Natural Glucocorticoid Receptor Mutants Causing Generalized Glucocorticoid Resistance: Molecular Genotype, Genetic Transmission and Clinical Phenotype
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The Journal of Clinical Endocrinology & Metabolism 89(4):1939 –1949 Copyright © 2004 by The Endocrine Society doi: 10.1210/jc.2003-030450
Natural Glucocorticoid Receptor Mutants Causing Generalized Glucocorticoid Resistance: Molecular Genotype, Genetic Transmission, and Clinical Phenotype EVANGELIA CHARMANDARI, TOMOSHIGE KINO, EMMANUIL SOUVATZOGLOU, ALESSANDRA VOTTERO, NISAN BHATTACHARYYA, AND GEORGE P. CHROUSOS Pediatric and Reproductive Endocrinology Branch (E.C., T.K., E.S., A.V., G.P.C.), National Institute of Child Health and Human Development, and Diabetes Branch (N.B.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 Glucocorticoid resistance is a rare, familial, or sporadic condition characterized by partial end-organ insensitivity to glucocorticoids. The clinical spectrum of the condition ranges from completely asymptomatic to severe hyperandrogenism, fatigue, and/or mineralocorticoid excess. The molecular basis of glucocorticoid resistance in several families and sporadic cases has been ascribed to mutations in the human glucocorticoid receptor-␣ (hGR␣) gene, which impair the ability of the receptor to transduce the glucocorticoid signal. We systematically investigated the molecular mechanisms through which natural, ligand-binding domain hGR␣ mutants, including hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, and hGR␣I747M, produce a defective signal and determined whether their differential effects on hGR␣ function might account for the type of genetic transmission of the disorder and the variable clinical phenotype of the affected subjects. Our findings suggest that all five mutant receptors studied have ligand-binding domains with decreased intrinsic tran-
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LUCOCORTICOIDS REGULATE A variety of biological processes and exert profound influences on many physiological functions by virtue of their diverse roles in growth, development, and maintenance of basal and stressrelated homeostasis (1). At the cellular level, their actions are mediated by an approximately 94-kDa intracellular receptor protein, the glucocorticoid receptor (GR). This receptor belongs to the superfamily of steroid/thyroid/retinoic acid receptor proteins that function as ligand-dependent transcription factors (2–5) (Fig. 1, A and B). Alternative splicing of the human (h) GR gene in exon 9 generates two highly homologous receptor isoforms, termed ␣ and . hGR␣ is ubiquitously expressed in almost all human tissues and cells and represents the classic GR that functions as Abbreviations: AF, Activation function; CBP, cAMP response elementbinding protein-binding protein; DBD, DNA-binding domain; FBS, fetal bovine serum; GFP, green fluorescent protein; GR, glucocorticoid receptor; GRE, glucocorticoid-response element; GRIP, GR-interacting protein 1; GST, glutathione-S-transferase; hGR␣, human glucocorticoid receptor-␣; hsp90, 90-kDa heat shock protein; LBD, ligand-binding domain; MMTV, mouse mammary tumor virus; NLS, nuclear localization signal; NRB, nuclear receptor-binding; RSV, Rous sarcoma virus; SV40, simian virus 40. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.
scriptional activity. Unlike hGR␣I559N and I747M previously shown to exert a dominant negative effect upon the transcriptional activity of hGR␣, hGR␣V571A, D641V, and V729I do not have such an effect. All five mutants studied demonstrate varying degrees of decreased affinity for the ligand in a standard dexamethasone binding assay, but preserve their ability to bind DNA. The nondominant negative mutants, hGR␣V571A, D641V, and V729I, show delayed translocation into the nucleus after exposure to ligand. Finally, hGR␣I559N, V571A, D641V, and V729I display an abnormal interaction with the glucocorticoid receptor-interacting protein-1 coactivator in vitro, as this was previously shown also for hGR␣I747M. We conclude that each of the above hGR␣ mutations imparts different functional defects upon the glucocorticoid signal transduction pathway, which explains the autosomal recessive or dominant transmission of the disorder, but might only explain in part its variable clinical phenotype. (J Clin Endocrinol Metab 89: 1939 –1949, 2004)
a ligand-dependent transcription factor. In the absence of ligand, hGR␣ 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 (hsp90), and several other proteins (6). The hsp90 molecules are thought to sequester hGR␣ in the cytoplasm of cells by maintaining the receptor in a conformation that masks or inactivates its nuclear localization signals (NLSs), but empowers it to interact with glucocorticoids. Upon hormone binding, the receptor undergoes an allosteric change, which results in dissociation from hsp90 and other proteins, and unmasking/activation of the NLSs. In its new conformation, the activated, ligand-bound GR translocates into the nucleus, where it binds as homodimer to glucocorticoid-response elements (GREs) located in the promoter region of target genes. The hGR␣ then communicates with the basal transcription machinery and regulates the expression of glucocorticoid-responsive genes positively or negatively, depending on the GRE sequence and promoter context (7). 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-B (8, 9). The ability of ligand-bound hGR␣ to trans-activate steroidresponsive genes depends on the presence of coactivators, nucleoproteins with chromatin-remodeling and other enzymatic activities, that are attracted to the promoter region of
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FIG. 1. A, Schematic representation of the structure of the hGR gene. Alternative splicing of the primary transcript gives rise to the two mRNA and protein isoforms, hGR␣ and hGR (15). B, Functional domains of the hGR␣. The functional domains and subdomains are indicated beneath the linearized protein structures. C, Schematic representation of the interaction of coactivators with the AF-1 and AF-2 domains of the GR and their role in transcriptional regulation. D, Location of the known mutations of the hGR␣ gene.
the target genes via the activation function-1 (AF-1) and AF-2 of hGR␣ (10 –12) (Fig. 1C). Several families of coactivators have been described, including the p160 coactivators, such as the steroid receptor coactivator-1 and the GR-interacting protein-1 (GRIP1), the p300/cAMP response element-binding protein-binding protein (CBP) cointegrators, the p300/ CBP-associated protein, the switching/sucrose nonfermenting complex, as well as the newly described vitamin D receptor-interacting protein/thyroid hormone-associated protein complex (10 –13). The p160 and p300/CBP coactivators have multiple amphipathic LXXLL signature motifs (coactivator motifs or nuclear receptor boxes), which serve as interfaces between these coactivators and a hydrophobic cleft formed by helices 3, 4, and 12 of the nuclear receptors (14). The p160, CBP/p300, and p300/CBP-associated protein all have histone acetylase activity, which loosens the DNA strands from the nucleosomes and allows the transcriptional preinitiation complex of RNA polymerase II and its ancillary components, including the TATA-binding protein and a host
of TATA-binding protein-associated proteins, to initiate and promote transcription (10 –12). Glucocorticoid resistance is a rare, familial or sporadic condition characterized by generalized, partial end-organ insensitivity to physiological glucocorticoid concentrations (15, 16). Patients have compensatory elevations in circulating cortisol and ACTH concentrations, which maintain circadian rhythmicity and appropriate responsiveness to stressors, albeit at higher hormone concentrations, and resistance of the hypothalamic-pituitary-adrenal axis to dexamethasone suppression, but no clinical evidence of hypo- or hypercortisolism. The clinical spectrum of the condition is broad, ranging from completely asymptomatic to severe cases of hyperandrogenism, fatigue, and/or mineralocorticoid excess (15, 16). The molecular basis of glucocorticoid resistance in several families and sporadic cases has been ascribed to mutations in the hGR␣ gene, which impair one or more of the molecular mechanisms of GR function, thus altering tissue sensitivity to glucocorticoids. Inactivating mutations mostly
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FIG. 1. Continued.
within the ligand-binding domain (LBD) as well as a 4-bp deletion at the 3⬘-boundary of exon 6 of the hGR␣ gene have been described in five kindreds and three sporadic cases (17–24) (Fig. 1D). The aim of our study was to investigate the molecular mechanisms of action of natural hGR␣ mutants located in the LBD of the receptor, including hGR␣I559N, V571A, D641V, V729I, and I747M, and to determine whether their differential effects on hGR␣ function may account for the variable
clinical phenotype and genetic transmission of familial glucocorticoid resistance. Materials and Methods Plasmids The pRShGR␣ plasmid expresses the human GR␣ isoform under the control of the Rous sarcoma virus (RSV) promoter. The plasmids pRShGR␣I559N, pRShGR␣V571A, pRShGR␣D641V, pRShGR␣V729I, and pRShGR␣I747M were constructed by introducing the indicated mutations
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J Clin Endocrinol Metab, April 2004, 89(4):1939 –1949
into the pRShGR␣ plasmid using PCR-assisted site-directed mutagenesis (Stratagene, La Jolla, CA). The pM-hGR␣-LBD, pM-hGR␣I559N-LBD, pMhGR␣V571A-LBD, pM-hGR␣D641V-LBD, pM-hGR␣V729I-LBD, and pMhGR␣I747M-LBD, which express, respectively, the LBD of pRShGR␣, pRShGR␣I559N, pRShGR␣V571A, pRShGR␣D641V, pRShGR␣V729I, and pRShGR␣I747M fused to the GAL4-DNA-binding domain (GAL4-DBD), were constructed by subcloning the corresponding cDNAs into the pM vector (Clontech, Palo Alto, CA). Green fluorescent protein (GFP)-fused plasmids expressing hGR␣, hGR␣V571A, hGR␣D641V, and hGR␣V729I were constructed by subcloning the corresponding cDNAs into the pF25GFP vector, which was a gift from Dr. G. Pavlakis (National Cancer Institute, National Institutes of Health, Frederick, MD). The pGEX4T3-GRIP1-(1–1462), pGEX4T3-GRIP1-(596 –774), and pGEX4T3-GRIP1-(740 –1217) plasmids, which express glutathione-Stransferase (GST) fusions of the full-length GRIP1, an AF-2-directed hGR␣ binding site [nuclear receptor-binding (NRB) site], and an AF-1directed, hGR␣-binding site with an auxiliary nuclear receptor-interacting domain, respectively, were constructed by subcloning the corresponding GRIP1 fragments of cDNA into the pGEX4T3 plasmid (Amersham Pharmacia Biotech, Piscataway, NJ) (24). The pSG5GRIP1(1–1462) plasmid was a gift from Dr. M. Stallcup (University of Southern California, Los Angeles, CA). The plasmid pRSV-erbA⫺1, which contains a thyroid receptor cDNA in inverse orientation, was used as negative control for all hGR␣-related plasmids. The pMMTV-luc plasmid expresses luciferase under the control of the glucocorticoid-inducible mouse mammary tumor virus (MMTV) promoter and was a gift from Dr. G. L. Hager (NCI, NIH, Bethesda, MD). The p17mer-tk-luc contains the luciferase gene under the control of the four GAL4-responsive elements cloned upstream of the proximal portion of the glucocorticoid-independent herpes simplex virus thymidine kinase promoter and was a gift from Dr. M. J. Tsai (Baylor College of Medicine). The pSV40--gal encodes the -galactosidase gene under the control of simian virus 40 (SV40) promoter (Promega Corp., Madison, WI).
Cell cultures CV-1 or COS-7 embryonic African green monkey kidney cells and HeLa human cervical carcinoma cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cells were incubated at 37 C in 5% CO2 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 at a concentration of 1.5 ⫻ 105 cells/well.
Transient transfection assays CV-1 cells were transfected using lipofectin (Life Technologies, Inc., Gaithersburg, MD) as previously described (25). Cells were cotransfected with 0.05 g/well pRShGR␣ or hGR␣-related plasmids, 0.5 g/ well pMMTV-luc or 17mer-tk-luc, and 0.1 g/well pSV40--gal. pRSVerbA-1 was added to maintain a constant amount of DNA. Twenty-four hours later, the transfection medium was replaced with supplemented DMEM. Forty-eight hours after transfection, dexamethasone (SigmaAldrich Corp., St Louis, MO) or vehicle (100% ethanol) was added to the medium at a concentration of 10⫺6 m.
Luciferase and -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.). Luciferase activity in the cell lysates was determined in a luminometer (Monolight 3010 Luminometer, BD PharMingen, San Diego, CA) as previously described (26). -Galactosidase activity was determined in the same samples using a -galactosidase enzyme assay system (Galacto-Light Plus, Tropix, Bedford, MA) according to the instructions of the manufacturer. Luciferase activity was divided by -galactosidase activity to account for transfection efficiency. All experiments were repeated at least three times.
Western blot analyses COS-7 cells were plated in 75-cm2 flasks and grown in supplemented DMEM. Subconfluent cells were transfected with hGR␣, hGR␣I559N,
hGR␣V571A, hGR␣D641V, hGR␣V729I, or hGR␣I747M (15 g/flask) using the lipofectin method (25), and 6 h later, the transfection medium was replaced with supplemented DMEM. Thirty hours after transfection, cells were washed with ice-cold PBS (three times), gently scraped from 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 inhibitor. The homogenates were centrifuged (75,000 ⫻ g at 4 C) for 30 min to obtain whole cell extracts. Whole cell extracts were mixed with Tris-glycine-sodium dodecyl sulfate sample buffer (2⫻; Invitrogen, Carlsbad, CA), heated to 95 C for 3–5 min, and electrophoresed alongside molecular weight prestained markers (SeeBlue, NOVEX, San Diego, CA) through 8% Tris-glycine gel (Invitrogen). After electroblotting (25 V; 0.8 mA/cm2) onto Hybond C membranes (Amersham Pharmacia Biotech, Little Chalfont, UK), proteins were incubated with blocking solution (5% milk powder, PBS, and 0.05% Tween 20) for 4 h. Immunoblotting was performed at 4 C overnight, using purified specific rabbit polyclonal antiglucocorticoid receptor antibody (Affinity BioReagents, Golden, CO) at 10 g/ml. After washing with PBS three times, membranes were incubated with horseradish peroxidaseconjugated goat antirabbit IgG at a 1:100 dilution at room temperature for 1 h. hGR␣, hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, and hGR␣I747M were visualized using the ECL Plus Western Blotting Detection System (Amersham Pharmacia Biotech) and were exposed to high performance chemiluminescence film (Hyperfilm ECL, Amersham Pharmacia Biotech).
Whole cell dexamethasone binding assays COS-7 cells were transfected using lipofectin (Life Technologies, Inc.) as previously described (25). Cells were transfected with the vectors expressing hGR␣, hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, or hGR␣I747M (1.5 g/well), and 6 h 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 Pharmacia Biotech) at 37 C in the presence or absence of a 500-fold molar excess of cold 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, and washed with PBS twice. Cells were transferred to scintillation vials, and radioactivity was measured using a -counter. Specific binding was calculated by subtracting nonspecific from total binding, and these data were analyzed using the Scatchard method. Binding capacity was expressed as fentomoles per 106 cells, and the apparent dissociation constant (Kd) was expressed in nanomoles. All experiments were performed in duplicate and repeated at least three times. One-way ANOVA with the Student-Newman-Keuls post hoc test was used to compare the mean apparent dissociation constant of the five mutant receptors and the wild-type hGR␣.
Detection and localization of GFP-fused GRs HeLa cells were plated on coated 25-mm diameter, glass-bottom dishes (MatTek Corp., Ashland, MA) in phenol red-free DMEM containing 10% charcoal-treated FBS (HyClone, Logan, UT). Twenty-four hours later, cells were transfected with GFP-fused plasmids expressing hGR␣, hGR␣V571A, hGR␣D641V, and hGR␣V729I. Forty-eight hours after transfection, dexamethasone was added to the transfection medium at a concentration of 10⫺6 m, and fluorescence was detected sequentially by an inverted fluorescence microscope (DM IRB, Leica, Wetzlar, Germany) as previously described (27). 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 Openlab software (Improvision, Boston, MA). All experiments were repeated at least three times.
EMSA COS-7 cells were transfected with 2.0 g/well of pRShGR␣ or the mutants hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, and hGR␣I747M. Twenty-four hours later the medium was replaced with either normal medium or medium containing dexamethasone (10⫺7 m). Forty-eight hours after transfection, cells were washed with PBS twice,
Charmandari et al. • hGR␣ Mutations in Glucocorticoid Resistance
and nuclear extracts were prepared as previously described (28). Four picomoles of the 22-bp double-stranded GRE probe (5⬘-GATCAGAACACAGTGTTCTCTA-3⬘; Stratagene, La Jolla, CA) were labeled using 4 U T4 polynucleotide kinase (Roche, Indianapolis, IN) and 25 Ci [␥-32P]ATP (6000 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ). Fifteen micrograms of undiluted nuclear extract protein were coincubated with 100,000 cpm 32P-labeled GRE probe for 20 min on ice and then for 15 min at room temperature. A specific anti-hGR␣ polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was included in reaction mixtures where indicated. PAGE was performed in a 0.5% Tris/borate/EDTA buffer at constant voltage (130 V) for 3 h. The gel was dried under vacuum and autoradiographed.
In vitro translation of hGRs and production of GST-fused GRIP1-related constructs In vitro translated and 35S-labeled hGR␣, hGR␣I559N, hGR␣V571A, hGR␣D641V, and hGR␣V729I were produced using the TNT-coupled reticulocyte lysate transcription/translation system (Promega Corp.) and pBK/CMV-hGR␣, pBK/CMV-hGR␣I559N, pBK/CMV-hGR␣V571A, pBK/CMV-hGR␣D641V, and pBK/CMV-hGR␣V729I, respectively, as templates. GST-fused GRIP1-(1–1462), GRIP1-(559–774), and GRIP1-(740– 1217) were bacterially produced, purified, and immobilized on the GST beads as previously described (29).
GST pull-down assay The in vitro interaction between hGR␣-related plasmids and GSTfused GRIP1-(1–1462), GRIP1-(559 –774), and GRIP1-(740 –1217), which contain binding sites for AF-1 and AF-2 of hGR␣, was tested using binding assays as previously described (29). Samples were loaded and electrophoresed on an 8% SDS-PAGE gel, and the gel was fixed, treated with Enlightning buffer (NEN Life Science Products, Inc., Boston, MA), and dried (29). Radioactivity was detected by exposing a film on the gel.
FIG. 2. Transcriptional activity of the LBDs of hGR␣, hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, and hGR␣I747M. CV-1 cells were cotransfected with p17mer-tk-luc (0.5 g/well) and a construct expressing the LBD of the wild-type (WT) or a mutant receptor (0.05 g/well). The GAL4-DBD-fused hGR␣-LBD stimulated the transcriptional activity of this promoter by 4- to 5-fold in response to dexamethasone. However, the dexamethasone-induced transcriptional activity of the GAL4-DBD fused-LBDs of all hGR␣ mutants was 2- to 4-fold lower than that of the wild-type receptor. Bars represent the mean ⫾ SEM of at least three independent experiments.
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Results The LBDs of hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, and hGR␣I747M demonstrate decreased intrinsic transcriptional activity compared with the LBD of hGR␣
To explore the mechanism(s) of action of the LBD hGR␣ mutants, we first tested whether the LBD of each mutant carrying the indicated mutation possesses defective intrinsic transcriptional activity. CV-1 cells were cotransfected with p17mer-tk-luc, and each of the constructs: pM-hGR␣-LBD, pMhGR␣I559N-LBD, pM-hGR␣V571A-LBD, pM-hGR␣D641VLBD, pM-hGR␣V729I-LBD, and pM-hGR␣I747M-LBD. The GAL4-DBD-fused hGR␣-LBD stimulated the transcriptional activity of this promoter by 4- to 5-fold in response to dexamethasone. The dexamethasone-induced transcriptional activity of the GAL4-DBD fused-LBDs of all five hGR␣ mutants was 2- to 4-fold lower than that of the wild-type receptor (Fig. 2). Mutant receptors hGR␣V571A, hGR␣D641V, and hGR␣V729I exert no dominant negative effect on the transcriptional activity of hGR␣
To determine whether the mutants pRShGR␣V571A, pRShGR␣D641V, and pRShGR␣V729I have a dominant negative effect on hGR␣-mediated trans-activation of the MMTV promoter, CV-1 cells were cotransfected with pRShGR␣, pMMTV-luc, and five different, progressively increasing concentrations of any of the above mutants, so that the ratio between hGR␣ and each mutant would range from 1:0 to 1:10 (1:0, 1:1, 1:3, 1:6, and 1:10). There was no evidence of a dose-dependent inhibition of hGR␣-mediated trans-activation of the MMTV promoter by any of the mutants, indicating that pRShGR␣V571A, pRShGR␣D641V, and pRShGR␣V729I have no dominant negative effect on hGR␣ transcriptional activity (Fig. 3). Western blot analyses demonstrated no differences in the
FIG. 3. The effect of hGR␣V729I on hGR␣-mediated trans-activation of the MMTV promoter. CV-1 cells were cotransfected with a constant quantity of wild-type hGR␣ (0.05 g/well) and increasing amounts of hGR␣V729I-expressing vector, so that the ratio between hGR␣ and hGR␣V729I would range from 1:0 to 1:10 (1:0, 1:1, 1:3, 1:6, and 1:10). Bars represent the mean ⫾ SEM of at least three independent experiments.
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FIG. 4. Nuclear translocation of GFPhGR␣ (A) and GFP-hGR␣V571A (B) after exposure to dexamethasone. HeLa cells transiently expressing GFP-hGR␣ or GFP-hGR␣V571A were cultured in steroid-free medium. Exposure to dexamethasone (10⫺6 M) induced translocation of the receptor from the cytoplasm to the nucleus. Images of the same cells were obtained at the indicated time points.
expression of all proteins studied, indicating that the abovedescribed alterations in hGR␣-mediated trans-activation of the MMTV promoter did not result from differences at the protein expression level. All mutant receptors hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, and hGR␣I747M demonstrate decreased affinity for ligand compared with hGR␣
Dexamethasone binding studies performed in COS-7 cells transfected with either the wild-type or the mutant receptors hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, and hGR␣I747M showed that all five mutants
had decreased specific binding affinity compared with the wild-type receptor. The apparent dissociation constants (Kd) of hGR␣I559N, hGR␣V571A, and hGR␣I747M were significantly higher than that of the wild-type receptor [hGR␣, 5.4 ⫾ 0.5 nmol; hGR␣I559N, 16.1 ⫾ 2.6 nmol (P ⫽ 0.004); hGR␣V571A, 13.8 ⫾ 2.4 nmol (P ⫽ 0.006); hGR␣I747M, 22.1 ⫾ 1.2 nmol (P ⬍ 0.001)]. The Kd values of hGR␣D641V and hGR␣V729I were also higher than the Kd of the wild-type receptor [hGR␣D641V, 7.9 ⫾ 1.3 (P ⫽ 0.9); hGR␣V729I, 10.2 ⫾ 1.9 nmol (P ⫽ 0.5)]; however, these differences did not reach statistical significance in the present study.
Charmandari et al. • hGR␣ Mutations in Glucocorticoid Resistance
FIG. 5. EMSA. The wild-type receptor hGR␣ and the mutant receptors hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, and hGR␣I747M, expressed in COS-7 cells in the presence or absence of dexamethasone (10⫺7 M), bound similarly to a radiolabeled doublestranded consensus GRE-containing oligonucleotide, indicating that all mutant receptors preserve their DNA-binding activity in both the presence and absence of ligand.
Subcellular localization of the GFP-fused mutant receptors hGR␣V571A, hGR␣D641V, and hGR␣V729I
Fusion of the wild-type and mutant receptors hGR␣V571A, hGR␣D641V, and hGR␣V729I with GFP enabled us to study their subcellular localization in HeLa cells in the absence or presence of dexamethasone (10⫺6 m). In the absence of dexamethasone, GFP-fused hGR␣ was primarily localized in the cytoplasm. Addition of 10⫺6 m dexamethasone resulted in translocation of the wild-type receptor into the nucleus within 12 min (Fig. 4A). The pathological mutant receptors GFPhGR␣V571A and GFP-hGR␣D641V were predominantly observed in the cytoplasm in the absence of ligand. Exposure to 10⫺6 m dexamethasone induced a slow translocation of these mutant receptors into the nucleus, which took 25 and 22 min, respectively (Fig. 4B). In contrast to the GFP-hGR␣V571A and GFP-hGR␣D641V, which, like the wild-type GFP-hGR␣ were predominantly cytoplasmic before addition of the ligand, the mutant receptor GFP-hGR␣V729I was observed predominantly in the nucleus in the absence of ligand, whereas further translocation from the cytoplasm into the nucleus required longer exposure (120 min) to the same concentration of dexamethasone. All mutant receptors hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, and hGR␣I747M preserve their DNA-binding activity
The wild-type receptor hGR␣ and the mutant receptors hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, and hGR␣I747M, expressed in COS-7 cells in the presence or absence of dexamethasone (10⫺7 m), bound similarly to a radiolabeled double-stranded consensus GRE-containing oligonucleotide in a gel mobility shift assay. These results indicate that all mutant receptors preserve their DNA-binding activity in both the presence and absence of ligand (Fig. 5). Mutant receptors hGR␣I559N, hGR␣V571A, hGR␣D641V, and hGR␣V729I display an abnormal interaction with the GRIP1 coactivator in vitro
To determine whether the mutant receptors hGR␣I559N, hGR␣V571A, hGR␣D641V, and hGR␣V729I have an abnor-
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mal interaction with p160 coactivators, which bind to both AF-1 and AF-2 of hGR␣ (30), we investigated the interaction between them and GRIP1 in a GST pull-down assay. GRIP1 contains two sites that bind to steroid receptors. One site, the NRB site, is located amino-terminally, between amino acids 542 and 745, and contains three LXXLL signature motifs through which it interacts with the AF-2 of hGR␣ in a liganddependent fashion. The other site is located carboxyl-terminally, between amino acids 1121 and 1250, and binds to the AF-1 of hGR␣ in a ligand-independent fashion (30 –32) (Fig. 6A). In vitro translated and 35S-labeled hGR␣, hGR␣I559N hGR␣V571A, hGR␣D641V, and hGR␣V729I were tested for binding to bacterially produced and purified GST-fused GRIP1-(1–1462), GRIP1-(597–774), and GRIP1-(740 –1217) in a GST pull-down assay. hGR␣ bound to full-length GRIP1 as well as the carboxyl-terminal GRIP1-(740 –1217) fragment independentlyofligand.hGR␣ alsointeractedwiththeaminoterminal GRIP1-(597–774) fragment in a ligand-dependent fashion. There was no interaction between hGR␣I559N and any of the fragments of GRIP1. hGR␣V571A interacted with both GRIP1-(1–1462) and GRIP1-(740 –1217), but showed a weak interaction with GRIP1-(559 –774). hGR␣V641D interacted with the amino-terminal, but not the carboxyl-terminal, fragment or the full-length GRIP1. Finally, hGR␣I729N demonstrated a weak, ligand-enhanced interaction with GRIP1(1–1462) and GRIP1-(740 –1217), but not with GRIP1-(559 – 774) (Fig. 6B). These results indicate that, like the previously described GRIP1 interaction-defective hGR␣I747M (24), hGR␣I559N, hGR␣V571A, hGR␣V641D, and hGR␣I729N have an abnormal interaction with this coactivator in vitro. Discussion
We explored the molecular mechanisms of action of various natural hGR␣ mutants and showed that 1) the LBDs of all five studied mutant receptors hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, and hGR␣I747M have decreased intrinsic transcriptional activity; 2) hGR␣V571A, hGR␣D641V, and hGR␣V729I do not exert a dominant negative effect on the transcriptional activity of hGR␣ and show delayed translocation into the nucleus after exposure to ligand; 3) all five mutant receptors hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, and hGR␣I747M demonstrate decreased affinity for ligand in a standard dexamethasone binding assay (see also Refs. 33 and 18 for hGR␣D641V and hGR␣V729I mutants, respectively) and preserve their ability to bind to DNA; and 4) hGR␣I559N, hGR␣V571A, hGR␣D641V, and hGR␣V729I display an abnormal interaction with the GRIP1 coactivator in vitro, as previously shown for hGR␣I747M (24). These results indicate that the process through which the above hGR␣ mutant receptors impair the physiological mechanisms of glucocorticoid action at the molecular level is multifactorial and involves impaired ability to bind ligand, aberrant nucleocytoplasmic trafficking, and abnormal interaction with the p160 coactivators. Although adequate compensation is achieved by elevated cortisol concentrations in the majority of patients with familial or sporadic generalized glucocorticoid resistance, the excess ACTH secretion also results in increased production of adrenal steroids with androgenic and/or mineralocorti-
1946
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Charmandari et al. • hGR␣ Mutations in Glucocorticoid Resistance
FIG. 6. A, Linearized GRIP1 molecule and distribution of its functional domains. HLH, Helixloop-helix; NIDaux, auxiliary nuclear receptor interacting domain; PAS, period arylhydrogen receptor and single-minded (24). B, GST pulldown assay. In vitro translated and 35S-labeled hGR␣, hGR␣I559N, hGR␣V571A, hGR␣D641V, and hGR␣V729I 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⫺6 M). Samples were run on an 8% SDS-PAGE gel, and gels were fixed and exposed to x-ray film.
coid activity (15, 16, 23). The former accounts for manifestations of androgen excess, such as precocious puberty, acne, hirsutism, and infertility in both sexes, sexual ambiguity at birth, male-pattern hair loss and menstrual irregularities in females, and adrenal rests in the testes and oligospermia in males. The latter accounts for symptoms and signs of mineralocorticoid excess, such as hypertension and hypokalemic alkalosis. A large number of subjects may be asymptomatic, displaying biochemical alterations only (15, 16). Treatment involves administration of high doses of mineralocorticoidsparing synthetic glucocorticoids, such as dexamethasone (1–3 mg/d), which activate the mutated and/or wild-type hGR␣, and suppress the endogenous secretion of ACTH (15, 16). Appropriate treatment is particularly important in cases of severe impairment of hGR␣ function, because longstanding corticotroph hyperstimulation in association with decreased glucocorticoid negative feedback might lead to the development of an ACTH-secreting adenoma (20). Since generalized glucocorticoid resistance was first described and investigated in detail (33–35), more than 10 kindreds and sporadic cases with the condition have been reported. Abnormalities of several hGR␣ characteristics, including cell concentrations, affinity for glucocorticoids, stability, and translocation into the nucleus, have been as-
sociated with this condition (16 –24). The molecular defects that have been elucidated in the reported cases are summarized in Table 1, whereas the corresponding mutations in the hGR␣ gene are shown in both Table 1 and Fig. 1D. We showed that the homozygous hGR␣ mutations V571A, D641V, and V729I did not exert a dominant negative effect on the transcriptional activity of hGR␣ in the heterozygotic state. Carriers of these mutations were asymptomatic, but mildly hypercortisolemic. By contrast, the heterozygous mutations I559N and I747M were previously 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 (22, 24). At a 1:1 ratio, the mutant receptors hGR␣I559N and hGR␣I747M inhibited approximately 25% of the transcriptional activity of the wild-type receptor. The dominant negative activity of these mutants in association with their severely impaired ability to activate gene expression resulted in a further decrement in the glucocorticoid sensitivity of affected individuals, given that their tissues sensed less than 40% of the glucocorticoid activity of normal cortisol concentrations (22, 24). The mode of genetic transmission in kindreds with these mutations was autosomal dominant, as the heterozygotic condition was sufficient to cause symptomatic disease. Dominant negative effects of
679 (G3 S)
1808 (T3 A)
1844 (C3 T)
2373 (T3 G)
1430 (G3 A)
2035 (G3 A)
Karl et al. (20) Kino et al. (22)
Mendonca et al. (23)
Vottero et al. (24)
Ruiz et al. (21)
a
477 (R3 H)
2317 (G3 A)
Malchoff et al. (18)
Affinity for ligand 2, trans-activation 2
Affinity for ligand 2, trans-activation 2, transdominance (⫹), nuclear translocation 2 Trans-activation 2
Affinity for ligand 2, trans-activation 2
Affinity for ligand 2, trans-activation 2, transdominance (⫹), nuclear translocation 2
hGR␣ number, 50% of control; inactivation of the affected allele Affinity for ligand 2, trans-activation 2
Affinity for ligand 2, trans-activation 2
Biochemical phenotype
Transcriptional activity of LBD 2, transdominance (⫺), nuclear translocation 2, DNA binding (⫹), abnormal interaction with GRIP1 Transcriptional activity of LBD 2, DNA binding (⫹), abnormal interaction with GRIP1 Transcriptional activity of LBD 2, transdominance (⫺), nuclear translocation 2, DNA binding (⫹) Transcriptional activity of LBD 2 DNA binding (⫹)
Transcriptional activity of LBD 2, transdominance (⫺), nuclear translocation 2, DNA binding (⫹), abnormal interaction with GRIP1
Present study
Heterozygous
Heterozygous
Heterozygous
Homozygous
Heterozygous
Homozygous
Heterozygous
Homozygous
Genotype
Sporadic
Sporadic
Autosomal dominant
Autosomal recessive
Sporadic
Autosomal recessive
Autosomal dominant
Autosomal recessive
Transmissiona
Hirsutism, fatigue, hypertension Hirsutism, fatigue, hypertension
Cystic acne, hirsutism, oligoamenorrhoea
Ambiguous genitalia at birth, hypertension, hyperandrogenism, hypokalemia
Hypertension, oligospermia, infertility
Hirsutism, male-pattern hair loss, menstrual irregularities Precocious puberty, hyperandrogenism
Hypertension, hypokalemic alkalosis
Phenotype
Autosomal recessive, a trait that is phenotypically expressed only in homozygotes; autosomal dominant, a trait that is phenotypically expressed in heterozygotes.
747 (I3 M)
571 (V3 A)
559 (I3 N)
729 (V3 I)
4-bp deletion in exon 6
Karl et al. (19)
641 (D3 V)
2054 (A3 T)
Amino acid
Mutation position
cDNA
Chrousos et al. (34) Hurley et al. (17)
Author (ref. no.)
TABLE 1. Mutations of the hGR gene causing glucocorticoid resistance
Charmandari et al. • hGR␣ Mutations in Glucocorticoid Resistance J Clin Endocrinol Metab, April 2004, 89(4):1939 –1949 1947
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mutant receptors have also been reported in the syndrome of thyroid hormone resistance (36), whereas dominant inhibitory actions of physiologically expressed receptor isoforms have been demonstrated for the A isoform of the human progesterone receptor (37) and the  isoform of hGR (38, 39). In a standard dexamethasone binding assay, all mutant receptors hGR␣I559N, hGR␣V571A, hGR␣D641V, hGR␣V729I, and hGR␣I747M had a reduction in the affinity for ligand compared with the wild-type hGR␣, as previously reported (17, 18, 23). This most likely reflects the location of these mutations in helices 3, 7, and 11, respectively, which all line the ligandbinding pocket and may therefore directly affect ligand binding. In addition to the decreased affinity for ligand, translocation of the GFP-fused mutants hGR␣V571A and hGR␣D641V from the cytoplasm into the nucleus after exposure to dexamethasone was slower than that of the wild-type receptor. After exposure to dexamethasone (10⫺6 m), GFP-hGR␣ was completely transported from the cytoplasm into the nucleus within 12 min, whereas the mutant receptors GFPhGR␣V571A and GFP-hGR␣D641V required 25 and 22 min, respectively. These findings indicate that the mutations V571A and D641V changed the receptors’ nucleocytoplasmic shuttling activity, probably through impairment of NL1 function. This impairment may be due to the decreased ligand binding affinity of these mutant receptors, which could prevent a proper ligand-induced conformation change and, hence, a normal interaction between NL1 and components of the importin system (22, 40). Binding of hsps to hGR␣ partially inactivates NL1 and might also explain the differences observed between hGR␣ and various mutants in the time required for entry into the nucleus (41– 43). The mutant receptor GFP-hGR␣V729I was observed predominantly in the nucleus in the absence of ligand, whereas further translocation from the cytoplasm into the nucleus required longer exposure to the same concentration of dexamethasone. This might indicate that this mutant interacts less closely with hsp90 and/or better with importin through its NL1 (41– 43) at the nonligand-bound state, and/or that the mutation at amino acid 729 suppresses nuclear export functions, resulting in increased retention of the unliganded receptor in the nucleus. Defective mechanisms that may relate to delayed nuclear export, such as the calreticulin export pathway, and certain motifs in the DBD that function as nuclear export signals might also account for the predominance in nuclear localization of unliganded hGR␣V729I (44 – 46). Although all five hGR␣ mutants preserved their ability to bind DNA in a gel-shift mobility assay, they demonstrated an abnormal interaction with the nuclear coactivator GRIP1. These results suggest that each mutant receptor may form a defective complex with GRIP1, which is partially or completely ineffective, because at least one important interaction site is either weak or nonexistent. That the mutations I559N and V729I are located very close to the AF-2 domain may indicate that the 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, could also be defective (10 –12). Based on our findings, we hypothesize that after exposure
Charmandari et al. • hGR␣ Mutations in Glucocorticoid Resistance
to ligand, hGR␣ molecules translocate into the nucleus as hGR␣ mutant-hGR␣ mutant homodimers (in case of a homozygous mutation) or hGR␣-hGR␣ mutant heterodimers (in case of a heterozygous mutation). These homodimers and heterodimers have severely compromised inherent transcriptional activity, and although they may preserve their ability to bind to GREs or transcription factors that normally interact with hGR␣, they produce functional impairment in one or more of the postligand-binding steps, i.e. translocation into the nucleus or interaction with coregulators and/or specific or general transcription factors (7). Therefore, each of the above mutations imparts different functional defects upon the GR signal transduction pathway, which explain the autosomal recessive or dominant transmission of the disorder and may in part explain its variable clinical phenotype. However, one must not underestimate the importance of background genetic and constitutional factors with epistatic actions on the expression of the disorder (15). Thus, factors that define the activity of the hypothalamic-pituitary-adrenal axis, renin-angiotensin-aldosterone system, and gonadal axis as well as the target tissue sensitivity to glucocorticoids, mineralocorticoids, and androgens are bound to play important roles in the clinical manifestations of this condition. Acknowledgments Received March 13, 2003. Accepted January 6, 2004. Address all correspondence and requests for reprints to: Dr. Evangelia Charmandari, Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, 10 Center Drive, Building 10, Room 9D42, Bethesda, Maryland 20892-1583.
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