natural glucocorticoid receptor mutants causing generalized glucocorticoid resistance: molecular...

Natural Glucocorticoid Receptor Mutants CausingGeneralized Glucocorticoid Resistance: MolecularGenotype, 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 andHuman 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 con-dition characterized by partial end-organ insensitivity to glu-cocorticoids. The clinical spectrum of the condition rangesfrom completely asymptomatic to severe hyperandrogenism,fatigue, and/or mineralocorticoid excess. The molecular basisof glucocorticoid resistance in several families and sporadiccases has been ascribed to mutations in the human glucocor-ticoid receptor-� (hGR�) gene, which impair the ability of thereceptor to transduce the glucocorticoid signal. We system-atically investigated the molecular mechanisms throughwhich natural, ligand-binding domain hGR� mutants, includ-ing hGR�I559N, hGR�V571A, hGR�D641V, hGR�V729I, andhGR�I747M, produce a defective signal and determinedwhether their differential effects on hGR� function mightaccount for the type of genetic transmission of the disorderand the variable clinical phenotype of the affected subjects.Our findings suggest that all five mutant receptors studiedhave ligand-binding domains with decreased intrinsic tran-

scriptional activity. Unlike hGR�I559N and I747M previouslyshown to exert a dominant negative effect upon the transcrip-tional activity of hGR�, hGR�V571A, D641V, and V729I do nothave such an effect. All five mutants studied demonstratevarying degrees of decreased affinity for the ligand in astandard dexamethasone binding assay, but preserve theirability to bind DNA. The nondominant negative mutants,hGR�V571A, D641V, and V729I, show delayed translocationinto the nucleus after exposure to ligand. Finally, hGR�I559N,V571A, D641V, and V729I display an abnormal interactionwith the glucocorticoid receptor-interacting protein-1 coac-tivator in vitro, as this was previously shown also forhGR�I747M. We conclude that each of the above hGR� muta-tions imparts different functional defects upon the glucocor-ticoid signal transduction pathway, which explains the auto-somal recessive or dominant transmission of the disorder, butmight only explain in part its variable clinical phenotype.(J Clin Endocrinol Metab 89: 1939–1949, 2004)

GLUCOCORTICOIDS REGULATE A variety of biolog-ical processes and exert profound influences on many

physiological functions by virtue of their diverse roles ingrowth, development, and maintenance of basal and stress-related homeostasis (1). At the cellular level, their actions aremediated by an approximately 94-kDa intracellular receptorprotein, the glucocorticoid receptor (GR). This receptor be-longs to the superfamily of steroid/thyroid/retinoic acidreceptor proteins that function as ligand-dependent tran-scription factors (2–5) (Fig. 1, A and B).

Alternative splicing of the human (h) GR gene in exon 9generates two highly homologous receptor isoforms, termed �and �. hGR� is ubiquitously expressed in almost all humantissues and cells and represents the classic GR that functions as

a ligand-dependent transcription factor. In the absence of li-gand, hGR� resides mostly in the cytoplasm of cells as part ofa large multiprotein complex, which consists of the receptorpolypeptide, two molecules of 90-kDa heat shock protein(hsp90), and several other proteins (6). The hsp90 molecules arethought to sequester hGR� in the cytoplasm of cells by main-taining the receptor in a conformation that masks or inactivatesits nuclear localization signals (NLSs), but empowers it to in-teract with glucocorticoids. Upon hormone binding, the recep-tor undergoes an allosteric change, which results in dissociationfrom hsp90 and other proteins, and unmasking/activation ofthe NLSs. In its new conformation, the activated, ligand-boundGR translocates into the nucleus, where it binds as homodimerto glucocorticoid-response elements (GREs) located in the pro-moter region of target genes. The hGR� then communicateswith the basal transcription machinery and regulates the ex-pression of glucocorticoid-responsive genes positively or neg-atively, depending on the GRE sequence and promoter context(7). The receptor can also modulate gene expression indepen-dently of GRE binding by physically interacting with othertranscription factors, such as activator protein-1 and nuclearfactor-�B (8, 9).

The ability of ligand-bound hGR� to trans-activate steroid-responsive genes depends on the presence of coactivators,nucleoproteins with chromatin-remodeling and other enzy-matic activities, that are attracted to the promoter region of

Abbreviations: AF, Activation function; CBP, cAMP response element-binding protein-binding protein; DBD, DNA-binding domain; FBS, fetalbovine 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, simianvirus 40.JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the en-docrine community.

0021-972X/04/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 89(4):1939–1949Printed in U.S.A. Copyright © 2004 by The Endocrine Society

doi: 10.1210/jc.2003-030450

1939

the target genes via the activation function-1 (AF-1) and AF-2of hGR� (10–12) (Fig. 1C). Several families of coactivatorshave been described, including the p160 coactivators, such asthe steroid receptor coactivator-1 and the GR-interactingprotein-1 (GRIP1), the p300/cAMP response element-bind-ing protein-binding protein (CBP) cointegrators, the p300/CBP-associated protein, the switching/sucrose nonferment-ing complex, as well as the newly described vitamin Dreceptor-interacting protein/thyroid hormone-associatedprotein complex (10–13). The p160 and p300/CBP coactiva-tors have multiple amphipathic LXXLL signature motifs (co-activator motifs or nuclear receptor boxes), which serve asinterfaces between these coactivators and a hydrophobic cleftformed by helices 3, 4, and 12 of the nuclear receptors (14).The p160, CBP/p300, and p300/CBP-associated protein allhave histone acetylase activity, which loosens the DNAstrands from the nucleosomes and allows the transcriptionalpreinitiation complex of RNA polymerase II and its ancillarycomponents, including the TATA-binding protein and a host

of TATA-binding protein-associated proteins, to initiate andpromote transcription (10–12).

Glucocorticoid resistance is a rare, familial or sporadiccondition characterized by generalized, partial end-organinsensitivity to physiological glucocorticoid concentrations(15, 16). Patients have compensatory elevations in circulatingcortisol and ACTH concentrations, which maintain circadianrhythmicity and appropriate responsiveness to stressors, al-beit at higher hormone concentrations, and resistance of thehypothalamic-pituitary-adrenal axis to dexamethasone sup-pression, but no clinical evidence of hypo- or hypercorti-solism. The clinical spectrum of the condition is broad, rang-ing from completely asymptomatic to severe cases ofhyperandrogenism, fatigue, and/or mineralocorticoid ex-cess (15, 16). The molecular basis of glucocorticoid resistancein several families and sporadic cases has been ascribed tomutations in the hGR� gene, which impair one or more of themolecular mechanisms of GR function, thus altering tissuesensitivity to glucocorticoids. Inactivating mutations mostly

FIG. 1. A, Schematic representation of the structure of the hGR gene. Alternative splicing of the primary transcript gives rise to the two mRNAand protein isoforms, hGR� and hGR� (15). B, Functional domains of the hGR�. The functional domains and subdomains are indicated beneaththe linearized protein structures. C, Schematic representation of the interaction of coactivators with the AF-1 and AF-2 domains of the GR andtheir role in transcriptional regulation. D, Location of the known mutations of the hGR� gene.

1940 J Clin Endocrinol Metab, April 2004, 89(4):1939–1949 Charmandari et al. • hGR� Mutations in Glucocorticoid Resistance

within the ligand-binding domain (LBD) as well as a 4-bpdeletion at the 3�-boundary of exon 6 of the hGR� gene havebeen described in five kindreds and three sporadic cases(17–24) (Fig. 1D).

The aim of our study was to investigate the molecularmechanisms of action of natural hGR� mutants located in theLBD of the receptor, including hGR�I559N, V571A, D641V,V729I, and I747M, and to determine whether their differen-tial effects on hGR� function may account for the variable

clinical phenotype and genetic transmission of familial glu-cocorticoid resistance.

Materials and MethodsPlasmids

The pRShGR� plasmid expresses the human GR� isoform under thecontrol of the Rous sarcoma virus (RSV) promoter. The plasmidspRShGR�I559N, pRShGR�V571A, pRShGR�D641V, pRShGR�V729I, andpRShGR�I747M were constructed by introducing the indicated mutations

FIG. 1. Continued.

Charmandari et al. • hGR� Mutations in Glucocorticoid Resistance J Clin Endocrinol Metab, April 2004, 89(4):1939–1949 1941

into the pRShGR� plasmid using PCR-assisted site-directed mutagenesis(Stratagene, La Jolla, CA). The pM-hGR�-LBD, pM-hGR�I559N-LBD, pM-hGR�V571A-LBD, pM-hGR�D641V-LBD, pM-hGR�V729I-LBD, and pM-hGR�I747M-LBD, which express, respectively, the LBD of pRShGR�,pRShGR�I559N, pRShGR�V571A, pRShGR�D641V, pRShGR�V729I, andpRShGR�I747M fused to the GAL4-DNA-binding domain (GAL4-DBD),were constructed by subcloning the corresponding cDNAs into the pMvector (Clontech, Palo Alto, CA). Green fluorescent protein (GFP)-fusedplasmids expressing hGR�, hGR�V571A, hGR�D641V, and hGR�V729Iwere constructed by subcloning the corresponding cDNAs into thepF25GFP vector, which was a gift from Dr. G. Pavlakis (National CancerInstitute, National Institutes of Health, Frederick, MD).

The pGEX4T3-GRIP1-(1–1462), pGEX4T3-GRIP1-(596–774), andpGEX4T3-GRIP1-(740–1217) plasmids, which express glutathione-S-transferase (GST) fusions of the full-length GRIP1, an AF-2-directedhGR� binding site [nuclear receptor-binding (NRB) site], and an AF-1-directed, hGR�-binding site with an auxiliary nuclear receptor-inter-acting domain, respectively, were constructed by subcloning the cor-responding GRIP1 fragments of cDNA into the pGEX4T3 plasmid(Amersham Pharmacia Biotech, Piscataway, NJ) (24). The pSG5-GRIP1(1–1462) plasmid was a gift from Dr. M. Stallcup (University ofSouthern California, Los Angeles, CA).

The plasmid pRSV-erbA�1, which contains a thyroid receptor cDNA ininverse orientation, was used as negative control for all hGR�-related plas-mids. The pMMTV-luc plasmid expresses luciferase under the control of theglucocorticoid-inducible mouse mammary tumor virus (MMTV) promoterand was a gift from Dr. G. L. Hager (NCI, NIH, Bethesda, MD). Thep17mer-tk-luc contains the luciferase gene under the control of the fourGAL4-responsive elements cloned upstream of the proximal portion of theglucocorticoid-independent herpes simplex virus thymidine kinase pro-moter and was a gift from Dr. M. J. Tsai (Baylor College of Medicine). ThepSV40-�-gal encodes the �-galactosidase gene under the control of simianvirus 40 (SV40) promoter (Promega Corp., Madison, WI).

Cell cultures

CV-1 or COS-7 embryonic African green monkey kidney cells andHeLa human cervical carcinoma cells were grown in DMEM supple-mented with 10% fetal bovine serum (FBS) and antibiotics. Cells wereincubated at 37 C in 5% CO2 and passaged every 3–4 d. Twenty-fourhours before transfection, subconfluent cells were removed from theirflasks by trypsinization, resuspended in supplemented medium, andplated 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 cotrans-fected 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. pRSV-erbA-1 was added to maintain a constant amount of DNA. Twenty-fourhours later, the transfection medium was replaced with supplementedDMEM. Forty-eight hours after transfection, dexamethasone (Sigma-Aldrich Corp., St Louis, MO) or vehicle (100% ethanol) was added to themedium at a concentration of 10�6 m.

Luciferase and �-galactosidase assays

Seventy-two hours after transfection, cells were washed with PBStwice 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) aspreviously described (26). �-Galactosidase activity was determined inthe same samples using a �-galactosidase enzyme assay system(Galacto-Light Plus, Tropix, Bedford, MA) according to the instructionsof the manufacturer. Luciferase activity was divided by �-galactosidaseactivity to account for transfection efficiency. All experiments wererepeated at least three times.

Western blot analyses

COS-7 cells were plated in 75-cm2 flasks and grown in supplementedDMEM. 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 mediumwas replaced with supplemented DMEM. Thirty hours after transfec-tion, cells were washed with ice-cold PBS (three times), gently scrapedfrom flasks, centrifuged briefly, and lysed using a lysis buffer thatconsisted 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 cellextracts were mixed with Tris-glycine-sodium dodecyl sulfate samplebuffer (2�; Invitrogen, Carlsbad, CA), heated to 95 C for 3–5 min, andelectrophoresed alongside molecular weight prestained markers (See-Blue, 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 in-cubated with blocking solution (5% milk powder, PBS, and 0.05% Tween20) for 4 h. Immunoblotting was performed at 4 C overnight, usingpurified specific rabbit polyclonal antiglucocorticoid receptor antibody(Affinity BioReagents, Golden, CO) at 10 �g/ml. After washing with PBSthree times, membranes were incubated with horseradish peroxidase-conjugated goat antirabbit IgG at a 1:100 dilution at room temperaturefor 1 h. hGR�, hGR�I559N, hGR�V571A, hGR�D641V, hGR�V729I, andhGR�I747M were visualized using the ECL Plus Western Blotting De-tection System (Amersham Pharmacia Biotech) and were exposed tohigh performance chemiluminescence film (Hyperfilm ECL, AmershamPharmacia 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 vectorsexpressing hGR�, hGR�I559N, hGR�V571A, hGR�D641V, hGR�V729I,or hGR�I747M (1.5 �g/well), and 6 h later, the transfection medium wasreplaced with DMEM supplemented with 10% FBS and antibiotics.Confluent cells were incubated in plain DMEM with six different con-centrations (1.56, 3.125, 6.25, 12.5, 25, and 50 nm) of [1,2,4,6,7-3H]dexa-methasone (Amersham Pharmacia Biotech) at 37 C in the presence orabsence of a 500-fold molar excess of cold dexamethasone for 1 h. Afterincubation, cells were washed with PBS (3 ml/well) twice to remove freesteroid. Cells were harvested, centrifuged at 1300 rpm for 15 min, andwashed with PBS twice. Cells were transferred to scintillation vials, andradioactivity was measured using a �-counter. Specific binding wascalculated by subtracting nonspecific from total binding, and these datawere analyzed using the Scatchard method. Binding capacity was ex-pressed as fentomoles per 106 cells, and the apparent dissociation con-stant (Kd) was expressed in nanomoles. All experiments were performedin duplicate and repeated at least three times. One-way ANOVA withthe Student-Newman-Keuls post hoc test was used to compare the meanapparent dissociation constant of the five mutant receptors and thewild-type hGR�.

Detection and localization of GFP-fused GRs

HeLa cells were plated on coated 25-mm diameter, glass-bottomdishes (MatTek Corp., Ashland, MA) in phenol red-free DMEM con-taining 10% charcoal-treated FBS (HyClone, Logan, UT). Twenty-fourhours later, cells were transfected with GFP-fused plasmids expressinghGR�, hGR�V571A, hGR�D641V, and hGR�V729I. Forty-eight hoursafter transfection, dexamethasone was added to the transfection me-dium at a concentration of 10�6 m, and fluorescence was detected se-quentially by an inverted fluorescence microscope (DM IRB, Leica, Wet-zlar, Germany) as previously described (27). Twelve-bit, black and whiteimages were captured using a digital charge-coupled device camera(Hamamatsu Photonics K.K., Hamamatsu, Japan). Image analysis andpresentation 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 themutants hGR�I559N, hGR�V571A, hGR�D641V, hGR�V729I, andhGR�I747M. Twenty-four hours later the medium was replaced witheither normal medium or medium containing dexamethasone (10�7 m).Forty-eight hours after transfection, cells were washed with PBS twice,


and nuclear extracts were prepared as previously described (28). Fourpicomoles of the 22-bp double-stranded GRE probe (5�-GATCAGAA-CACAGTGTTCTCTA-3�; Stratagene, La Jolla, CA) were labeled using 4U 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 co-incubated with 100,000 cpm 32P-labeled GRE probe for 20 min on ice andthen for 15 min at room temperature. A specific anti-hGR� polyclonalantibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was includedin 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 wasdried under vacuum and autoradiographed.

In vitro translation of hGRs and production of GST-fusedGRIP1-related constructs

In vitro translated and 35S-labeled hGR�, hGR�I559N, hGR�V571A,hGR�D641V, and hGR�V729I were produced using the TNT-coupledreticulocyte 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, astemplates. GST-fused GRIP1-(1–1462), GRIP1-(559–774), and GRIP1-(740–1217) were bacterially produced, purified, and immobilized on the GSTbeads as previously described (29).

GST pull-down assay

The in vitro interaction between hGR�-related plasmids and GST-fused GRIP1-(1–1462), GRIP1-(559–774), and GRIP1-(740–1217), whichcontain binding sites for AF-1 and AF-2 of hGR�, was tested usingbinding assays as previously described (29). Samples were loaded andelectrophoresed on an 8% SDS-PAGE gel, and the gel was fixed, treatedwith Enlightning buffer (NEN Life Science Products, Inc., Boston, MA),and dried (29). Radioactivity was detected by exposing a film on the gel.

ResultsThe LBDs of hGR�I559N, hGR�V571A, hGR�D641V,hGR�V729I, and hGR�I747M demonstrate decreasedintrinsic transcriptional activity compared with theLBD of hGR�

To explore the mechanism(s) of action of the LBD hGR�mutants, we first tested whether the LBD of each mutantcarrying the indicated mutation possesses defective intrinsictranscriptional activity. CV-1 cells were cotransfected withp17mer-tk-luc, and each of the constructs: pM-hGR�-LBD, pM-hGR�I559N-LBD, pM-hGR�V571A-LBD, pM-hGR�D641V-LBD, pM-hGR�V729I-LBD, and pM-hGR�I747M-LBD. TheGAL4-DBD-fused hGR�-LBD stimulated the transcriptional ac-tivity of this promoter by 4- to 5-fold in response to dexameth-asone. The dexamethasone-induced transcriptional activity ofthe GAL4-DBD fused-LBDs of all five hGR� mutants was 2- to4-fold lower than that of the wild-type receptor (Fig. 2).

Mutant receptors hGR�V571A, hGR�D641V, andhGR�V729I exert no dominant negative effect on thetranscriptional activity of hGR�

To determine whether the mutants pRShGR�V571A,pRShGR�D641V, and pRShGR�V729I have a dominant neg-ative effect on hGR�-mediated trans-activation of the MMTVpromoter, CV-1 cells were cotransfected with pRShGR�, pM-MTV-luc, and five different, progressively increasing con-centrations of any of the above mutants, so that the ratiobetween 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 adose-dependent inhibition of hGR�-mediated trans-activa-tion of the MMTV promoter by any of the mutants, indicatingthat pRShGR�V571A, pRShGR�D641V, and pRShGR�V729Ihave no dominant negative effect on hGR� transcriptionalactivity (Fig. 3).

Western blot analyses demonstrated no differences in the

FIG. 2. Transcriptional activity of the LBDs of hGR�, hGR�I559N,hGR�V571A, hGR�D641V, hGR�V729I, and hGR�I747M. CV-1 cellswere cotransfected with p17mer-tk-luc (0.5 �g/well) and a constructexpressing the LBD of the wild-type (WT) or a mutant receptor (0.05�g/well). The GAL4-DBD-fused hGR�-LBD stimulated the transcrip-tional activity of this promoter by 4- to 5-fold in response to dexa-methasone. However, the dexamethasone-induced transcriptional ac-tivity of the GAL4-DBD fused-LBDs of all hGR� mutants was 2- to4-fold lower than that of the wild-type receptor. Bars represent themean � SEM of at least three independent experiments.

FIG. 3. The effect of hGR�V729I on hGR�-mediated trans-activationof the MMTV promoter. CV-1 cells were cotransfected with a constantquantity of wild-type hGR� (0.05 �g/well) and increasing amounts ofhGR�V729I-expressing vector, so that the ratio between hGR� andhGR�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 exper-iments.


expression of all proteins studied, indicating that the above-described alterations in hGR�-mediated trans-activation ofthe MMTV promoter did not result from differences at theprotein expression level.

All mutant receptors hGR�I559N, hGR�V571A,hGR�D641V, hGR�V729I, and hGR�I747M demonstratedecreased affinity for ligand compared with hGR�

Dexamethasone binding studies performed in COS-7cells transfected with either the wild-type or the mutantreceptors hGR�I559N, hGR�V571A, hGR�D641V,hGR�V729I, and hGR�I747M showed that all five mutants

had decreased specific binding affinity compared with thewild-type receptor. The apparent dissociation constants(Kd) of hGR�I559N, hGR�V571A, and hGR�I747M weresignificantly 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 valuesof hGR�D641V and hGR�V729I were also higher than theKd 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, thesedifferences did not reach statistical significance in thepresent study.

FIG. 4. Nuclear translocation of GFP-hGR� (A) and GFP-hGR�V571A (B) afterexposure to dexamethasone. HeLa cellstransiently expressing GFP-hGR� orGFP-hGR�V571A were cultured insteroid-free medium. Exposure to dexa-methasone (10�6 M) induced transloca-tion of the receptor from the cytoplasm tothe nucleus. Images of the same cellswere obtained at the indicated timepoints.


Subcellular localization of the GFP-fused mutant receptorshGR�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 studytheir subcellular localization in HeLa cells in the absence orpresence of dexamethasone (10�6 m). In the absence of dexa-methasone, GFP-fused hGR� was primarily localized in thecytoplasm. Addition of 10�6 m dexamethasone resulted intranslocation of the wild-type receptor into the nucleus within12 min (Fig. 4A). The pathological mutant receptors GFP-hGR�V571A and GFP-hGR�D641V were predominantly ob-served in the cytoplasm in the absence of ligand. Exposure to10�6 m dexamethasone induced a slow translocation of thesemutant receptors into the nucleus, which took 25 and 22 min,respectively (Fig. 4B). In contrast to the GFP-hGR�V571A andGFP-hGR�D641V, which, like the wild-type GFP-hGR� werepredominantly cytoplasmic before addition of the ligand, themutant receptor GFP-hGR�V729I was observed predomi-nantly in the nucleus in the absence of ligand, whereas furthertranslocation from the cytoplasm into the nucleus requiredlonger exposure (120 min) to the same concentration ofdexamethasone.

All mutant receptors hGR�I559N, hGR�V571A,hGR�D641V, hGR�V729I, and hGR�I747M preserve theirDNA-binding activity

The wild-type receptor hGR� and the mutant receptorshGR�I559N, hGR�V571A, hGR�D641V, hGR�V729I, andhGR�I747M, expressed in COS-7 cells in the presence or ab-sence of dexamethasone (10�7 m), bound similarly to a radio-labeled double-stranded consensus GRE-containing oligonu-cleotide in a gel mobility shift assay. These results indicate thatall mutant receptors preserve their DNA-binding activity inboth the presence and absence of ligand (Fig. 5).

Mutant receptors hGR�I559N, hGR�V571A, hGR�D641V,and hGR�V729I display an abnormal interaction with theGRIP1 coactivator in vitro

To determine whether the mutant receptors hGR�I559N,hGR�V571A, hGR�D641V, and hGR�V729I have an abnor-

mal interaction with p160 coactivators, which bind to bothAF-1 and AF-2 of hGR� (30), we investigated the interactionbetween them and GRIP1 in a GST pull-down assay. GRIP1contains two sites that bind to steroid receptors. One site, theNRB site, is located amino-terminally, between amino acids542 and 745, and contains three LXXLL signature motifsthrough which it interacts with the AF-2 of hGR� in a ligand-dependent fashion. The other site is located carboxyl-termi-nally, between amino acids 1121 and 1250, and binds to theAF-1 of hGR� in a ligand-independent fashion (30–32) (Fig.6A).

In vitro translated and 35S-labeled hGR�, hGR�I559NhGR�V571A, hGR�D641V, and hGR�V729I were tested forbinding to bacterially produced and purified GST-fusedGRIP1-(1–1462), GRIP1-(597–774), and GRIP1-(740–1217) ina GST pull-down assay. hGR� bound to full-length GRIP1 aswell as the carboxyl-terminal GRIP1-(740–1217) fragmentindependentlyofligand.hGR�alsointeractedwiththeamino-terminal GRIP1-(597–774) fragment in a ligand-dependentfashion. There was no interaction between hGR�I559N andany of the fragments of GRIP1. hGR�V571A interacted withboth GRIP1-(1–1462) and GRIP1-(740–1217), but showed aweak interaction with GRIP1-(559–774). hGR�V641D inter-acted with the amino-terminal, but not the carboxyl-terminal,fragment or the full-length GRIP1. Finally, hGR�I729N dem-onstrated 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 previouslydescribed GRIP1 interaction-defective hGR�I747M (24),hGR�I559N, hGR�V571A, hGR�V641D, and hGR�I729Nhave an abnormal interaction with this coactivator in vitro.

Discussion

We explored the molecular mechanisms of action of variousnatural hGR� mutants and showed that 1) the LBDs of allfive studied mutant receptors hGR�I559N, hGR�V571A,hGR�D641V, hGR�V729I, and hGR�I747M have decreased in-trinsic transcriptional activity; 2) hGR�V571A, hGR�D641V,and hGR�V729I do not exert a dominant negative effect on thetranscriptional activity of hGR� and show delayed transloca-tion into the nucleus after exposure to ligand; 3) all five mutantreceptors hGR�I559N, hGR�V571A, hGR�D641V, hGR�V729I,and hGR�I747M demonstrate decreased affinity for ligand in astandard dexamethasone binding assay (see also Refs. 33 and18 for hGR�D641V and hGR�V729I mutants, respectively) andpreserve their ability to bind to DNA; and 4) hGR�I559N,hGR�V571A, hGR�D641V, and hGR�V729I display an abnor-mal interaction with the GRIP1 coactivator in vitro, as previ-ously shown for hGR�I747M (24). These results indicate that theprocess through which the above hGR� mutant receptors im-pair the physiological mechanisms of glucocorticoid action atthe molecular level is multifactorial and involves impaired abil-ity to bind ligand, aberrant nucleocytoplasmic trafficking, andabnormal interaction with the p160 coactivators.

Although adequate compensation is achieved by elevatedcortisol concentrations in the majority of patients with fa-milial or sporadic generalized glucocorticoid resistance, theexcess ACTH secretion also results in increased productionof adrenal steroids with androgenic and/or mineralocorti-

FIG. 5. EMSA. The wild-type receptor hGR� and the mutant recep-tors hGR�I559N, hGR�V571A, hGR�D641V, hGR�V729I, andhGR�I747M, expressed in COS-7 cells in the presence or absence ofdexamethasone (10�7 M), bound similarly to a radiolabeled double-stranded consensus GRE-containing oligonucleotide, indicating thatall mutant receptors preserve their DNA-binding activity in both thepresence and absence of ligand.


coid activity (15, 16, 23). The former accounts for manifes-tations of androgen excess, such as precocious puberty, acne,hirsutism, and infertility in both sexes, sexual ambiguity atbirth, male-pattern hair loss and menstrual irregularities infemales, and adrenal rests in the testes and oligospermia inmales. The latter accounts for symptoms and signs of min-eralocorticoid excess, such as hypertension and hypokalemicalkalosis. A large number of subjects may be asymptomatic,displaying biochemical alterations only (15, 16). Treatmentinvolves administration of high doses of mineralocorticoid-sparing synthetic glucocorticoids, such as dexamethasone(1–3 mg/d), which activate the mutated and/or wild-typehGR�, and suppress the endogenous secretion of ACTH(15, 16). Appropriate treatment is particularly important incases of severe impairment of hGR� function, because long-standing corticotroph hyperstimulation in association withdecreased glucocorticoid negative feedback might lead to thedevelopment of an ACTH-secreting adenoma (20).

Since generalized glucocorticoid resistance was first de-scribed and investigated in detail (33–35), more than 10 kin-dreds and sporadic cases with the condition have been re-ported. 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 defectsthat have been elucidated in the reported cases are summa-rized in Table 1, whereas the corresponding mutations in thehGR� 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 effecton the transcriptional activity of hGR� in the heterozygoticstate. Carriers of these mutations were asymptomatic, butmildly hypercortisolemic. By contrast, the heterozygous mu-tations I559N and I747M were previously shown to have adominant negative effect on the wild-type receptor and toproduce disease even in the presence of a normally func-tioning allele (22, 24). At a 1:1 ratio, the mutant receptorshGR�I559N and hGR�I747M inhibited approximately 25%of the transcriptional activity of the wild-type receptor. Thedominant negative activity of these mutants in associationwith their severely impaired ability to activate gene expres-sion resulted in a further decrement in the glucocorticoidsensitivity of affected individuals, given that their tissuessensed less than 40% of the glucocorticoid activity of normalcortisol concentrations (22, 24). The mode of genetic trans-mission in kindreds with these mutations was autosomaldominant, as the heterozygotic condition was sufficient tocause symptomatic disease. Dominant negative effects of

FIG. 6. A, Linearized GRIP1 molecule and dis-tribution of its functional domains. HLH, Helix-loop-helix; NIDaux, auxiliary nuclear receptorinteracting domain; PAS, period arylhydrogenreceptor and single-minded (24). B, GST pull-down assay. In vitro translated and 35S-labeledhGR�, hGR�I559N, hGR�V571A, hGR�D641V,and hGR�V729I were incubated with bacteri-ally produced GST-fused GRIP1-(1–1462),GRIP1-(597–774), and GRIP1-(740–1217) in theabsence or presence of dexamethasone (10�6 M).Samples were run on an 8% SDS-PAGE gel, andgels were fixed and exposed to x-ray film.


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mutant receptors have also been reported in the syndrome ofthyroid hormone resistance (36), whereas dominant inhibi-tory actions of physiologically expressed receptor isoformshave been demonstrated for the A isoform of the humanprogesterone receptor (37) and the � isoform of hGR (38, 39).

In a standard dexamethasone binding assay, all mutant re-ceptors hGR�I559N, hGR�V571A, hGR�D641V, hGR�V729I,and hGR�I747M had a reduction in the affinity for ligand com-pared with the wild-type hGR�, as previously reported (17, 18,23). This most likely reflects the location of these mutations inhelices 3, 7, and 11, respectively, which all line the ligand-binding pocket and may therefore directly affect ligandbinding.

In addition to the decreased affinity for ligand, transloca-tion of the GFP-fused mutants hGR�V571A and hGR�D641Vfrom the cytoplasm into the nucleus after exposure to dexa-methasone was slower than that of the wild-type receptor.After exposure to dexamethasone (10�6 m), GFP-hGR� wascompletely transported from the cytoplasm into the nucleuswithin 12 min, whereas the mutant receptors GFP-hGR�V571A and GFP-hGR�D641V required 25 and 22 min,respectively. These findings indicate that the mutationsV571A and D641V changed the receptors’ nucleocytoplasmicshuttling activity, probably through impairment of NL1function. This impairment may be due to the decreasedligand binding affinity of these mutant receptors, whichcould prevent a proper ligand-induced conformation changeand, hence, a normal interaction between NL1 and compo-nents of the importin system (22, 40). Binding of hsps tohGR� partially inactivates NL1 and might also explain thedifferences observed between hGR� and various mutants inthe time required for entry into the nucleus (41–43). Themutant receptor GFP-hGR�V729I was observed predomi-nantly in the nucleus in the absence of ligand, whereas fur-ther translocation from the cytoplasm into the nucleus re-quired longer exposure to the same concentration ofdexamethasone. This might indicate that this mutant inter-acts less closely with hsp90 and/or better with importinthrough its NL1 (41–43) at the nonligand-bound state,and/or that the mutation at amino acid 729 suppresses nu-clear export functions, resulting in increased retention of theunliganded receptor in the nucleus. Defective mechanismsthat may relate to delayed nuclear export, such as the cal-reticulin export pathway, and certain motifs in the DBD thatfunction as nuclear export signals might also account for thepredominance in nuclear localization of unligandedhGR�V729I (44–46).

Although all five hGR� mutants preserved their ability tobind DNA in a gel-shift mobility assay, they demonstratedan abnormal interaction with the nuclear coactivator GRIP1.These results suggest that each mutant receptor may form adefective complex with GRIP1, which is partially or com-pletely ineffective, because at least one important interactionsite is either weak or nonexistent. That the mutations I559Nand V729I are located very close to the AF-2 domain mayindicate that the interaction with other AF-2-associated pro-teins, such as p300/CBP and components of the vitamin Dreceptor-interacting protein/thyroid hormone-associatedprotein complex, could also be defective (10–12).

Based on our findings, we hypothesize that after exposure

to ligand, hGR� molecules translocate into the nucleus ashGR� mutant-hGR� mutant homodimers (in case of a ho-mozygous mutation) or hGR�-hGR� mutant heterodimers(in case of a heterozygous mutation). These homodimers andheterodimers have severely compromised inherent tran-scriptional activity, and although they may preserve theirability to bind to GREs or transcription factors that normallyinteract with hGR�, they produce functional impairment inone or more of the postligand-binding steps, i.e. translocationinto the nucleus or interaction with coregulators and/or spe-cific or general transcription factors (7). Therefore, each of theabove mutations imparts different functional defects uponthe GR signal transduction pathway, which explain the au-tosomal recessive or dominant transmission of the disorderand may in part explain its variable clinical phenotype. How-ever, one must not underestimate the importance of back-ground genetic and constitutional factors with epistatic ac-tions on the expression of the disorder (15). Thus, factors thatdefine the activity of the hypothalamic-pituitary-adrenalaxis, renin-angiotensin-aldosterone system, and gonadal axisas well as the target tissue sensitivity to glucocorticoids,mineralocorticoids, and androgens are bound to play im-portant 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. Evan-

gelia Charmandari, Pediatric and Reproductive Endocrinology Branch,National Institute of Child Health and Human Development, NationalInstitutes of Health, 10 Center Drive, Building 10, Room 9D42, Bethesda,Maryland 20892-1583.

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