DOI: 10.1111/eci.12470
ORIGINAL ARTICLE A novel mutation of the hGR gene causing Chrousos syndrome Nicolas C. Nicolaides*,†, Eliza B. Geer‡, Dimitrios Vlachakis§, Michael L. Roberts*,†, Anna-Maria G. Psarra¶, Paraskevi Moutsatsou**, Amalia Sertedaki*,†, Sophia Kossida§,†† and Evangelia Charmandari*,† *
Division of Endocrinology, Metabolism and Diabetes, First Department of Pediatrics, ‘Aghia Sophia’ Children’s Hospital, University of Athens Medical School, Athens, Greece, †Division of Endocrinology and Metabolism, Clinical Research Center, Biomedical Research Foundation of the Academy of Athens, Athens, Greece, ‡Division of Endocrinology, Diabetes, and Bone Diseases, Icahn School of Medicine at Mount Sinai School, New York, NY, USA, §Bioinformatics and Medical Informatics Team, Biomedical Research Foundation of the Academy of Athens, Athens, Greece, ¶Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece, **Department of Clinical Biochemistry, ‘Attiko’ Hospital, University of Athens Medical School, Athens, Greece, ††IMGT, The International ImMunoGeneTics Information System, Institute of Human Genetics, Montpellier, France
ABSTRACT Background Natural mutations in the human glucocorticoid receptor (hGR, NR3C1) gene cause Chrousos syndrome, a rare condition characterized by generalized, partial, target-tissue insensitivity to glucocorticoids. Objective To present a new case of Chrousos syndrome caused by a novel mutation in the hGR gene, and to elucidate the molecular mechanisms through which the natural mutant receptor affects glucocorticoid signal transduction. Design and Results The index case presented with hirsutism, acne, alopecia, anxiety, fatigue and irregular menstrual cycles, but no clinical manifestations suggestive of Cushing’s syndrome. Endocrinologic evaluation revealed elevated 08:00 h plasma adrenocorticotropic hormone, serum cortisol and androstenedione concentrations and increased urinary free cortisol excretion. The patient harbored a novel A > G transition at nucleotide position 2177, which resulted in histidine (H) to arginine (R) substitution at amino acid position 726 of the receptor (c.2177A > G, p.H726R). Compared with the wild-type receptor, the mutant receptor hGRaH726R demonstrated decreased ability to transactivate glucocorticoid-responsive genes and to transrepress the nuclear factor-jB signalling pathway, displayed 55% lower affinity for the ligand and a four-fold delay in nuclear translocation, and interacted with the glucocorticoid receptor-interacting protein 1 coactivator mostly through its activation function-1 domain. Finally, a 3-dimensional molecular modelling study of the H726R mutation revealed a significant structural shift in the rigidity of helix 10 of the receptor, which resulted in reduced flexibility and decreased affinity of the mutant receptor for binding to the ligand. Conclusions The natural mutant receptor hGRaH726R impairs multiple steps of glucocorticoid signal transduction, thereby decreasing tissue sensitivity to glucocorticoids. Keywords Chrousos syndrome, glucocorticoid receptor, glucocorticoid signalling, mutations. Eur J Clin Invest 2015; 45 (8): 782–791
Introduction Mutations in the human GR gene identified in human patients impair the molecular mechanisms of hGR action and alter tissue sensitivity to glucocorticoids [1,2]. The majority of these mutations have been associated with Chrousos syndrome, which is a rare, familial or sporadic condition characterized by generalized, partial, target-tissue insensitivity to glucocorticoids (Table 1) [3–26]. Affected subjects have compensatory
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activation of the hypothalamic–pituitary–adrenal (HPA) axis and elevated circulating 24-h serum cortisol and plasma adrenocorticotropic hormone (ACTH) concentrations, which maintain circadian rhythmicity and appropriate responsiveness to stressors, as well as resistance of the HPA axis to dexamethasone suppression without any clinical manifestations of hypercortisolism [3–26]. The increased secretion of ACTH often results in increased production of adrenal steroids with androgenic and/or mineralocorticoid activities and the
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Table 1 Mutations of the human glucocorticoid receptor gene causing Chrousos syndrome Author (References)
cDNA
Amino acid
Genotype
Phenotype
Chrousos et al. [10]; Hurley et al. [11]; Charmandari et al. [8]
1922 (A?T)
641 (D?V)
Homozygous
Hypertension, Hypokalemic alkalosis
Karl et al. [12]
4 bp deletion in exon-intron 6
Heterozygous
Hirsutism, Male-pattern hair-loss, Menstrual irregularities
Malchoff et al. [13.]; Charmandari et al. [8]
2185 (G?A)
729 (V?I)
Homozygous
Precocious puberty, Hyperandrogenism
Karl et al. [14]; Kino et al. [15]; Charmandari et al. [8]
1676 (T?A)
559 (I?N)
Heterozygous
Hypertension, Oligospermia, Infertility
Ruiz et al. [16]; Charmandari et al. [17]
1430 (G?A)
477 (R?H)
Heterozygous
Hirsutism, Fatigue, Hypertension
Ruiz et al. [16]; Charmandari et al. [17]
2035 (G?A)
679 (G?S)
Heterozygous
Hirsutism, Fatigue, Hypertension
Mendonca et al. [18]; Charmandari et al. [8]
1712 (T?C)
571 (V?A)
Homozygous
Ambiguous genitalia, Hypertension, Hypokalaemia, Hyperandrogenism
Vottero et al. [19]; Charmandari et al. [8]
2241 (T?G)
747 (I?M)
Heterozygous
Cystic acne, Hirsutism, Oligo-amenorrhoea
Charmandari et al. [20]
2318 (T?C)
773 (L?P)
Heterozygous
Fatigue, Anxiety, Acne, Hirsutism, Hypertension
Charmandari et al. [21]
2209 (T?C)
737 (F?L)
Heterozygous
Hypertension, Hypokalaemia
McMahon et al. [22]
2 bp deletion at nt 2318-9
773
Homozygous
Hypoglycaemia, Fatigability with feeding, Hypertension
Nader et al. [23]
2141 (G?A)
714 (R?Q)
Heterozygous
Hypoglycaemia, Hypokalaemia, Hypertension, Mild clitoromegaly, Advanced bone age, Precocious pubarche
Zhu Hui-juan et al. [24]
1667 (G?T)
556 (T?I)
Heterozygous
Adrenal incidentaloma
Roberts et al. [25]
1268 (T?C)
423 (V?A)
Heterozygous
Fatigue, Anxiety, Hypertension
Nicolaides et al. [26]
1724 (T?G)
575 (V?G)
Heterozygous
Melanoma, Asymptomatic daughters
Numbers in the parentheses following authors’ names indicate the corresponding references.
corresponding clinical phenotype [3–26]. The clinical spectrum of the condition is broad, ranging from most severe to mild cases of mineralocorticoid and/or androgen excess, while a number of patients may be asymptomatic, displaying biochemical alterations only [3–26]. In this study, we identified a case of Chrousos syndrome caused by a novel heterozygous point mutation in the hGR gene and investigated the molecular mechanisms through which the natural mutant receptor impairs glucocorticoid signal transduction.
Case report A 30-year-old woman presented with a long-standing history of hirsutism, acne, diffuse alopecia, fatigue, anxiety and irregular menstrual cycles. She was otherwise asymptomatic and had no
clinical manifestations of Cushing’s syndrome. Hormonal evaluation revealed elevated 08:00 h plasma ACTH [207 pg/mL, normal range (nr) < 52)], serum cortisol (26 lg/dL) and androstenedione (252 ng/dL, nr < 235) concentrations and increased urinary free cortisol (UFC) excretion (97–122 lg/day; nr: < 50). Resistance of the HPA axis to overnight dexamethasone suppression was found. A pituitary magnetic resonance imaging scan was normal. The above clinical, hormonal and imaging findings suggested the diagnosis of Chrousos syndrome. Written informed consent was obtained from the patient, and further molecular studies were undertaken. Following treatment with dexamethasone at a dose of 1 mg per os at night, the clinical manifestations of the condition subsided, and the concentrations of plasma ACTH and serum androgens were normalized.
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Materials and methods Sequencing of the patient’s hGR gene Genomic DNA was isolated from peripheral blood lymphocytes employing the Maxwell 16 instrument for automated DNA extraction (Promega Corporation, Madison, WI, USA). The entire coding region of the hGR gene (NR3C1; NM 001018074.1), exons 2–9 and their intronic flanking sequences, were PCR amplified and bidirectionally sequenced using the Big Dye Terminator cycle sequencing kit 3.1 (Applied Biosystems, Life Technologies, Foster City, CA, USA). The sequencing reactions were analysed on the automated genetic analyzer ABI 3500 (Applied Biosystems, Life Technologies). Three in silico software were used to predict the impact of the amino acid change on the protein function: PolyPhen-2 (Polymorphism Phenotyping v2), Mutation Taster and SIFT (Sorting Intolerant From Tolerant).
Plasmids The plasmids used in the following studies included pRShGRa, pF25GFP-hGRa, pMMTV-luc, pGL473[hRluc/SV40], pRSVerbA 1, pGEX4T3-GRIP1(1–1462), pGEX4T3-GRIP1(596–774), pGEX4T3-GRIP1(740–1217), pRSVC(p50)-NF-jB, pRSVC(p65)RelA and p(IjB)3-luc, and have been previously described [20,26]. The plasmids pRShGRaH726R and pF25GFPhGRaH726R were created by introducing the H726R mutation into the pRShGRa and pF25GFP-hGRa plasmids, respectively, using the QuickChange II XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA).
Transactivation and transrepression assays CV-1 cells were transiently cotransfected with pRShGRa or pRShGRaH726R (0.05 lg/well), pMMTV-luc (0.5 lg/well) and pGL473[hRluc/SV40] (0.1 lg/well) for the transactivation assays, or with pRSVC(p50)-NF-jB (0.05 lg/well), pRSVC (p65)-RelA (0.05 lg/well), p(IjB)3-luc (0.5 lg/well) for the transrepression assays, using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA). Forty-eight hours after transfection, cells were incubated with increasing concentrations of dexamethasone for an additional 24 h. Cells were then lysed with 59 Reporter Lysis Buffer (Promega Corporation), and firefly and renilla luciferase activities were, respectively, determined using the Dual-Luciferase Reporter Assay System (Promega) [20,26].
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brane. The latter was incubated with anti-hGR and anti-actin antibodies. Western blot analyses were performed as previously described [20,26].
Dexamethasone-binding assays COS-7 cells were transiently transfected with pRShGRa or pRShGRaH726R (1.5 lg/well) using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were incubated with six progressively increasing concentrations of [1,2,4,6,7-3H] dexamethasone (PerkinElmer Waltham, MA, USA) in the presence or absence of a 500-fold molar excess of nonradioactive dexamethasone for 1 h. Dexamethasone-binding assays were performed as previously described [20,26].
Subcellular localization and nuclear translocation studies HeLa cells were transiently transfected with pF25GFP-hGRa or pF25GFP-hGRaH726R (2 lg/well) using FuGENE 6 (Roche Diagnostics Corporation, Indianapolis, IN, USA). The subcellular localization and cytoplasmic-to-nuclear translocation of the wild-type and mutant receptors were examined before and after the addition of 10 6 M dexamethasone under a fluorescence microscope (DM IRB; Leica, Wetzlar, Germany). Representative images were captured using a digital charge-coupled device camera (Hamamatsu Photonics K.K., Hamamatsu, Japan) [20,26].
In vitro binding assays for evaluation of the GR/GRE association HCT116 cells were transiently transfected with pRShGRa or pRShGRaH726R (15 lg/flask) using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were incubated with 10 6 M dexamethasone for 3 h. Nuclear extracts were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA). The association of hGRa or hGRaH726R to glucocorticoid-response elements (GREs) was investigated by in vitro binding assays using the TRansAMTM GR Kit (Active Motif) according to the instructions of the manufacturer.
Glutathione S-transferase (GST) pull-down assays GST-fused glucocorticoid receptor-interacting protein 1 (GRIP1) (1–1462), GRIP1 (559–774) and GRIP1 (740–1217) were bacterially produced, purified and immobilized on GST beads. The in vitro interaction of pRShGRa and pRShGRaH726R with the GST-fused GRIP1 proteins was tested as previously described [17,20,21,25,26].
Western blot analyses CV-1 and COS-7 cells were transiently transfected with pRShGRa or pRShGRaH726R (15 lg/flask) using Lipofectamine 2000 (Invitrogen). Equal amounts of cell lysates were run on 8% SDS-PAGE gel and blotted onto nitrocellulose mem-
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Structural biology studies Three-dimentional (3D) coordinates of the wild-type hGRa were obtained from the X-ray solved, crystal structure with Research Collaboratory for Structural Bioinformatics (RCSB)
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code: 3CLD, which is the ligand-binding domain of the hGRa complexed with fluticasone furoate [27]. The resolution of the X-ray structures is 284 A°. The hGRaH726R mutant structure was manually mutated using molecular operating environment (MOE) [28]. The hGRa and hGRaH726R molecular systems were initially subjected to energy minimization using the Gromacs-implemented force-fields to remove the geometrical strain [29,30]. The 3D structures were subsequently solvated with simple point charge (SPC) water using the truncated from the model. Molecular octahedron box extending to 7 A dynamics were performed after that at 300K, 1 atm with 2 fs step size, and for a total of one microsecond, using the number, volume, and temperature (NVT) ensemble in a canonical environment; NVT stands for number of atoms, volume and temperature that remain constant throughout the simulation. The 2D maps were drawn using the built-in module of MOE.
Results Identification of a novel heterozygous A ? G mutation in the patient’s hGR gene Direct sequencing of the entire coding region and the exon– intron boundaries of the hGRa gene revealed a novel heterozygous A > G transition at nucleotide position 2177, which resulted in histidine (His, H) to arginine (Arg, R) substitution at amino acid position 726 in exon 8 (c.2177A > G, p.H726R), in helix 10 of the ligand-binding domain (LBD) of the receptor (Fig. 1a,b). The 3 in silico software employed to predict the
effect of the amino acid change indicated that the p.H726R is probably damaging (Polyphen-2, score 0999), disease causing (Mutation Taster, prob: 0999999999296905) and affects protein function (SIFT, score 003).
The hGRaH726R displays decreased ability to transactivate glucocorticoid-responsive genes compared with the wild-type hGRa and does not exert a dominant negative effect upon the hGRamediated transcriptional activity The transactivation assays showed that the hGRaH726R displayed a statistically significant reduction (40%) in its ability to transactivate the glucocorticoid-inducible mouse mammary tumour virus (MMTV) promoter in response to increasing concentrations of dexamethasone, compared with the wild-type hGRa (Fig. 2a). In addition, the mutant receptor did not exert a dominant negative effect upon the hGRa-mediated transcriptional activity (Fig. 2b). Western blot analyses demonstrated no differences in the expression of hGRa and hGRaH726R proteins, indicating that the above-described alterations in hGRmediated transactivation of the MMTV promoter did not reflect differences at the protein expression level.
The hGRaH726R demonstrates decreased ability to transrepress the nuclear factor (NF)-jB-responsive genes compared with the wild-type hGRa Compared with the wild-type receptor, the hGRaH726R demonstrated significantly decreased ability (~30%) to transrepress
(a) aa
Figure 1 (a) Sequencing of the NR3C1 gene revealed a heterozygous A > G transition at nucleotide position 2177 (indicated by the black arrow), which resulted in the replacement of histidine (His, H) to arginine (Arg, R) at amino acid position 726, c.2177A > G, p.H726R, in the helix 10 of the LBD of the receptor. (b) Crystal structure of the LBD of hGRa. The yellow arrow indicates the position of the mutation in helix 10 of the agonist-bound conformation of the receptor.
722
723
724
725
726
727
728
729
(b)
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Relative luciferase activity
(a) 0·1
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The hGRaH726R displays a four-fold delay in the cytoplasmic-to-nuclear translocation compared with the wild-type hGRa
CV-1 MMTV
0·08
WT - - - - H726R
0·06 0·04 0·02 0
10–8 10–12 10–10 10–6 Dexamethasone (M)
0
Relative luciferase activity
(b) 0·12 0·1
10–5
CV-1 MMTV
The hGRaH726R preserves its ability to bind to DNA
0·08
In vitro binding assays revealed no significant difference in the association of hGRaH726R to GREs following exposure to dexamethasone compared with the hGRa (Fig. 4). These findings indicate that the mutant receptor preserves its ability to bind to DNA.
0·06 0·04 0·02
Dexamethasone 0 WT:H726R Ratio
The hGRaH726R interacts with the GRIP1 coactivator mostly through its activation function (AF)-1 domain –
+ 1:0
– 1:1
+
– 1:3
+
– 1:6
+
–
+
1:10
Figure 2 (a) The mutant receptor hGRaH726R had decreased ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to increasing concentrations of dexamethasone, compared with the wildtype hGRa. Bars represent mean SEM of at least five independent experiments. (b) The hGRaH726R did not exert a dominant negative effect upon the hGRa-mediated transcriptional activity. Bars represent mean SEM of at least five independent experiments.
the transcriptional activity of NF-jB in response to increasing concentrations of dexamethasone.
The hGRaH726R has a lower affinity for the ligand compared with the wild-type hGRa Dexamethasone-binding assays showed that the affinity of the hGRaH726R for the ligand was 55% lower than that of the wildtype hGRa (Kd = 20.7 3.25 nM vs. 9.2 3.8 nM). No difference in the number of hGR-binding sites was noted between the wild-type and mutant receptors. Western blot analyses demonstrated similar expression of hGRa and hGRaH726R proteins in COS-7 cells.
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In the absence of ligand, the hGRa was primarily localized in the cytoplasm of cells. Exposure to dexamethasone at a concentration of 10 6 M resulted in translocation of the wild-type receptor into the nucleus within 15 min (mean SE, 15.4 0.4 min) (Fig. 3a). The mutant receptor hGRaH726R was also observed in the cytoplasm of cells in the absence of ligand. However, ligand-induced activation of the mutant receptor resulted in slower translocation into the nucleus, which required 60 min (mean SE, 63.1 1.6 min) (Fig. 3b). These findings indicate that the mutant receptor hGRaH726R displays a four-fold delay in nuclear translocation compared with the wild-type hGRa.
We investigated the in vitro interaction among the mutant receptor and the GRIP1 coactivator in GST pull-down assays. GRIP1 contains two sites that bind to steroid receptors. One site, the nuclear receptor-binding (NRB) site, is located between amino acids 542 and 745 and interacts with the AF-2 of hGRa in a ligand-dependent fashion. The other site is located at the carboxyterminus of GRIP1, between amino acids 1121 and 1250, and binds to the AF-1 of hGRa in a ligand-independent fashion [31–33]. The wild-type receptor bound to full-length GRIP1, NRB site of GRIP1 and the carboxyterminal fragment of GRIP1. However, the mutant receptor hGRaH726R interacted with the full length and carboxyterminal fragment of GRIP1 but not with the NRB fragment of GRIP1 (Fig. 5a). These findings indicate that the hGRaH726R interacts with the GRIP1 coactivator in vitro mostly through its AF-1 domain due to a defective interaction through its AF-2 domain (Fig. 5b).
Helix 10 in the LBD of the hGRaH726R displays reduced flexibility due to the formation of stronger hydrogen bonds The 3D molecular modelling study of the H726R mutation revealed a significant structural shift in the rigidity of helix 10. The conjugated imidazole ring of the wild-type histidine is well balanced via pi-stacking and hydrophobic interactions with Lys771. Moreover, the = N- atom of the imidazole ring acts as a
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(a)
0 min
3 min
6 min
9 min
12 min
15 min
(b)
0 min
Figure 3 (a) Following dexamethasoneinduced activation, the wild-type hGRa translocated into the nucleus within 15 min. (b) The cytoplasmic-to-nuclear translocation of the mutant receptor hGRaH726R required 60 min.
hydrogen acceptor via a side chain bond from Tyr602. These bonds are strong enough to stabilize the side chain of His726, but still allow enough degrees of freedom to the overall structural arrangement of helix 10. A dihedral energy plot of the His726 wild-type residue revealed that there are more than one allowed rotamer conformations for this amino acid, which confirms the hypothesis of structural flexibility for the latter (Fig. 6a–c and b–d). On the other hand, the arginine residue of hGRaH726R has a rather longer side chain compared with the histidine side chain. As a result, the amino groups of the arginine’s side chain venture far deeper into the core of the protein and pick up two new hydrogen bonds (Fig. 6f). The first one is a backbone donor bond from Leu772, and the second one is a side chain acceptor bond with Ser674. These two hydrogen bonds are far stronger than the hydrophobic interactions of His726 and therefore stabilize the arginine residue in the core of the protein (Fig. 6e,f). Consequently, helix 10 is fixed in space, having lost its flexibility that is very important for the formation of the interface for protein–protein interaction of these
10 min
40 min
20 min
50 min
30 min
60 min
proteins. The dihedral energy plot of the Arg726 mutant residue confirmed that there is only one rotamer conformation for the longer arginine side chain that can be accommodated in the original His726 position (Fig. S1).
Discussion In the present study, we present the clinical manifestations and hormonal findings of a patient with Chrousos syndrome harboring a novel heterozygous point mutation in the hGR gene. The A > G transition at nucleotide position 2177 resulted in His to Arg substitution at amino acid position 726 of the LBD of the receptor. Functional characterization studies demonstrated that the mutant receptor hGRaH726R displayed decreased transcriptional and transrepressive activities, had 55% lower affinity for the ligand, required longer time to translocate into the nucleus, and interacted with the GRIP1 coactivator mostly through its AF-1 domain. The hGRaH726R did not exert a dominant negative effect upon the hGRa-mediated
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NS
hGR activation (OD 450 nm)
1·2 1 0·8 0·6 0·4 0·2
0 Dexamethasone hGRα
–
+
–
+
WT
WT
H726R
H726R
–
+
–
GRIP1 (559-774)
GRIP1 (1-1462)
(a)
GRIP1 (740-1217)
Figure 4 The hGRaH726R preserves its ability to associate to GREs following dexamethasone-induced activation. NS: nonstatistically significant.
+
–
+
WT H726R
(b)
hGRαH726R
GRIP1 NRB/AF2
C-terminal/AF1
Figure 5 (a) GST pull-down assay. In the presence of dexamethasone, the mutant receptor hGRaH726R did not interact with the NRB site of GRIP1 coactivator through its AF-2 domain. (b) Linearized GRIP1 coactivator and interaction sites with the mutant receptor hGRaH726R. AF-1: activation function 1 domain; AF-2: activation function 2 domain; C-terminal: carboxyterminal fragment of GRIP1; NRB: nuclear receptorbinding site of GRIP1.
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transcriptional activity and preserved its ability to bind to GREs. These findings indicate that the hGRaH726R causes Chrousos syndrome by affecting multiple steps in the glucocorticoid signalling cascade. The decreased affinity of the mutant receptor for the ligand most likely reflects the location of the mutation H726R in the LBD of hGRa. The structure of the hGR LBD contains 12 ahelices and four small b-strands that fold into a three-layer helical domain [34,35]. 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 of the protein, thereby creating a cavity in the bottom half of the LBD, where the agonist molecule is bound. Helix 12 packs against helices 3, 4 and 10 as an integrated part of the domain structure and plays a critical role in the formation of both the ligand-binding pocket and the AF-2 surface, which facilitates the interaction of the receptor with coactivators. Upon ligand-induced activation, the receptor undergoes major conformational changes that alter the position of helices 10, 11 and 12, and generate an interaction surface that allows coactivators to bind to AF-2 through their LXXLL motifs [34,35]. The substitution of His to Arg at amino acid position 726 of helix 10 in our patient resulted in the formation of two new strong hydrogen bonds with neighbouring amino acids fixing helix 10 in space. Consequently, helix 10 lost its molecular flexibility and became less able to interact with other molecules, including the ligand or other proteins. These conformational changes may explain the reduced affinity of the mutant receptor for the ligand, its decreased ability to transrepress NF-jBresponsive genes (possibly owing to a weaker interaction with the p65 subunit of the NF-jB transcription factor), as well as its impaired interaction with the GRIP1 coactivator in vitro. Furthermore, the mutant receptor hGRaH726R may also display an abnormal interaction with other AF-2-associated proteins, such as the p300/CBP cointegrators and components of the DRIP/ TRAP complex [1,2,6,10]. Finally, our nuclear translocation studies demonstrated a significant delay in the translocation of the hGRaH726R into the nucleus compared with the hGRa. This is likely to reflect either a defective nuclear localization sequence (NLS) 1 and/or NLS 2, or the lower affinity of the mutant receptor for the ligand. Our findings of the molecular mechanisms through which the mutation H726R impairs hGRa function concur with previous reports of other mutations in helix 10 of the receptor and highlight the importance of an integral structure of the LBD of hGRa in normal glucocorticoid signal transduction [23]. The patient was treated with high doses of dexamethasone, which ameliorated the clinical manifestations of the condition. Based on our in vitro findings, the exposure of the patient’s glucocorticoid-responsive tissues to high concentrations of dexamethasone resulted in the activation of the defective
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Figure 6 3D modelling study of the wildtype hGRa structure (turquoise colour, left column) and the hGRaH726R model (orange colour, right column). (a) The full structure of hGRa. The histidine region has been highlighted in the transparent cloud. (b) The 3D model of hGRaH726R. The area in close proximity to the introduced arginine residue has been highlighted in the transparent cloud. (c) The histidine 3D interaction scheme in the hGRa. (d) The arginine 3D interaction scheme in the hGRaH726R. (e) The histidine 2D interaction plot in the hGRa. (f) The arginine 2D interaction plot in the hGRaH726R.
(a)
(b)
(c)
(d)
(e)
(f)
hGRaH726R, which effectively restored the activity of the negative feedback loops of the HPA axis leading to normalization of plasma ACTH and serum androgens concentrations. Indeed, all the clinical manifestations due to androgen excess subsided, while the reported anxiety, possibly due to the inferred increased concentrations of CRH, improved substantially. In summary, the mutant receptor hGRaH726R causes Chrousos syndrome by affecting multiple steps in the molecular mechanisms of glucocorticoid signal transduction through reduced flexibility of helix 10 of the LBD of the receptor. Acknowledgements This work was supported by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the
National Strategic Reference Framework (NSRF) – Research Funding Program: THALIS – University of Athens (UOA), Athens, Greece. Author contributions EBG was responsible for the clinical management of the patient. NCN, DV, MLR, SK and EC conceived and designed the experiments. NCN, DV, MLR and AS performed the experiments. NCN, DV, AMGP, PM, AS, SK and EC analysed the data. NCN and EC wrote the manuscript. Address Division of Endocrinology, Metabolism and Diabetes, First Department of Pediatrics, University of Athens Medical School, ‘Aghia Sophia’ Children’s Hospital, Athens 11527, Greece (N. C. Nicolaides, M. L. Roberts, A. Sertedaki, E. Charmandari);
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Division of Endocrinology and Metabolism, Clinical Research Center, Biomedical Research Foundation of the Academy of Athens, Athens 11527, Greece (N. C. Nicolaides, M. L. Roberts, A. Sertedaki, E. Charmandari); Division of Endocrinology, Diabetes, and Bone Diseases, Icahn School of Medicine at Mount Sinai School, New York, NY 10029, USA (E. B. Geer); Bioinformatics and Medical Informatics Team, Biomedical Research Foundation of the Academy of Athens, Athens 11527, Greece (D. Vlachakis, S. Kossida); Department of Biochemistry and Biotechnology, University of Thessaly, Larissa 41221, Greece (A.-M. G. Psarra); Department of Clinical Biochemistry, University of Athens Medical School, ‘Attiko’ Hospital, Athens 12462, Greece (P. Moutsatsou); IMGT, the international ImMunoGeneTics information system, Institute of Human Genetics, 34396 Montpellier Cedex 5, France (S. Kossida). Correspondence to: Nicolas C. Nicolaides, Division of Endocrinology and Metabolism, Clinical Research Center, Biomedical Research Foundation of the Academy of Athens, 4 Soranou tou Efessiou Street, Athens 11527, Greece. Tel.: +30 210 6597041; fax: +30 210 6597545; e-mail: nnicolaides@ bioacademy.gr Received 6 April 2015; accepted 26 May 2015
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References 1 Nicolaides NC, Charmandari E, Chrousos GP, Kino T. Recent advances in the molecular mechanisms determining tissue sensitivity to glucocorticoids: novel mutations, circadian rhythm and ligand-induced repression of the human glucocorticoid receptor. BMC Endocr Disord 2014;14:71. 2 Nicolaides NC, Galata Z, Kino T, Chrousos GP, Charmandari E. The human glucocorticoid receptor: molecular basis of biologic function. Steroids 2010;75:1–12. 3 Charmandari E, Kino T, Chrousos GP. Primary generalized familial and sporadic glucocorticoid resistance and hypersensitivity. Endocr Dev 2013;24:67–85. 4 Charmandari E. Primary generalized glucocorticoid resistance and hypersensitivity: the end-organ involvement in the stress response. Sci Signal 2012;5:pt5. 5 Charmandari E. Primary generalized glucocorticoid resistance and hypersensitivity. Horm Res Paediatr 2011;76:145–55. 6 Charmandari E, Kino T, Ichijo T, Chrousos GP. Generalized glucocorticoid resistance: clinical aspects, molecular mechanisms, and implications of a rare genetic disorder. J Clin Endocrinol Metab 2008;93:1563–72. 7 Chrousos G. Q&A: primary generalized glucocorticoid resistance. BMC Med 2011;9:27. 8 Charmandari E, Kino T, Souvatzoglou E, Vottero A, Bhattacharyya N, Chrousos GP. Natural glucocorticoid receptor mutants causing generalized glucocorticoid resistance: molecular genotype, genetic transmission, and clinical phenotype. J Clin Endocrinol Metab 2004;89:1939–49. 9 Charmandari E, Chrousos GP, Kino T. Identification of natural human glucocorticoid receptor (hGR) mutations or polymorphisms
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18
19
20
21
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and their functional consequences at the hormone-receptor interaction level. Methods Mol Biol 2009;590:33–60. Chrousos GP, Vingerhoeds A, Brandon D, Eil C, Pugeat M, DeVroede M et al. Primary cortisol resistance in man: a glucocorticoid receptor-mediated disease. J Clin Invest 1982;69:1261–9. Hurley DM, Accili D, Stratakis CA, Karl M, Vamvakopoulos N, Rorer E et al. Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance. J Clin Invest 1991;87:680–6. Karl M, Lamberts SW, Detera-Wadleigh SD, Encio IJ, Stratakis CA, Hurley DM et al. Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene. J Clin Endocrinol Metab 1993;76:683–9. Malchoff DM, Brufsky A, Reardon G, McDermott P, Javier EC, Bergh CH et al. A mutation of the glucocorticoid receptor in primary cortisol resistance. J Clin Invest 1993;91:1918–25. Karl M, Lamberts SW, Koper JW, Katz DA, Huizenga NE, Kino T et al. Cushing’s disease preceded by generalized glucocorticoid resistance: clinical consequences of a novel, dominant-negative glucocorticoid receptor mutation. Proc Assoc Am Physicians 1996;108:296–307. Kino T, Stauber RH, Resau JH, Pavlakis GN, Chrousos GP. Pathologic human GR mutant has a transdominant negative effect on the wild-type GR by inhibiting its translocation into the nucleus: importance of the ligand-binding domain for intracellular GR trafficking. J Clin Endocrinol Metab 2001;86:5600–8. Ruiz M, Lind U, Gafvels M, Eggertsen G, Carlstedt-Duke J, Nilsson L et al. Characterization of two novel mutations in the glucocorticoid receptor gene in patients with primary cortisol resistance. Clin Endocrinol 2001;55:363–71. Charmandari E, Kino T, Ichijo T, Zachman K, Alatsatianos A, Chrousos GP. Functional characterization of the natural human glucocorticoid receptor (hGR) mutants hGRaR477H and hGRaG679S associated with generalized glucocorticoid resistance. J Clin Endocrinol Metab 2006;91:1535–43. Mendonca BB, Leite MV, de Castro M, Kino T, Elias LL, Bachega TA et al. Female pseudohermaphroditism caused by a novel homozygous missense mutation of the GR gene. J Clin Endocrinol Metab 2002;87:1805–9. Vottero A, Kino T, Combe H, Lecomte P, Chrousos GP. A novel, C-terminal dominant negative mutation of the GR causes familial glucocorticoid resistance through abnormal interactions with p160 steroid receptor coactivators. J Clin Endocrinol Metab 2002;87:2658–67. Charmandari E, Raji A, Kino T, Ichijo T, Tiulpakov A, Zachman K et al. 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. J Clin Endocrinol Metab 2005;90:3696–705. Charmandari E, Kino T, Ichijo T, Jubiz W, Mejia L, Zachman K et al. A novel point mutation in helix 11 of the ligand-binding domain of the human glucocorticoid receptor gene causing generalized glucocorticoid resistance. J Clin Endocrinol Metab 2007;92:3986–90. McMahon SK, Pretorius CJ, Ungerer JP, Salmon NJ, Conwell LS, Pearen MA et al. Neonatal complete generalized glucocorticoid resistance and growth hormone deficiency caused by a novel homozygous mutation in Helix 12 of the ligand binding domain of the glucocorticoid receptor gene (NR3C1). J Clin Endocrinol Metab 2010;95:297–302.
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23 Nader N, Bachrach BE, Hurt DE, Gajula S, Pittman A, Lescher R et al. A novel point mutation in the helix 10 of the human glucocorticoid receptor causes Generalized Glucocorticoid Resistance by disrupting the structure of the ligand-binding domain. J Clin Endocrinol Metab 2010;95:2281–5. 24 Zhu HJ, Dai YF, Wang O, Li M, Lu L, Zhao WG et al. Generalized glucocorticoid resistance accompanied with an adrenocortical adenoma and caused by a novel point mutation of human glucocorticoid receptor gene. Chin Med J 2011;124: 551–5. 25 Roberts ML, Kino T, Nicolaides NC, Hurt DE, Katsantoni E, Sertedaki A et al. A novel point mutation in the DNA-binding domain (DBD) of the human glucocorticoid receptor causes primary generalized glucocorticoid resistance by disrupting the hydrophobic structure of its DBD. J Clin Endocrinol Metab 2013;98: E790–5. 26 Nicolaides NC, Roberts ML, Kino T, Braatvedt G, Hurt DE, Katsantoni E et al. A novel point mutation of the human glucocorticoid receptor gene causes primary generalized glucocorticoid resistance through impaired interaction with the LXXLL motif of the p160 coactivators: dissociation of the transactivating and transreppressive activities. J Clin Endocrinol Metab 2014;99:E902–7. 27 Biggadike K1, Bledsoe RK, Hassell AM, Kirk BE, McLay IM, Shewchuk LM et al. X-ray crystal structure of the novel enhancedaffinity glucocorticoid agonist fluticasone furoate in the glucocorticoid receptor-ligand binding domain. J Med Chem 2008;51:3349–52. 28 Molecular Operating Environment (MOE 2014.09) Montreal, Quebec, Canada: Chemical Computing Group, Inc.; Available at: http://www.chemcomp.com. 29 Hess B, Kutzner C, van der Spoel D, Lindahl E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput 2008;4:435–47.
30 Van der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ. GROMACS: fast, flexible, and free. J Comput Chem 2005;26:1701–18. 31 Ding XF, Anderson CM, Ma H, Hong H, Uht RM, Kushner PJ et al. Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1): multiple motifs with different binding specificities. Mol Endocrinol 1998;12:302–13. 32 Hong H, Kohli K, Garabedian MJ, Stallcup MR. GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. Mol Cell Biol 1997;17:2735–44. 33 Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR. GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 1996;93:4948–52. 34 Bledsoe RK, Montana VG, Stanley TB, Delves CJ, Apolito CJ, McKee DD et al. Crystal structure of the glucocorticoid receptor ligandbinding domain reveals a novel mode of receptor dimerization and coactivator recognition. Cell 2002;110:93–105. 35 Kauppi B, Jakob C, F€arneg ardh M, Yang J, Ahola H, Alarcon M et al. The three-dimensional structures of antagonistic and agonistic forms of the glucocorticoid receptor ligand-binding domain: RU-486 induces a transconformation that leads to active antagonism. J Biol Chem 2003;278:22748–54.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Dihedral energy plots.
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ORIGINAL ARTICLE A novel mutation of the hGR gene causing Chrousos syndrome Nicolas C. Nicolaides*,†, Eliza B. Geer‡, Dimitrios Vlachakis§, Michael L. Roberts*,†, Anna-Maria G. Psarra¶, Paraskevi Moutsatsou**, Amalia Sertedaki*,†, Sophia Kossida§,†† and Evangelia Charmandari*,† *
Division of Endocrinology, Metabolism and Diabetes, First Department of Pediatrics, ‘Aghia Sophia’ Children’s Hospital, University of Athens Medical School, Athens, Greece, †Division of Endocrinology and Metabolism, Clinical Research Center, Biomedical Research Foundation of the Academy of Athens, Athens, Greece, ‡Division of Endocrinology, Diabetes, and Bone Diseases, Icahn School of Medicine at Mount Sinai School, New York, NY, USA, §Bioinformatics and Medical Informatics Team, Biomedical Research Foundation of the Academy of Athens, Athens, Greece, ¶Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece, **Department of Clinical Biochemistry, ‘Attiko’ Hospital, University of Athens Medical School, Athens, Greece, ††IMGT, The International ImMunoGeneTics Information System, Institute of Human Genetics, Montpellier, France
ABSTRACT Background Natural mutations in the human glucocorticoid receptor (hGR, NR3C1) gene cause Chrousos syndrome, a rare condition characterized by generalized, partial, target-tissue insensitivity to glucocorticoids. Objective To present a new case of Chrousos syndrome caused by a novel mutation in the hGR gene, and to elucidate the molecular mechanisms through which the natural mutant receptor affects glucocorticoid signal transduction. Design and Results The index case presented with hirsutism, acne, alopecia, anxiety, fatigue and irregular menstrual cycles, but no clinical manifestations suggestive of Cushing’s syndrome. Endocrinologic evaluation revealed elevated 08:00 h plasma adrenocorticotropic hormone, serum cortisol and androstenedione concentrations and increased urinary free cortisol excretion. The patient harbored a novel A > G transition at nucleotide position 2177, which resulted in histidine (H) to arginine (R) substitution at amino acid position 726 of the receptor (c.2177A > G, p.H726R). Compared with the wild-type receptor, the mutant receptor hGRaH726R demonstrated decreased ability to transactivate glucocorticoid-responsive genes and to transrepress the nuclear factor-jB signalling pathway, displayed 55% lower affinity for the ligand and a four-fold delay in nuclear translocation, and interacted with the glucocorticoid receptor-interacting protein 1 coactivator mostly through its activation function-1 domain. Finally, a 3-dimensional molecular modelling study of the H726R mutation revealed a significant structural shift in the rigidity of helix 10 of the receptor, which resulted in reduced flexibility and decreased affinity of the mutant receptor for binding to the ligand. Conclusions The natural mutant receptor hGRaH726R impairs multiple steps of glucocorticoid signal transduction, thereby decreasing tissue sensitivity to glucocorticoids. Keywords Chrousos syndrome, glucocorticoid receptor, glucocorticoid signalling, mutations. Eur J Clin Invest 2015; 45 (8): 782–791
Introduction Mutations in the human GR gene identified in human patients impair the molecular mechanisms of hGR action and alter tissue sensitivity to glucocorticoids [1,2]. The majority of these mutations have been associated with Chrousos syndrome, which is a rare, familial or sporadic condition characterized by generalized, partial, target-tissue insensitivity to glucocorticoids (Table 1) [3–26]. Affected subjects have compensatory
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activation of the hypothalamic–pituitary–adrenal (HPA) axis and elevated circulating 24-h serum cortisol and plasma adrenocorticotropic hormone (ACTH) concentrations, which maintain circadian rhythmicity and appropriate responsiveness to stressors, as well as resistance of the HPA axis to dexamethasone suppression without any clinical manifestations of hypercortisolism [3–26]. The increased secretion of ACTH often results in increased production of adrenal steroids with androgenic and/or mineralocorticoid activities and the
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Table 1 Mutations of the human glucocorticoid receptor gene causing Chrousos syndrome Author (References)
cDNA
Amino acid
Genotype
Phenotype
Chrousos et al. [10]; Hurley et al. [11]; Charmandari et al. [8]
1922 (A?T)
641 (D?V)
Homozygous
Hypertension, Hypokalemic alkalosis
Karl et al. [12]
4 bp deletion in exon-intron 6
Heterozygous
Hirsutism, Male-pattern hair-loss, Menstrual irregularities
Malchoff et al. [13.]; Charmandari et al. [8]
2185 (G?A)
729 (V?I)
Homozygous
Precocious puberty, Hyperandrogenism
Karl et al. [14]; Kino et al. [15]; Charmandari et al. [8]
1676 (T?A)
559 (I?N)
Heterozygous
Hypertension, Oligospermia, Infertility
Ruiz et al. [16]; Charmandari et al. [17]
1430 (G?A)
477 (R?H)
Heterozygous
Hirsutism, Fatigue, Hypertension
Ruiz et al. [16]; Charmandari et al. [17]
2035 (G?A)
679 (G?S)
Heterozygous
Hirsutism, Fatigue, Hypertension
Mendonca et al. [18]; Charmandari et al. [8]
1712 (T?C)
571 (V?A)
Homozygous
Ambiguous genitalia, Hypertension, Hypokalaemia, Hyperandrogenism
Vottero et al. [19]; Charmandari et al. [8]
2241 (T?G)
747 (I?M)
Heterozygous
Cystic acne, Hirsutism, Oligo-amenorrhoea
Charmandari et al. [20]
2318 (T?C)
773 (L?P)
Heterozygous
Fatigue, Anxiety, Acne, Hirsutism, Hypertension
Charmandari et al. [21]
2209 (T?C)
737 (F?L)
Heterozygous
Hypertension, Hypokalaemia
McMahon et al. [22]
2 bp deletion at nt 2318-9
773
Homozygous
Hypoglycaemia, Fatigability with feeding, Hypertension
Nader et al. [23]
2141 (G?A)
714 (R?Q)
Heterozygous
Hypoglycaemia, Hypokalaemia, Hypertension, Mild clitoromegaly, Advanced bone age, Precocious pubarche
Zhu Hui-juan et al. [24]
1667 (G?T)
556 (T?I)
Heterozygous
Adrenal incidentaloma
Roberts et al. [25]
1268 (T?C)
423 (V?A)
Heterozygous
Fatigue, Anxiety, Hypertension
Nicolaides et al. [26]
1724 (T?G)
575 (V?G)
Heterozygous
Melanoma, Asymptomatic daughters
Numbers in the parentheses following authors’ names indicate the corresponding references.
corresponding clinical phenotype [3–26]. The clinical spectrum of the condition is broad, ranging from most severe to mild cases of mineralocorticoid and/or androgen excess, while a number of patients may be asymptomatic, displaying biochemical alterations only [3–26]. In this study, we identified a case of Chrousos syndrome caused by a novel heterozygous point mutation in the hGR gene and investigated the molecular mechanisms through which the natural mutant receptor impairs glucocorticoid signal transduction.
Case report A 30-year-old woman presented with a long-standing history of hirsutism, acne, diffuse alopecia, fatigue, anxiety and irregular menstrual cycles. She was otherwise asymptomatic and had no
clinical manifestations of Cushing’s syndrome. Hormonal evaluation revealed elevated 08:00 h plasma ACTH [207 pg/mL, normal range (nr) < 52)], serum cortisol (26 lg/dL) and androstenedione (252 ng/dL, nr < 235) concentrations and increased urinary free cortisol (UFC) excretion (97–122 lg/day; nr: < 50). Resistance of the HPA axis to overnight dexamethasone suppression was found. A pituitary magnetic resonance imaging scan was normal. The above clinical, hormonal and imaging findings suggested the diagnosis of Chrousos syndrome. Written informed consent was obtained from the patient, and further molecular studies were undertaken. Following treatment with dexamethasone at a dose of 1 mg per os at night, the clinical manifestations of the condition subsided, and the concentrations of plasma ACTH and serum androgens were normalized.
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Materials and methods Sequencing of the patient’s hGR gene Genomic DNA was isolated from peripheral blood lymphocytes employing the Maxwell 16 instrument for automated DNA extraction (Promega Corporation, Madison, WI, USA). The entire coding region of the hGR gene (NR3C1; NM 001018074.1), exons 2–9 and their intronic flanking sequences, were PCR amplified and bidirectionally sequenced using the Big Dye Terminator cycle sequencing kit 3.1 (Applied Biosystems, Life Technologies, Foster City, CA, USA). The sequencing reactions were analysed on the automated genetic analyzer ABI 3500 (Applied Biosystems, Life Technologies). Three in silico software were used to predict the impact of the amino acid change on the protein function: PolyPhen-2 (Polymorphism Phenotyping v2), Mutation Taster and SIFT (Sorting Intolerant From Tolerant).
Plasmids The plasmids used in the following studies included pRShGRa, pF25GFP-hGRa, pMMTV-luc, pGL473[hRluc/SV40], pRSVerbA 1, pGEX4T3-GRIP1(1–1462), pGEX4T3-GRIP1(596–774), pGEX4T3-GRIP1(740–1217), pRSVC(p50)-NF-jB, pRSVC(p65)RelA and p(IjB)3-luc, and have been previously described [20,26]. The plasmids pRShGRaH726R and pF25GFPhGRaH726R were created by introducing the H726R mutation into the pRShGRa and pF25GFP-hGRa plasmids, respectively, using the QuickChange II XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA).
Transactivation and transrepression assays CV-1 cells were transiently cotransfected with pRShGRa or pRShGRaH726R (0.05 lg/well), pMMTV-luc (0.5 lg/well) and pGL473[hRluc/SV40] (0.1 lg/well) for the transactivation assays, or with pRSVC(p50)-NF-jB (0.05 lg/well), pRSVC (p65)-RelA (0.05 lg/well), p(IjB)3-luc (0.5 lg/well) for the transrepression assays, using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA). Forty-eight hours after transfection, cells were incubated with increasing concentrations of dexamethasone for an additional 24 h. Cells were then lysed with 59 Reporter Lysis Buffer (Promega Corporation), and firefly and renilla luciferase activities were, respectively, determined using the Dual-Luciferase Reporter Assay System (Promega) [20,26].
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brane. The latter was incubated with anti-hGR and anti-actin antibodies. Western blot analyses were performed as previously described [20,26].
Dexamethasone-binding assays COS-7 cells were transiently transfected with pRShGRa or pRShGRaH726R (1.5 lg/well) using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were incubated with six progressively increasing concentrations of [1,2,4,6,7-3H] dexamethasone (PerkinElmer Waltham, MA, USA) in the presence or absence of a 500-fold molar excess of nonradioactive dexamethasone for 1 h. Dexamethasone-binding assays were performed as previously described [20,26].
Subcellular localization and nuclear translocation studies HeLa cells were transiently transfected with pF25GFP-hGRa or pF25GFP-hGRaH726R (2 lg/well) using FuGENE 6 (Roche Diagnostics Corporation, Indianapolis, IN, USA). The subcellular localization and cytoplasmic-to-nuclear translocation of the wild-type and mutant receptors were examined before and after the addition of 10 6 M dexamethasone under a fluorescence microscope (DM IRB; Leica, Wetzlar, Germany). Representative images were captured using a digital charge-coupled device camera (Hamamatsu Photonics K.K., Hamamatsu, Japan) [20,26].
In vitro binding assays for evaluation of the GR/GRE association HCT116 cells were transiently transfected with pRShGRa or pRShGRaH726R (15 lg/flask) using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were incubated with 10 6 M dexamethasone for 3 h. Nuclear extracts were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA). The association of hGRa or hGRaH726R to glucocorticoid-response elements (GREs) was investigated by in vitro binding assays using the TRansAMTM GR Kit (Active Motif) according to the instructions of the manufacturer.
Glutathione S-transferase (GST) pull-down assays GST-fused glucocorticoid receptor-interacting protein 1 (GRIP1) (1–1462), GRIP1 (559–774) and GRIP1 (740–1217) were bacterially produced, purified and immobilized on GST beads. The in vitro interaction of pRShGRa and pRShGRaH726R with the GST-fused GRIP1 proteins was tested as previously described [17,20,21,25,26].
Western blot analyses CV-1 and COS-7 cells were transiently transfected with pRShGRa or pRShGRaH726R (15 lg/flask) using Lipofectamine 2000 (Invitrogen). Equal amounts of cell lysates were run on 8% SDS-PAGE gel and blotted onto nitrocellulose mem-
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Structural biology studies Three-dimentional (3D) coordinates of the wild-type hGRa were obtained from the X-ray solved, crystal structure with Research Collaboratory for Structural Bioinformatics (RCSB)
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code: 3CLD, which is the ligand-binding domain of the hGRa complexed with fluticasone furoate [27]. The resolution of the X-ray structures is 284 A°. The hGRaH726R mutant structure was manually mutated using molecular operating environment (MOE) [28]. The hGRa and hGRaH726R molecular systems were initially subjected to energy minimization using the Gromacs-implemented force-fields to remove the geometrical strain [29,30]. The 3D structures were subsequently solvated with simple point charge (SPC) water using the truncated from the model. Molecular octahedron box extending to 7 A dynamics were performed after that at 300K, 1 atm with 2 fs step size, and for a total of one microsecond, using the number, volume, and temperature (NVT) ensemble in a canonical environment; NVT stands for number of atoms, volume and temperature that remain constant throughout the simulation. The 2D maps were drawn using the built-in module of MOE.
Results Identification of a novel heterozygous A ? G mutation in the patient’s hGR gene Direct sequencing of the entire coding region and the exon– intron boundaries of the hGRa gene revealed a novel heterozygous A > G transition at nucleotide position 2177, which resulted in histidine (His, H) to arginine (Arg, R) substitution at amino acid position 726 in exon 8 (c.2177A > G, p.H726R), in helix 10 of the ligand-binding domain (LBD) of the receptor (Fig. 1a,b). The 3 in silico software employed to predict the
effect of the amino acid change indicated that the p.H726R is probably damaging (Polyphen-2, score 0999), disease causing (Mutation Taster, prob: 0999999999296905) and affects protein function (SIFT, score 003).
The hGRaH726R displays decreased ability to transactivate glucocorticoid-responsive genes compared with the wild-type hGRa and does not exert a dominant negative effect upon the hGRamediated transcriptional activity The transactivation assays showed that the hGRaH726R displayed a statistically significant reduction (40%) in its ability to transactivate the glucocorticoid-inducible mouse mammary tumour virus (MMTV) promoter in response to increasing concentrations of dexamethasone, compared with the wild-type hGRa (Fig. 2a). In addition, the mutant receptor did not exert a dominant negative effect upon the hGRa-mediated transcriptional activity (Fig. 2b). Western blot analyses demonstrated no differences in the expression of hGRa and hGRaH726R proteins, indicating that the above-described alterations in hGRmediated transactivation of the MMTV promoter did not reflect differences at the protein expression level.
The hGRaH726R demonstrates decreased ability to transrepress the nuclear factor (NF)-jB-responsive genes compared with the wild-type hGRa Compared with the wild-type receptor, the hGRaH726R demonstrated significantly decreased ability (~30%) to transrepress
(a) aa
Figure 1 (a) Sequencing of the NR3C1 gene revealed a heterozygous A > G transition at nucleotide position 2177 (indicated by the black arrow), which resulted in the replacement of histidine (His, H) to arginine (Arg, R) at amino acid position 726, c.2177A > G, p.H726R, in the helix 10 of the LBD of the receptor. (b) Crystal structure of the LBD of hGRa. The yellow arrow indicates the position of the mutation in helix 10 of the agonist-bound conformation of the receptor.
722
723
724
725
726
727
728
729
(b)
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Relative luciferase activity
(a) 0·1
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The hGRaH726R displays a four-fold delay in the cytoplasmic-to-nuclear translocation compared with the wild-type hGRa
CV-1 MMTV
0·08
WT - - - - H726R
0·06 0·04 0·02 0
10–8 10–12 10–10 10–6 Dexamethasone (M)
0
Relative luciferase activity
(b) 0·12 0·1
10–5
CV-1 MMTV
The hGRaH726R preserves its ability to bind to DNA
0·08
In vitro binding assays revealed no significant difference in the association of hGRaH726R to GREs following exposure to dexamethasone compared with the hGRa (Fig. 4). These findings indicate that the mutant receptor preserves its ability to bind to DNA.
0·06 0·04 0·02
Dexamethasone 0 WT:H726R Ratio
The hGRaH726R interacts with the GRIP1 coactivator mostly through its activation function (AF)-1 domain –
+ 1:0
– 1:1
+
– 1:3
+
– 1:6
+
–
+
1:10
Figure 2 (a) The mutant receptor hGRaH726R had decreased ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to increasing concentrations of dexamethasone, compared with the wildtype hGRa. Bars represent mean SEM of at least five independent experiments. (b) The hGRaH726R did not exert a dominant negative effect upon the hGRa-mediated transcriptional activity. Bars represent mean SEM of at least five independent experiments.
the transcriptional activity of NF-jB in response to increasing concentrations of dexamethasone.
The hGRaH726R has a lower affinity for the ligand compared with the wild-type hGRa Dexamethasone-binding assays showed that the affinity of the hGRaH726R for the ligand was 55% lower than that of the wildtype hGRa (Kd = 20.7 3.25 nM vs. 9.2 3.8 nM). No difference in the number of hGR-binding sites was noted between the wild-type and mutant receptors. Western blot analyses demonstrated similar expression of hGRa and hGRaH726R proteins in COS-7 cells.
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In the absence of ligand, the hGRa was primarily localized in the cytoplasm of cells. Exposure to dexamethasone at a concentration of 10 6 M resulted in translocation of the wild-type receptor into the nucleus within 15 min (mean SE, 15.4 0.4 min) (Fig. 3a). The mutant receptor hGRaH726R was also observed in the cytoplasm of cells in the absence of ligand. However, ligand-induced activation of the mutant receptor resulted in slower translocation into the nucleus, which required 60 min (mean SE, 63.1 1.6 min) (Fig. 3b). These findings indicate that the mutant receptor hGRaH726R displays a four-fold delay in nuclear translocation compared with the wild-type hGRa.
We investigated the in vitro interaction among the mutant receptor and the GRIP1 coactivator in GST pull-down assays. GRIP1 contains two sites that bind to steroid receptors. One site, the nuclear receptor-binding (NRB) site, is located between amino acids 542 and 745 and interacts with the AF-2 of hGRa in a ligand-dependent fashion. The other site is located at the carboxyterminus of GRIP1, between amino acids 1121 and 1250, and binds to the AF-1 of hGRa in a ligand-independent fashion [31–33]. The wild-type receptor bound to full-length GRIP1, NRB site of GRIP1 and the carboxyterminal fragment of GRIP1. However, the mutant receptor hGRaH726R interacted with the full length and carboxyterminal fragment of GRIP1 but not with the NRB fragment of GRIP1 (Fig. 5a). These findings indicate that the hGRaH726R interacts with the GRIP1 coactivator in vitro mostly through its AF-1 domain due to a defective interaction through its AF-2 domain (Fig. 5b).
Helix 10 in the LBD of the hGRaH726R displays reduced flexibility due to the formation of stronger hydrogen bonds The 3D molecular modelling study of the H726R mutation revealed a significant structural shift in the rigidity of helix 10. The conjugated imidazole ring of the wild-type histidine is well balanced via pi-stacking and hydrophobic interactions with Lys771. Moreover, the = N- atom of the imidazole ring acts as a
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(a)
0 min
3 min
6 min
9 min
12 min
15 min
(b)
0 min
Figure 3 (a) Following dexamethasoneinduced activation, the wild-type hGRa translocated into the nucleus within 15 min. (b) The cytoplasmic-to-nuclear translocation of the mutant receptor hGRaH726R required 60 min.
hydrogen acceptor via a side chain bond from Tyr602. These bonds are strong enough to stabilize the side chain of His726, but still allow enough degrees of freedom to the overall structural arrangement of helix 10. A dihedral energy plot of the His726 wild-type residue revealed that there are more than one allowed rotamer conformations for this amino acid, which confirms the hypothesis of structural flexibility for the latter (Fig. 6a–c and b–d). On the other hand, the arginine residue of hGRaH726R has a rather longer side chain compared with the histidine side chain. As a result, the amino groups of the arginine’s side chain venture far deeper into the core of the protein and pick up two new hydrogen bonds (Fig. 6f). The first one is a backbone donor bond from Leu772, and the second one is a side chain acceptor bond with Ser674. These two hydrogen bonds are far stronger than the hydrophobic interactions of His726 and therefore stabilize the arginine residue in the core of the protein (Fig. 6e,f). Consequently, helix 10 is fixed in space, having lost its flexibility that is very important for the formation of the interface for protein–protein interaction of these
10 min
40 min
20 min
50 min
30 min
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proteins. The dihedral energy plot of the Arg726 mutant residue confirmed that there is only one rotamer conformation for the longer arginine side chain that can be accommodated in the original His726 position (Fig. S1).
Discussion In the present study, we present the clinical manifestations and hormonal findings of a patient with Chrousos syndrome harboring a novel heterozygous point mutation in the hGR gene. The A > G transition at nucleotide position 2177 resulted in His to Arg substitution at amino acid position 726 of the LBD of the receptor. Functional characterization studies demonstrated that the mutant receptor hGRaH726R displayed decreased transcriptional and transrepressive activities, had 55% lower affinity for the ligand, required longer time to translocate into the nucleus, and interacted with the GRIP1 coactivator mostly through its AF-1 domain. The hGRaH726R did not exert a dominant negative effect upon the hGRa-mediated
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hGR activation (OD 450 nm)
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0 Dexamethasone hGRα
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Figure 4 The hGRaH726R preserves its ability to associate to GREs following dexamethasone-induced activation. NS: nonstatistically significant.
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hGRαH726R
GRIP1 NRB/AF2
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Figure 5 (a) GST pull-down assay. In the presence of dexamethasone, the mutant receptor hGRaH726R did not interact with the NRB site of GRIP1 coactivator through its AF-2 domain. (b) Linearized GRIP1 coactivator and interaction sites with the mutant receptor hGRaH726R. AF-1: activation function 1 domain; AF-2: activation function 2 domain; C-terminal: carboxyterminal fragment of GRIP1; NRB: nuclear receptorbinding site of GRIP1.
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transcriptional activity and preserved its ability to bind to GREs. These findings indicate that the hGRaH726R causes Chrousos syndrome by affecting multiple steps in the glucocorticoid signalling cascade. The decreased affinity of the mutant receptor for the ligand most likely reflects the location of the mutation H726R in the LBD of hGRa. The structure of the hGR LBD contains 12 ahelices and four small b-strands that fold into a three-layer helical domain [34,35]. 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 of the protein, thereby creating a cavity in the bottom half of the LBD, where the agonist molecule is bound. Helix 12 packs against helices 3, 4 and 10 as an integrated part of the domain structure and plays a critical role in the formation of both the ligand-binding pocket and the AF-2 surface, which facilitates the interaction of the receptor with coactivators. Upon ligand-induced activation, the receptor undergoes major conformational changes that alter the position of helices 10, 11 and 12, and generate an interaction surface that allows coactivators to bind to AF-2 through their LXXLL motifs [34,35]. The substitution of His to Arg at amino acid position 726 of helix 10 in our patient resulted in the formation of two new strong hydrogen bonds with neighbouring amino acids fixing helix 10 in space. Consequently, helix 10 lost its molecular flexibility and became less able to interact with other molecules, including the ligand or other proteins. These conformational changes may explain the reduced affinity of the mutant receptor for the ligand, its decreased ability to transrepress NF-jBresponsive genes (possibly owing to a weaker interaction with the p65 subunit of the NF-jB transcription factor), as well as its impaired interaction with the GRIP1 coactivator in vitro. Furthermore, the mutant receptor hGRaH726R may also display an abnormal interaction with other AF-2-associated proteins, such as the p300/CBP cointegrators and components of the DRIP/ TRAP complex [1,2,6,10]. Finally, our nuclear translocation studies demonstrated a significant delay in the translocation of the hGRaH726R into the nucleus compared with the hGRa. This is likely to reflect either a defective nuclear localization sequence (NLS) 1 and/or NLS 2, or the lower affinity of the mutant receptor for the ligand. Our findings of the molecular mechanisms through which the mutation H726R impairs hGRa function concur with previous reports of other mutations in helix 10 of the receptor and highlight the importance of an integral structure of the LBD of hGRa in normal glucocorticoid signal transduction [23]. The patient was treated with high doses of dexamethasone, which ameliorated the clinical manifestations of the condition. Based on our in vitro findings, the exposure of the patient’s glucocorticoid-responsive tissues to high concentrations of dexamethasone resulted in the activation of the defective
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Figure 6 3D modelling study of the wildtype hGRa structure (turquoise colour, left column) and the hGRaH726R model (orange colour, right column). (a) The full structure of hGRa. The histidine region has been highlighted in the transparent cloud. (b) The 3D model of hGRaH726R. The area in close proximity to the introduced arginine residue has been highlighted in the transparent cloud. (c) The histidine 3D interaction scheme in the hGRa. (d) The arginine 3D interaction scheme in the hGRaH726R. (e) The histidine 2D interaction plot in the hGRa. (f) The arginine 2D interaction plot in the hGRaH726R.
(a)
(b)
(c)
(d)
(e)
(f)
hGRaH726R, which effectively restored the activity of the negative feedback loops of the HPA axis leading to normalization of plasma ACTH and serum androgens concentrations. Indeed, all the clinical manifestations due to androgen excess subsided, while the reported anxiety, possibly due to the inferred increased concentrations of CRH, improved substantially. In summary, the mutant receptor hGRaH726R causes Chrousos syndrome by affecting multiple steps in the molecular mechanisms of glucocorticoid signal transduction through reduced flexibility of helix 10 of the LBD of the receptor. Acknowledgements This work was supported by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the
National Strategic Reference Framework (NSRF) – Research Funding Program: THALIS – University of Athens (UOA), Athens, Greece. Author contributions EBG was responsible for the clinical management of the patient. NCN, DV, MLR, SK and EC conceived and designed the experiments. NCN, DV, MLR and AS performed the experiments. NCN, DV, AMGP, PM, AS, SK and EC analysed the data. NCN and EC wrote the manuscript. Address Division of Endocrinology, Metabolism and Diabetes, First Department of Pediatrics, University of Athens Medical School, ‘Aghia Sophia’ Children’s Hospital, Athens 11527, Greece (N. C. Nicolaides, M. L. Roberts, A. Sertedaki, E. Charmandari);
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Division of Endocrinology and Metabolism, Clinical Research Center, Biomedical Research Foundation of the Academy of Athens, Athens 11527, Greece (N. C. Nicolaides, M. L. Roberts, A. Sertedaki, E. Charmandari); Division of Endocrinology, Diabetes, and Bone Diseases, Icahn School of Medicine at Mount Sinai School, New York, NY 10029, USA (E. B. Geer); Bioinformatics and Medical Informatics Team, Biomedical Research Foundation of the Academy of Athens, Athens 11527, Greece (D. Vlachakis, S. Kossida); Department of Biochemistry and Biotechnology, University of Thessaly, Larissa 41221, Greece (A.-M. G. Psarra); Department of Clinical Biochemistry, University of Athens Medical School, ‘Attiko’ Hospital, Athens 12462, Greece (P. Moutsatsou); IMGT, the international ImMunoGeneTics information system, Institute of Human Genetics, 34396 Montpellier Cedex 5, France (S. Kossida). Correspondence to: Nicolas C. Nicolaides, Division of Endocrinology and Metabolism, Clinical Research Center, Biomedical Research Foundation of the Academy of Athens, 4 Soranou tou Efessiou Street, Athens 11527, Greece. Tel.: +30 210 6597041; fax: +30 210 6597545; e-mail: nnicolaides@ bioacademy.gr Received 6 April 2015; accepted 26 May 2015
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References 1 Nicolaides NC, Charmandari E, Chrousos GP, Kino T. Recent advances in the molecular mechanisms determining tissue sensitivity to glucocorticoids: novel mutations, circadian rhythm and ligand-induced repression of the human glucocorticoid receptor. BMC Endocr Disord 2014;14:71. 2 Nicolaides NC, Galata Z, Kino T, Chrousos GP, Charmandari E. The human glucocorticoid receptor: molecular basis of biologic function. Steroids 2010;75:1–12. 3 Charmandari E, Kino T, Chrousos GP. Primary generalized familial and sporadic glucocorticoid resistance and hypersensitivity. Endocr Dev 2013;24:67–85. 4 Charmandari E. Primary generalized glucocorticoid resistance and hypersensitivity: the end-organ involvement in the stress response. Sci Signal 2012;5:pt5. 5 Charmandari E. Primary generalized glucocorticoid resistance and hypersensitivity. Horm Res Paediatr 2011;76:145–55. 6 Charmandari E, Kino T, Ichijo T, Chrousos GP. Generalized glucocorticoid resistance: clinical aspects, molecular mechanisms, and implications of a rare genetic disorder. J Clin Endocrinol Metab 2008;93:1563–72. 7 Chrousos G. Q&A: primary generalized glucocorticoid resistance. BMC Med 2011;9:27. 8 Charmandari E, Kino T, Souvatzoglou E, Vottero A, Bhattacharyya N, Chrousos GP. Natural glucocorticoid receptor mutants causing generalized glucocorticoid resistance: molecular genotype, genetic transmission, and clinical phenotype. J Clin Endocrinol Metab 2004;89:1939–49. 9 Charmandari E, Chrousos GP, Kino T. Identification of natural human glucocorticoid receptor (hGR) mutations or polymorphisms
790
17
18
19
20
21
22
and their functional consequences at the hormone-receptor interaction level. Methods Mol Biol 2009;590:33–60. Chrousos GP, Vingerhoeds A, Brandon D, Eil C, Pugeat M, DeVroede M et al. Primary cortisol resistance in man: a glucocorticoid receptor-mediated disease. J Clin Invest 1982;69:1261–9. Hurley DM, Accili D, Stratakis CA, Karl M, Vamvakopoulos N, Rorer E et al. Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance. J Clin Invest 1991;87:680–6. Karl M, Lamberts SW, Detera-Wadleigh SD, Encio IJ, Stratakis CA, Hurley DM et al. Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene. J Clin Endocrinol Metab 1993;76:683–9. Malchoff DM, Brufsky A, Reardon G, McDermott P, Javier EC, Bergh CH et al. A mutation of the glucocorticoid receptor in primary cortisol resistance. J Clin Invest 1993;91:1918–25. Karl M, Lamberts SW, Koper JW, Katz DA, Huizenga NE, Kino T et al. Cushing’s disease preceded by generalized glucocorticoid resistance: clinical consequences of a novel, dominant-negative glucocorticoid receptor mutation. Proc Assoc Am Physicians 1996;108:296–307. Kino T, Stauber RH, Resau JH, Pavlakis GN, Chrousos GP. Pathologic human GR mutant has a transdominant negative effect on the wild-type GR by inhibiting its translocation into the nucleus: importance of the ligand-binding domain for intracellular GR trafficking. J Clin Endocrinol Metab 2001;86:5600–8. Ruiz M, Lind U, Gafvels M, Eggertsen G, Carlstedt-Duke J, Nilsson L et al. Characterization of two novel mutations in the glucocorticoid receptor gene in patients with primary cortisol resistance. Clin Endocrinol 2001;55:363–71. Charmandari E, Kino T, Ichijo T, Zachman K, Alatsatianos A, Chrousos GP. Functional characterization of the natural human glucocorticoid receptor (hGR) mutants hGRaR477H and hGRaG679S associated with generalized glucocorticoid resistance. J Clin Endocrinol Metab 2006;91:1535–43. Mendonca BB, Leite MV, de Castro M, Kino T, Elias LL, Bachega TA et al. Female pseudohermaphroditism caused by a novel homozygous missense mutation of the GR gene. J Clin Endocrinol Metab 2002;87:1805–9. Vottero A, Kino T, Combe H, Lecomte P, Chrousos GP. A novel, C-terminal dominant negative mutation of the GR causes familial glucocorticoid resistance through abnormal interactions with p160 steroid receptor coactivators. J Clin Endocrinol Metab 2002;87:2658–67. Charmandari E, Raji A, Kino T, Ichijo T, Tiulpakov A, Zachman K et al. 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. J Clin Endocrinol Metab 2005;90:3696–705. Charmandari E, Kino T, Ichijo T, Jubiz W, Mejia L, Zachman K et al. A novel point mutation in helix 11 of the ligand-binding domain of the human glucocorticoid receptor gene causing generalized glucocorticoid resistance. J Clin Endocrinol Metab 2007;92:3986–90. McMahon SK, Pretorius CJ, Ungerer JP, Salmon NJ, Conwell LS, Pearen MA et al. Neonatal complete generalized glucocorticoid resistance and growth hormone deficiency caused by a novel homozygous mutation in Helix 12 of the ligand binding domain of the glucocorticoid receptor gene (NR3C1). J Clin Endocrinol Metab 2010;95:297–302.
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23 Nader N, Bachrach BE, Hurt DE, Gajula S, Pittman A, Lescher R et al. A novel point mutation in the helix 10 of the human glucocorticoid receptor causes Generalized Glucocorticoid Resistance by disrupting the structure of the ligand-binding domain. J Clin Endocrinol Metab 2010;95:2281–5. 24 Zhu HJ, Dai YF, Wang O, Li M, Lu L, Zhao WG et al. Generalized glucocorticoid resistance accompanied with an adrenocortical adenoma and caused by a novel point mutation of human glucocorticoid receptor gene. Chin Med J 2011;124: 551–5. 25 Roberts ML, Kino T, Nicolaides NC, Hurt DE, Katsantoni E, Sertedaki A et al. A novel point mutation in the DNA-binding domain (DBD) of the human glucocorticoid receptor causes primary generalized glucocorticoid resistance by disrupting the hydrophobic structure of its DBD. J Clin Endocrinol Metab 2013;98: E790–5. 26 Nicolaides NC, Roberts ML, Kino T, Braatvedt G, Hurt DE, Katsantoni E et al. A novel point mutation of the human glucocorticoid receptor gene causes primary generalized glucocorticoid resistance through impaired interaction with the LXXLL motif of the p160 coactivators: dissociation of the transactivating and transreppressive activities. J Clin Endocrinol Metab 2014;99:E902–7. 27 Biggadike K1, Bledsoe RK, Hassell AM, Kirk BE, McLay IM, Shewchuk LM et al. X-ray crystal structure of the novel enhancedaffinity glucocorticoid agonist fluticasone furoate in the glucocorticoid receptor-ligand binding domain. J Med Chem 2008;51:3349–52. 28 Molecular Operating Environment (MOE 2014.09) Montreal, Quebec, Canada: Chemical Computing Group, Inc.; Available at: http://www.chemcomp.com. 29 Hess B, Kutzner C, van der Spoel D, Lindahl E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput 2008;4:435–47.
30 Van der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ. GROMACS: fast, flexible, and free. J Comput Chem 2005;26:1701–18. 31 Ding XF, Anderson CM, Ma H, Hong H, Uht RM, Kushner PJ et al. Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1): multiple motifs with different binding specificities. Mol Endocrinol 1998;12:302–13. 32 Hong H, Kohli K, Garabedian MJ, Stallcup MR. GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. Mol Cell Biol 1997;17:2735–44. 33 Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR. GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 1996;93:4948–52. 34 Bledsoe RK, Montana VG, Stanley TB, Delves CJ, Apolito CJ, McKee DD et al. Crystal structure of the glucocorticoid receptor ligandbinding domain reveals a novel mode of receptor dimerization and coactivator recognition. Cell 2002;110:93–105. 35 Kauppi B, Jakob C, F€arneg ardh M, Yang J, Ahola H, Alarcon M et al. The three-dimensional structures of antagonistic and agonistic forms of the glucocorticoid receptor ligand-binding domain: RU-486 induces a transconformation that leads to active antagonism. J Biol Chem 2003;278:22748–54.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Dihedral energy plots.
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