasmid expresses hGR{alpha} under control of the Rous sarcoma virus (RSV) promoter. The plasmid pRShGR{alpha}L773P was constructed by introducing the indicated mutation into the pRShGR{alpha} plasmid using PCR-assisted, site-directed mutagenesis (Stratagene, La Jolla, CA). The green fluorescent protein (GFP)-fused plasmid expressing hGR{alpha} was constructed by subcloning the corresponding cDNA into the pF25GFP vector, as previously described (11). The pBK/CMV-hGR{alpha} plasmid was constructed by subcloning the corresponding cDNA into the pBK-CMV vector (Stratagene) (13). The GFP-fused hGR{alpha}L773P and pBK/CMV-hGR{alpha}L773P plasmids were constructed by introducing the indicated mutation into the pF25GFP-hGR{alpha} and pBK/CMV-hGR{alpha}, respectively, using PCR-assisted site-directed mutagenesis (Stratagene). Successful introduction of the L773P mutation into pRShGR{alpha}, pF25GFP-hGR{alpha}, and pBK/CMV-hGR{alpha} plasmids was confirmed by sequencing.

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 by subcloning the corresponding GRIP1 fragments of cDNA into the pGEX4T3 vector (Amersham Biosciences, Piscataway, NJ), as previously described (25).

The plasmid pRSV-erbA–1, which contains a thyroid receptor cDNA in inverse orientation, was used as a negative control in the appropriate experiments. The pMMTV-luc plasmid, which expresses luciferase (luc) under the control of the glucocorticoid-inducible mouse mammary tumor virus (MMTV) promoter, was a gift from Dr. G. L. Hager (National Cancer Institute, National Institutes of Health, Bethesda, MD). The pSV40-ß-gal plasmid encodes the ß-galactosidase (ß-gal) gene under the control of simian virus 40 (SV40) promoter (Promega Corp.).

Cell cultures

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% FBS and antibiotics. HCT-116 human colon carcinoma cells stably transfected with pMAM-nc-Luc (Clontech), which contains the full-length 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 by trypsinization, resuspended in supplemented medium, and plated in six-well plates (CV-1 and COS-7), 75-cm2 flasks (CV-1 and COS-7), 35-mm diameter dishes (HeLa), or 150-mm diameter dishes (HCT-116) at concentrations of 1.5 x 105 cells/well, 1 x 106 cells/flask, 1.5 x 105 cells/35-mm dish, and 2.5 x 106 cells/150-mm dish, respectively.

Transient transfection assays

CV-1, COS-7, and HCT-116 cells were transfected using the lipofectin method (Invitrogen Life Technologies, Inc., Gaithersburg, MD) as previously described (26). HeLa cells were transfected using FuGene 6 reagent according to the instructions of the manufacturer (Roche, Indianapolis, IN). The FuGene 6 to transfected DNA ratio was 2:1.

Transactivation assays

CV-1 cells were seeded in six-well plates at a concentration of 1.5 x 105 cells/well. Twenty-four hours later, cells were cotransfected with pRShGR{alpha}, pRShGR{alpha}L773P, 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). For experiments designed to determine whether the mutant receptor exerts a dominant negative effect on 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}L773P, so that the ratio between the wild-type and mutant receptor 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 (Sigma-Aldrich Corp.) 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 Corp.). Assay buffer solution (350 µl; 25 mM Gly-Gly, 10 mM ATP, 25 mM MgSO4, and 1% Triton X-100, pH 8.0) was added to 50 µl cell lysates. Luc activity in the cell lysates was determined in a Monolight 3010 Luminometer (BD Pharmingen, San Diego, CA) using as substrate 100 µl 1 mM D-luciferin sodium salt solution, as previously described (8, 27). ß-Gal activity was determined in the same samples using a ß-gal enzyme assay system (Galacto-Light Plus, Tropix, Bedford, MA) according to the instructions of the manufacturer. Luc activity was divided by ß-gal activity to account for transfection efficiency. All experiments were 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/flask and grown in supplemented DMEM. Subconfluent cells were transfected with hGR{alpha} or hGR{alpha}L773P (15 µg/flask) using the lipofectin method (26). Twenty-four hours (CV-1) or 6 h (COS-7) after transfection, the transfection medium was replaced with supplemented DMEM. Twenty-four hours later, cells were washed with ice-cold PBS three times, gently scraped from their flasks, centrifuged briefly, and lysed using a lysis buffer that consisted of 100 mM Tris-HCl (pH 8.5), 250 mM NaCl, 1% Nonidet P-40 (pH 7.2), and protease inhibitors (one tablet per 50 ml lysate; Complete, Roche). The homogenates were centrifuged (1500 rpm at 4 C) for 10 min to obtain whole cell extracts. Whole cell extracts were mixed with an equal amount of Tris-glycine-sodium dodecyl sulfate (SDS) sample buffer (2x; Invitrogen Life Technologies, Inc.), heated to 95 C for 3 min, and electrophoresed alongside molecular weight prestained markers (SeeBlue Plus 2 Prestained Protein Standard, Invitrogen Life Technologies, Inc.) through 8% Tris-glycine gel (Invitrogen Life Technologies, Inc.). After electroblotting (25 V/0.8 mA/cm2) onto Hybond C nitrocellulose membranes (Amersham Biosciences), proteins were incubated with blocking solution [5% milk powder/50 mM 1 M Tris-HCl (pH 8.5), 10 mM 5 M NaCl, and 0.5% Tween 20] for 4 h. Immunoblotting was performed at 4 C overnight, using purified specific rabbit polyclonal anti-hGR{alpha} antibody (Affinity BioReagents, Golden, CO) at 10 µg/ml. After washing with 50 mM 1 M Tris-HCl (pH 8.5), 10 mM 5 M NaCl, and 0.5% Tween 20 three times, membranes were incubated with horseradish peroxidase-conjugated goat antirabbit IgG at a 1:1000 dilution at room temperature for 1 h. hGR{alpha} and hGR{alpha}L773P were visualized using the ECL Plus Western Blotting Detection System (Amersham Biosciences) and exposed to high performance chemiluminescence film (Hyperfilm ECL, Amersham Biosciences).

Whole cell dexamethasone binding assays on COS-7 cells

COS-7 cells were seeded in six-well plates (1.5 x 105 cells/well) and transfected with hGR{alpha} or hGR{alpha}L773P (1.5 µg/well) using lipofectin (Invitrogen Life Technologies, Inc.) (26). Six hours later, the transfection medium was replaced with DMEM supplemented with 10% FBS and antibiotics. 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 Biosciences) at 37 C in the presence or absence of a 500-fold molar excess of nonradioactive dexamethasone for 1 h. After incubation, cells were washed with PBS (3 ml/well) twice to remove free steroid. Cells were harvested, centrifuged at 1300 rpm for 15 min, washed with PBS twice, then transferred to scintillation vials, and radioactivity was measured using a ß-counter (LS6000IC counter, Beckman Coulter). 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 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 Kd of the mutant receptor 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/well) in supplemented DMEM. Twenty-fours hours later, cells were transfected with GFP-fused hGR{alpha}- or GFP-fused hGR{alpha}L773P-expressing plasmids (2 µg/dish) using FuGene 6 (Roche). In additional experiments designed to determine the effect of the mutant receptor on 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}L773P (1 µg/dish). Forty-eight hours after transfection, the medium was replaced by phenol red-free DMEM supplemented with 10% charcoal-treated FBS (HyClone Laboratories, Inc., Logan, UT) and antibiotics. Sixteen hours later, cells were exposed to dexamethasone (10–6 M), and fluorescence was detected sequentially by an inverted fluorescence microscope (DM IRB, Leica, Wetzlar, Germany) as previously described (28). Twelve-bit black and white images were captured using a digital charge-coupled device camera (Hamamatsu Photonics K.K., Hamamatsu, Japan). Image analysis and presentation were performed using the Openlab software (Improvision, Boston, MA). All experiments were repeated at least three times, and a population of 7–10 cells was examined in each experiment. Results are expressed as the mean ± SE.

Chromatin immunoprecipitation assays

HCT-116 human colon carcinoma cells stably transfected with MMTV were seeded in 150-mm diameter dishes (2.5 x 106 cells/dish) and grown in supplemented McCoy’s 5A medium. Subconfluent cells were transiently transfected with pRShGR{alpha} or pRShGR{alpha}L773P (15 µg/dish) using lipofectin (Invitrogen Life Technologies, Inc.) (26). Three hours later, the transfection medium was replaced with supplemented McCoy’s 5A medium. Sixteen hours after transfection, cells were treated with dexamethasone (10–6 M) or vehicle (100% ethanol) for 24 h. Cells were fixed using 1% formaldehyde for 10 min and suspended in ice-cold lysis buffer [10 mM Tris-HCl (pH 7.5), 3 mM CaCl2, and 2 mM MgCl2]. Swollen cells were resuspended with equal volumes of lysis buffer and Nonidet P-40 (NP-40) lysis buffer [10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, and 0.5% NP-40], homogenized in a Dounce homogenizer (Kontes Co., Vineland, NJ), and centrifuged at 1400 rpm for 5 min at 4 C. Nuclear pellets were stored in a buffer containing 50 mM Tris-HCl (pH 8.0), 25% glycerol, 5 mM (CH3COO)2Mg, and 0.1 mM EDTA at –80 C (29).

Equal amounts of nuclei (100 µg) were used for each immunoprecipitation experiment. Nuclei were diluted in 500 µl chromatin immunoprecipitation dilution buffer [16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, and protease inhibitors], sonicated eight times for 10 sec each time (at 10-sec intervals; Misonix, Farmingdale, NY), and centrifuged at 14,000 rpm for 5 min. Supernatants were precleared, and the amount of DNA-protein complexes present in each sample was measured at 260 nm. Equal amounts of chromatosomes were immunoprecipitated with anti-hGR{alpha} antibody (Affinity Bioreagents) for 14 h at 4 C. Immunocomplexes were captured on protein A-agarose, washed sequentially with low salt wash buffer [20 mM Tris-HCl (pH 8.1), 150 mM NaCl, 0.1% SDS, 1% Triton X-100, and 2 mM EDTA], high salt wash buffer [20 mM Tris-HCl (pH 8.1), 500 mM NaCl, 0.1% SDS, 1% Triton X-100, and 2 mM EDTA], and LiCl wash buffer [10 mM Tris-HCl (pH 8.1), 0.25 M LiCl, 1% NP40, 1% deoxycholate, and 1 mM EDTA] and twice with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. Samples were eluted with freshly prepared elution buffer (100 mM NaHCO3 and 1% SDS) at room temperature for 15 min twice, and after adding 20 µl 5 M NaCl, cross-linking was reverted at 65 C for 4 h. After treatment with 0.5 M EDTA, 1 M Tris-HCl (pH 6.5), and proteinase K (10 mg/ml) for 1 h at 45 C, genomic DNA fragments were extracted with phenol/chloroform and precipitated with ethanol.

Primer sets were designed to amplify the MMTV promoter region, which contains two GREs and is located approximately 250 bp upstream of the transcription initiation site (forward primer, 5'-AACCTTGCGGTTCCCAG-3'; reverse primer, 5'-GCATTTACATAAGATTTGG-3'). Equal volumes of DNA were used for PCR amplification of the MMTV promoter region. Initiation was performed at 94 C for 7 min, followed by 30 cycles of denaturation at 94 C for 1 min, annealing at 50 C for 1 min, and extension at 72 C for 1 min, with a final period of extension at 72 C for 7 min. PCR-amplified products (173 bp) were electrophoresed on 2% agarose gel and visualized by ethidium bromide staining.

GST pull-down assay

GST fusion protein expression vectors [GST-fused GRIP1-(1–1462), GRIP1-(559–774), and GRIP1-(740–1217)] were grown in Escherichia coli BL21 cells for 3–4 h and induced with isopropyl ß-D-thiogalactoside (0.5 mM) for an additional 2 h. Cells were resuspended in 900 µl PBS, and extracts were generated by pulse sonication on ice (three times at 15-sec intervals). Cell debris was then pelleted at 14,000 rpm for 10 min at 4 C. A 50% slurry of glutathione-Sepharose beads (60 µl) was added to the cell extracts and allowed to bind by gentle agitation for 2 h at 4 C in binding buffer [50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.1% NP-40, 1 mM EDTA, 10% glycerol, and 1 mg/ml albumin bovine fraction (pH 7.0)]. The beads with the bound GST fusion protein were then pelleted, washed three times with 1 ml of the same buffer, and analyzed by SDS-PAGE for the amount of protein bound to the GST beads (30).

Coupled in vitro transcription/translation reactions (TNT Quick Coupled Transcription/Translation System, Promega Corp.) were used to produce 35S-labeled hGR{alpha} and hGR{alpha}L773P in rabbit reticulocyte lysate using pBK/CMV-hGR{alpha} and pBK/CMV-hGR{alpha}L773P, respectively, as templates. 35S-Labeled hGR{alpha} and hGR{alpha}L773P (4 µl crude translated protein) were incubated with equal amounts of GST fusion proteins bound to glutathione-Sepharose beads, washed, eluted, and fractionated by SDS-PAGE. Samples were loaded and electrophoresed on an 8% SDS-PAGE gel. The percentage of starting material loaded in input lanes was 10% or 20%. The gel was fixed, treated with Enlightning buffer (NEN Life Science Products, Inc., Boston, MA), and dried. Radioactivity was detected by exposing a film on the gel.


   Results
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 Abstract
 Introduction
 Case Reports
 Materials and Methods
 Results
Discussion
 References
 
Thymidine incorporation and dexamethasone binding studies of peripheral lymphocytes and EBV-transformed B lymphocytes

The thymidine incorporation assay performed on fresh peripheral lymphocytes revealed resistance to dexamethasone-induced suppression of phytohemagglutinin-stimulated thymidine incorporation in the patient compared with a matched control subject (Fig. 1Go). The dexamethasone binding assay was performed on EBV-transformed lymphocytes and showed that the affinity of the GR for the ligand was 2.7-fold lower in the patient than in the control cells (Kd, 23.6 vs. 8.8 nM). No difference was noted in the number of binding sites between the two subjects.



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  		FIG. 1. Thymidine incorporation assay performed on fresh peripheral lymphocytes. There was increased resistance to dexamethasone-induced suppression of phytohemagglutinin-stimulated thymidine incorporation in the patient compared with the control subject.

 
Sequencing of the hGR{alpha} gene

The exons of the entire coding region of the hGR{alpha} gene, including the intron/exon junctions, were amplified by PCR and sequenced. A single heterozygous thymine to cytosine (T->C) substitution was identified at nucleotide position 2318 (exon 9{alpha}), resulting in leucine to proline substitution (CTG->CCG) at amino acid 773 in the LBD of the receptor (Fig. 2AGo). RNA extraction from peripheral lymphocytes, RT-PCR amplification of the coding region of the gene, and sequencing confirmed these findings. Using the three-dimensional crystal structure of the LBD of hGR{alpha}, we determined that the L773P mutation is located 13 amino acids downstream of helix 12 (31) (Fig. 2BGo).



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  		FIG. 2. A, Sequencing of the entire coding region of the hGR{alpha} gene revealed a single heterozygous thymine to cytosine (T->C) substitution at nucleotide position 2318 (exon 9), resulting in leucine to proline substitution (CTG->CCG) at amino acid 773 in the LBD of the receptor. B, Crystal structure of the LBD of hGR{alpha}. The left arrow indicates the position of helix 12 in the agonist-bound form of the receptor. The right arrow indicates the position of the L773P mutation (in yellow), which is located 13 amino acids downstream of helix 12.

 
The mutant receptor hGR{alpha}L773P demonstrates decreased transcriptional activity compared with the wild-type hGR{alpha}

In transient transfection assays performed on CV-1 cells, which are devoid of endogenous GR expression, the mutant receptor demonstrated a 2-fold reduction in its ability to transactivate the glucocorticoid-inducible MMTV promoter in response to dexamethasone compared with the wild-type receptor. The concentration of dexamethasone required to achieve 50% of transactivation was 10–9 M for the wild-type receptor and 10–8 M for the mutant receptor (Fig. 3AGo).



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  		FIG. 3. A, Transcriptional activity of the wild-type hGR{alpha}, the mutant receptor hGR{alpha}L773P, and a control plasmid. Compared with the wild-type receptor, the mutant receptor demonstrated a 2-fold reduction in the ability to transactivate the MMTV promoter in response to dexamethasone (10–12–10–5 M). Bars represent mean ± SEM of at least three independent experiments. B, Dominant negative effect of the mutant receptor on the wild-type hGR{alpha}. Cotransfection with a constant amount of hGR{alpha} and five progressively increasing concentrations of hGR{alpha}L773P revealed a dose-dependent inhibition of hGR{alpha}-mediated transactivation of the MMTV promoter. Bars represent mean ± SEM of at least three independent experiments.

 
The mutant receptor hGR{alpha}L773P exerts a dominant negative effect on the wild-type hGR{alpha}

In transient transfection assays performed on CV-1 cells, cotransfection with a constant amount of the wild-type hGR{alpha} and five progressively increasing concentrations of the mutant receptor hGR{alpha}L773P showed a dose-dependent inhibition of hGR{alpha}-mediated transactivation of the MMTV promoter, which ranged from 23% at the 1:1 hGR{alpha}/hGR{alpha}L773P ratio to 59% at the 1:10 hGR{alpha}/hGR{alpha}L773P ratio (Fig. 3BGo). These findings suggest that the mutant receptor exerts a dominant negative effect on the wild-type receptor.

Western blot analyses demonstrated no differences in the expression of hGR{alpha} and hGR{alpha}L773P proteins studied in CV-1 cells, indicating that the above-described alterations in hGR{alpha}-mediated transactivation of the MMTV promoter did not reflect differences at the protein expression level (data not shown).

The mutant receptor hGR{alpha}L773P demonstrates decreased affinity for ligand compared with the wild-type hGR{alpha}

Dexamethasone binding studies performed on COS-7 cells, which are devoid of endogenous GR expression, transfected with either hGR{alpha} or hGR{alpha}L773P showed that the apparent Kd of hGR{alpha}L773P was significantly higher than that of the wild-type receptor (24.4 ± 5.0 vs. 9.4 ± 0.8 nM; P = 0.03), suggesting that the affinity of the mutant receptor hGR{alpha}L773P for the ligand was 2.6-fold lower than that of the wild-type hGR{alpha}. No difference in the number of GR-binding sites was noted between the wild-type and mutant receptors (Fig. 4Go). Western blot analyses demonstrated similar expression of hGR{alpha} and hGR{alpha}L773P proteins in COS-7 cells (data not shown).



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  		FIG. 4. A, Whole cell dexamethasone binding studies performed on COS-7 cells. The mutant receptor hGR{alpha}L773P demonstrated decreased affinity for ligand compared with the wild-type receptor. B, Scatchard analysis of whole cell ligand binding assays showed that the apparent Kd of hGR{alpha}L773P was higher than that of the wild-type receptor. No difference in the number of GR-binding sites was noted between the wild-type and mutant receptors.

 
The mutant receptor hGR{alpha}L773P demonstrates 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 two receptors. In the absence of dexamethasone, GFP-fused hGR{alpha} was primarily localized in the cytoplasm of cells. Addition of dexamethasone at a concentration of 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. 5AGo). The pathological mutant receptor GFP-hGR{alpha}L773P was 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 this receptor into the nucleus, which took 30 min (mean ± SE, 30.00 ± 1.18 min; Fig. 5BGo). These findings suggest that the mutant receptor shows a 2.5-fold delay in nuclear translocation compared with the wild-type receptor. Coexpression of both receptors at a 1:1 ratio had no apparent effect on the nuclear translocation of the wild-type hGR{alpha} (data not shown).



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  		FIG. 5. Nuclear translocation of GFP-hGR{alpha} (A) and GFP-hGR{alpha}L773P (B) after exposure to dexamethasone. HeLa cells transiently expressing GFP-hGR{alpha} or GFP-hGR{alpha}L773P 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}L773P preserves its ability to bind to DNA

We investigated the ability of the mutant receptor to bind to DNA in a chromatin immunoprecipitation assay. HCT-116 cells stably transformed with the MMTV promoter were transiently transfected with the wild-type receptor, the mutant receptor, or a control plasmid. Both wild-type and mutant receptors coprecipitated with MMTV GREs similarly in a ligand-dependent fashion, suggesting that the mutant receptor preserves its ability to bind to DNA (Fig. 6Go).



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  		FIG. 6. Chromatin immunoprecipitation assay performed on HCT-116 cells stably transfected with MMTV. Both the wild-type and mutant receptors coprecipitated with MMTV GREs in a ligand-dependent fashion, indicating that the mutant receptor preserves its ability to bind to DNA. MW, Molecular weight.

 
The mutant receptor hGR{alpha}L773P interacts with the GRIP1 coactivator in vitro only through its AF-1

We investigated the in vitro interaction between the mutant receptor hGR{alpha}L773P 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. The other site is located at the carboxyl terminus of GRIP1, between amino acids 1121 and 1250, and binds to the AF-1 of hGR{alpha} in a ligand-independent fashion (32, 33, 34) (Fig. 7AGo).



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  		FIG. 7. 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 aryl hydrogen receptor and single-minded (adapted from Ref. 13 ). B, GST pull-down assay. In vitro translated and 35S-labeled hGR{alpha} and hGR{alpha}L773P were 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 and the NRB fragment of GRIP1 in vitro, indicating that the AF-2 domain of hGR{alpha}L773P is ineffective.

 
In vitro translated and 35S-radiolabeled hGR{alpha} and hGR{alpha}L773P were tested for binding to bacterially produced and purified GST-fused full-length GRIP1 [GRIP1-(1–1462)], the NRB fragment of GRIP1 [GRIP1-(559–774)], and the carboxyl-terminal fragment of GRIP1 [GRIP1-(740–1217)]. Both wild-type and mutant receptors bound to full-length GRIP1 and the carboxyl-terminal fragment of GRIP1. However, although the wild-type hGR{alpha} interacted with the NRB fragment of GRIP1 in a ligand-dependent fashion, there was no interaction between the mutant receptor and this fragment of GRIP1 (Fig. 7BGo). These results suggest that the mutant receptor interacts with the GRIP1 coactivator in vitro only through its AF-1.


   Discussion
Top
 Abstract
 Introduction
 Case Reports
 Materials and Methods
 Results
 Discussion
References
 
The mutant receptor hGR{alpha}L773P had decreased affinity for dexamethasone, delayed nuclear translocation, and decreased transcriptional activity and exerted a dominant negative effect on the wild-type receptor. It preserved its ability to bind to DNA, but displayed an abnormal interaction with the GRIP1 coactivator in vitro. These findings suggest that the L773P mutation affects multiple functions of the GR and highlight the importance of the C terminus of the LBD of the hGR (downstream of helix 12) in conferring transactivational activity and preserving normal function of this composite transcription factor.

The dominant negative effect of hGR{alpha}L773P on the wild-type hGR{alpha} may contribute to the manifestations of the disease at the heterozygote state. At a 1:1 ratio, the mutant receptor inhibited approximately 25% of the transcriptional activity of the wild-type receptor. Similar findings were previously reported for the hGR{alpha}I559N and hGR{alpha}I747M natural mutants, which were shown to have a dominant negative effect on the wild-type receptor and to produce disease even in the presence of a normally functioning allele (11, 13). The dominant negative activity of these mutants, in association with their severely impaired ability to transactivate glucocorticoid-responsive genes, resulted in an additional decrement in the glucocorticoid sensitivity of affected subjects (11, 13). Dominant negative effects of mutant receptors have also been reported in the syndrome of thyroid hormone resistance (35), whereas dominant inhibitory actions of physiologically expressed receptor isoforms have been demonstrated for the A isoform of the human progesterone receptor (36) and the ß isoform of hGR (37, 38, 39).

The mutant receptor hGR{alpha}L773P had a decreased affinity for the ligand compared with the wild-type hGR{alpha}. This is most likely due to the location of the mutation at the C terminus of the LBD of the receptor, close to helix 12. The structure of the hGR LBD contains 12 {alpha}-helices and four small ß-strands that fold into a three-layer helical domain (30). Helices 1 and 3 form one side of a helical sandwich, whereas helices 7 and 10 form the other side. The middle layer of helices (helices 4, 5, 8, and 9) are present in the top half, but not in the bottom half, of the protein. This arrangement of helices creates a cavity in the bottom half of the LBD where the agonist molecule is bound. Helix 12, which plays an essential function in ligand-dependent activation, adopts the so-called agonist-bound conformation where it packs against helices 3, 4, and 10 as an integrated part of the domain structure. After helix 12, 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 helix 12 in the active conformation (31). Deletion of the last few residues that form the ß-strand lead to alterations in hormone binding specificity and significant reduction in the receptor-mediated transactivation of target genes (40), suggesting that the C-terminal region of hGR{alpha}, downstream of helix 12, is essential for ligand binding specificity and agonist potential, although it does not appear to confer differential hormone-binding capacities to the receptor (40, 41).

In addition to decreased affinity for ligand, the mutant receptor demonstrated delayed nuclear translocation 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 receptor GFP-hGR{alpha}L773P required approximately 30 min. These findings indicate that the mutation L773P affects the nucleocytoplasmic shuttling of hGR{alpha}, probably through impairment of nuclear localization sequence 1 (NL1) and/or NL2 function. Impairment of NL1 function may arise as a 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 (11, 42). Impairment of the NL2 function, in contrast, might be specifically dependent upon the conformation of the LBD induced by the ligand and could also be due to the mutation (42). Differential binding of the mutant and wild-type receptors to heat shock proteins, which partially inactivate NL1 and NL2, might also explain the differences observed between the times required for entry into the nucleus (43, 44, 45).

Although hGR{alpha}L773P preserved its ability to bind to DNA GREs, it interacted with the GRIP1 coactivator in vitro only through its AF-1. The abnormal interaction between the AF-2 of the mutant receptor and GRIP1 might have been predicted by the location of the mutation 13 amino acids downstream of helix 12 of the LBD. Helix 12 plays an important role in protein-protein interactions between the GR and its coactivators. In the agonist-bound conformational state of the GR, helix 12 adopts a position over the ligand-binding pocket, allowing coactivators to interact within the coactivator cavity, thus forming a transcriptionally active receptor. In contrast, in the antagonist-bound structure of the GR, the glucocorticoid antagonist physically prevents helix 12 from adopting the characteristic agonist position, as in the agonist-bound allosteric conformation of the receptor. Instead, helix 12 is translocated and covers the coactivator cavity, thus preventing the receptor from recruiting coactivators (41). That the mutant receptor hGR{alpha}L773P did not interact with the GRIP1 coactivator in vitro through its AF-2 may indicate that the presence of the L773P mutation in the C terminus of hGR{alpha} prevents helix 12 from adopting its characteristic agonist position and interferes with the interaction between the receptor and the GRIP1 coactivator. Furthermore, the mutant receptor may also display an abnormal interaction with other AF-2-associated proteins, such as p300/CBP and components of the vitamin D receptor-interacting protein/thyroid hormone-associated protein complex (20, 21, 22, 23).

The profound anxiety in our patient is being described for the first time as a possible presenting feature of generalized glucocorticoid resistance. We hypothesize that this may arise as a result of the compensatory elevations of hypothalamic CRH secretion (46, 47, 48), and that it might have been missed in previously reported cases as a nonspecific symptom. We also hypothesize that in this family the mode of transmission is autosomal dominant, as previously described for two other kindreds (5).

Administration of high doses of dexamethasone resulted in suppression of the endogenous secretion of ACTH and cortisol and a significant improvement in the clinical manifestations, including fatigue, anxiety, hyperandrogenism, and hypertension (2, 3, 4, 5). It should be emphasized that treatment with adequate doses of mineralocorticoid-sparing synthetic glucocorticoids, such as dexamethasone, is recommended in cases characterized by severe impairment of the transactivational activity of the receptor, not only as a rational target therapy for the disorder, but also because long-standing corticotroph hyperstimulation in association with decreased glucocorticoid negative feedback may lead to the development of an ACTH-secreting adenoma (8).


   Footnotes
 
First Published Online March 15, 2005

Abbreviations: AF, Activation function; CBP, cAMP response element-binding protein-binding protein; CMV, cytomegalovirus; EBV, Epstein-Barr virus; FBS, fetal bovine serum; ß-gal, ß-galactosidase; GFP, green fluorescent protein; GR, glucocorticoid receptor; GRE, glucocorticoid response element; GRIP1, glucocorticoid receptor-interacting protein 1; GST, glutathione-S-transferase; hGR{alpha}, human glucocorticoid receptor-{alpha}; LBD, ligand-binding domain; luc, luciferase; MMTV, mouse mammary tumor virus; nr, normal range; NL, nuclear localization sequence; NP-40, Nonidet P-40; NRB, nuclear receptor binding; RSV, Rous sarcoma virus; SDS, sodium dodecyl sulfate; SV40, simian virus 40.

Received October 5, 2004.

Accepted March 7, 2005.


   References
Top
 Abstract
 Introduction
 Case Reports
 Materials and Methods
 Results
 Discussion
 References
 

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