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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 Transreppre

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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
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  A Novel Point Mutation of the Human GlucocorticoidReceptor Gene Causes Primary GeneralizedGlucocorticoid Resistance Through ImpairedInteraction With the LXXLL Motif of the p160Coactivators: Dissociation of the Transactivating andTransreppressive Activities Nicolas C. Nicolaides,* Michael L. Roberts,* Tomoshige Kino, Geoffrey Braatvedt,Darrell E. Hurt, Eleni Katsantoni, Amalia Sertedaki, George P. Chrousos,and Evangelia Charmandari Division of Endocrinology, Metabolism, and Diabetes (N.C.N., M.L.R., A.S., G.P.C., E.C.), First Department ofPediatrics, University of Athens Medical School, “Aghia Sophia” Children’s Hospital, and Divisions ofEndocrinology and Metabolism (N.C.N., M.L.R., A.S., G.P.C., E.C.) and Hematology (E.K.), Clinical ResearchCenter, Biomedical Research Foundation of the Academy of Athens, Athens 11527, Greece; Unit onMolecular Hormone Action (T.K.), Program in Reproductive and Adult Endocrinology,  Eunice Kennedy Shriver  National Institute of Child Health and Human Development, National Institutes of Health, and Bioinformaticsand Computational Biosciences Branch (D.E.H.), Office of Cyber Infrastructure and Computational Biology,National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892;and Department of Medicine (G.B.), University of Auckland, 1142 Auckland, New Zealand Context: Primarygeneralizedglucocorticoidresistanceisararegeneticdisordercharacterizedbygen-eralized,partial,target-tissueinsensitivitytoglucocorticoids.Themolecularbasisoftheconditionhasbeen ascribed to inactivating mutations in the human glucocorticoid receptor ( hGR ) gene. Objective:  The objective of the study was to present three new cases caused by a novel mutationin the  hGR  gene and to delineate the molecular mechanisms through which the mutant receptorimpairs glucocorticoid signal transduction. Design and Results:  The index case (father) and his two daughters presented with increased urinaryfree cortisol excretion and resistance of the hypothalamic-pituitary-adrenal axis to dexamethasonesuppression in the absence of clinical manifestations suggestive of Cushing syndrome. All subjectsharboredanovel,heterozygous,pointmutation(T 3  G)atnucleotideposition1724ofthe hGR gene,which resulted in substitution of valine by glycine at amino acid 575 of the receptor. Compared withthe wild-type receptor, the hGR  V575G demonstrated a significant (33%) reduction in its ability totransactivate the mouse mammary tumor virus promoter in response to dexamethasone, a 50% de-crease in its affinity for the ligand, and a 2.5-fold delay in nuclear translocation. Although it did notexert a dominant negative effect on the wild-type receptor and preserved its ability to bind to DNA,hGR  V575G displayed significantly enhanced (  80%) ability to transrepress the nuclear factor-   signalingpathway.Finally,themutantreceptorhGR  V575Gdemonstratedimpairedinteractionwiththe LXXLL motif of the glucocorticoid receptor-interacting protein 1 coactivator in vitro and in com-puter-based structural simulation via its defective activation function-2 (AF-2) domain. Conclusions:  The natural mutant receptor hGR  V575G causes primary generalized glucocorticoidresistancebyaffectingmultiplestepsintheglucocorticoidsignalingcascade,includingtheaffinityfor the ligand, the time required for nuclear translocation, and the interaction with the gluco-corticoid-interacting protein-1 coactivator.  (  J Clin Endocrinol Metab  99: E902–E907, 2014) ISSN Print 0021-972X ISSN Online 1945-7197Printed in U.S.A.Copyright © 2014 by the Endocrine SocietyReceived July 29, 2013. Accepted January 24, 2014.First Published Online January 31, 2014* N.C.N. and M.L.R. contributed equally to this project.Abbreviations:AF,activationfunction;ChIP,chromatinimmunoprecipitation;Gly,glycine;GRIP1,glucocorticoidreceptor-interactingprotein1;GST,glutathione-S-transferase;hGR,human glucocorticoid receptor; HPA, hypothalamic-pituitary-adrenal; I  B, inhibitory-  B;LBD, ligand-binding domain; NF-  B, nuclear factor-  B; nr, normal range; Val, valine. J C E M O N L I N EA d v a n c e s i n G e n e t i c s — E n d o c r i n e R e s e a r c h E902  jcem.endojournals.org J Clin Endocrinol Metab, May 2014, 99(5):E902–E907 doi: 10.1210/jc.2013-3005 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 06 May 2014. at 13:52 For personal use only. No other uses without permission. . All rights reserved.  P rimary generalized glucocorticoid resistance is a rare,familial, or sporadic genetic condition characterizedby generalized, partial, target-tissue insensitivity to gluco-corticoids, compensatory elevations in circulating cortisoland ACTH concentrations, and resistance of the hypotha-lamic-pituitary-adrenal (HPA) axis to dexamethasone sup-pression. The molecular basis of the condition has been as-cribed to mutations in the human glucocorticoid receptor( hGR ) gene, which impair the molecular mechanisms of hGR action (1–7). In the present study, we describe threenewcasescausedbyanovelheterozygousmutationofthe hGR geneandpresentthemolecularmechanismsthroughwhich the mutant receptor alters glucocorticoid signaltransduction. Case reports A 70-year-old man presented with bilateral adrenal hy-perplasiadetectedduringmelanomasurveillance.Hewasotherwise asymptomatic, normotensive (130/64 mm Hg)and had no clinical manifestations suggestive of Cushingsyndrome. Endocrinological evaluation revealed elevated8:00  AM  serum cortisol concentrations [661 nmol/L; nor-mal range (nr) 220–525 nmol/L] and increased urinaryfree cortisol excretion (507–749 nmol/d; nr 100–380nmol/d).Alow-dosedexamethasonesuppressiontest(0.5mgdexamethasoneevery6hfor48h)revealedresistanceof the HPA axis to dexamethasone suppression (8:00  AM serum cortisol, 98 nmol/L; nr  50 nmol/L). A pituitarymagnetic resonance imaging scan was normal.His two daughters, aged 41 and 47 years, presentedwith mild hirsutism and increased 24-hour urinary freecortisol excretion (556 and 687 nmol/d, respectively).They had normal blood pressure of 130/60 mm Hg and120/62 mm Hg, respectively. An overnight dexametha-sone suppression test (1 mg) revealed resistance of theHPA axis to dexamethasone suppression (8:00  AM  serumcortisol of 351 and 175 nmol/L, respectively; nr    50nmol/L).Apituitarymagneticresonanceimagingscanwasnormalandacomputedtomographyscanrevealednoad-renal hyperplasia.The above clinical findings suggested the diagnosis of primarygeneralizedglucocorticoidresistance.Writtenin-formed consent was obtained and additional molecularstudies were undertaken. Materials and Methods Amplification and sequencing of  hGR  gene Genomic DNA was isolated from peripheral blood lympho-cytes,andthecodingsequencesandintron-exonjunctionsofthe hGR  gene were PCR amplified and sequenced (8). Plasmids The plasmids used included pRShGR  , pF25GFP-hGR  ,pBK/CMV-hGR  ,pMMTV-luc,pGL4.73[hRluc/SV40],pRSV-erbA-1, pGEX4T3-GRIP1(1–1462), pGEX4T3-GRIP1 (596–774), and pGEX4T3-GRIP1(740–1217) (8). The plasmidspRShGR  V575G, pF25GFP-hGR  V575G, and pBK/CMV-hGR  V575G were constructed by introducing the V575G mu-tation into the pRShGR  , pF25GFP-hGR  , and pBK/CMV-hGR  , respectively. The plasmids pRSVC(p50)-NF-  B andpRSVC(p65)-RelA express, respectively, the p50 and the p65subunitsofnuclearfactor-  B(NF-  B)undercontroloftheRoussarcoma virus promoter. The p(I  B)3-luc expresses luciferaseunder the control of a NF-  B-inducible promoter (9). Transactivation and transrepression assays CV-1 cells were cotransfected with pRShGR   orpRShGR  V575G (0.0125   g/well) and pGL4.73[hRluc/SV40](0.02   g/well) together with pMMTV-luc (0.125   g/well) (fortransactivation assays) or pRSVC(p50)-NF-  B (0.0125   g/ well), pRSVC(p65)-RelA (0.0125   g/well), and p(I  B)3-luc(0.125  g/well)(fortransrepressionassays)usingLipofectin(In-vitrogen). Forty-eight hours later, cells were exposed to dexa-methasone or vehicle for 24 hours. Firefly and renilla luciferaseactivities were determined in the cell lysates (10). Western blot analyses CV-1 and COS-7 cells were transfected with pRShGR   orpRShGR  V575G (15   g/flask) using Lipofectin (Invitrogen).Westernblotanalyseswereperformedaspreviouslydescribed(8). Dexamethasone-binding assays COS-7 cells were transfected with pRShGR   orhGR  V575G (1.5   g/well) using Lipofectin (Invitrogen) andincubated with six different concentrations of [ 3 H]-dexa-methasone at 37°C in the presence or absence of a 500-foldmolar excess of nonradioactive dexamethasone for 1 hour.Dexamethasone-bindingassayswereperformedaspreviouslydescribed (8). Nuclear translocation studies HeLa cells were transfected with pF25GFP-hGR   orpF25GFP-hGR  V575G (2   g/dish) using FuGENE 6. Nucleartranslocation studies were performed as previously described (8). Chromatin immunoprecipitation (ChIP) assays HCT-116 cells were transfected with pRShGR   orpRShGR  V575G expressing plasmids using Lipofectamine2000 (Invitrogen). ChIP assays were performed as previouslydescribed (10). 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 GSTbeads.TheinvitrointeractionofpRShGR  andpRShGR  V575Gwith the GST-fused GRIP1 proteins was tested as previously de-scribed (10). doi: 10.1210/jc.2013-3005 jcem.endojournals.org  E903 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 06 May 2014. at 13:52 For personal use only. No other uses without permission. . All rights reserved.  Structural biology studies MoleculardynamicssimulationsforthehGRligand-bindingdomain(LBD;PDBID[ProteinDataBankID]:1M2Z)withandwithout the V575G mutation were performed in NAMD 2.7usingtheCHARMMforcefieldpackagedwithinVMD1.8.7aspreviously described (10). Results Sequencing of the  hGR  gene AsingleheterozygousT 3  Gsubstitutionwasidentifiedatnucleotideposition1724inallthreesubjects.Thismu-tationresultedinvaline(Val)toglycine(Gly)substitution(GTG 3  GGG)ataminoacidposition575(exon5)intheLBD of the receptor (Figure 1A). hGR  V575G demonstrates decreasedtransactivational activity but does not exertdominant negative effect upon the wild-typehGR  Comparedwiththewild-typereceptor,thehGR  V575Gdemonstrated a significant reduction (33%) in its ability totransactivatethemousemammarytumorviruspromoterinresponsetodexamethasone(Figure1B).CotransfectionwithaconstantamountofhGR  andincreasingconcentrationsof hGR  V575G showed that hGR  V575G did not exert adominantnegativeeffectuponthehGR  .Westernblotanal-ysesdemonstratednodifferencesintheexpressionofhGR  and hGR  V575G proteins. hGR  V575G Displays Enhanced Ability toTransrepress the NF-   B Signaling PathwayCompared with the Wild-type hGR  Compared with the hGR  , the hGR  V575G demon-strated significantly enhanced ability (  80%) to transre-press the activity of NF-  B on the inhibitory-  B (I  B)-3promoter in response to dexamethasone. hGR  V575G demonstrates lower affinity for theligand compared with the wild-type hGR  TheapparentdissociationconstantofhGR  V575wassignificantlyhigherthanthatofhGR  (11.6  1.5nMvs6.0  0.6 nM,  P  .05), indicating that the affinity of themutant receptor for the ligand was 50% lower than thatof the wild-type receptor. No difference in the number of hGR binding sites was noted between hGR   andhGR  V575G. A 0 10 -12 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 Dexamethasone(M)    R   e    l   a      v   e   L   u   c   i    f   e   r   a   s   e   A   c      v   i   t   y B *** C 0 min9 min6 min3 min12 min15 min D 0 min6 min12 min18 min24 min36 min Figure 1.  A, Sequencing of the entire coding region of the  hGR  generevealed a novel heterozygous T 3  G substitution at nucleotide position1724, resulting in replacement of Val by Gly (GTG 3  GGG) at aminoacid position 575 (exon 5) in the LBD of the receptor. B, Transcriptionalactivity of the wild-type (WT) hGR   and the mutant receptorhGR  V575G. Compared with the wild-type receptor, the mutantreceptor demonstrated an approximately 33% reduction in its ability totransactivate the glucocorticoid-inducible mouse mammary tumor viruspromoter in response to increasing concentrations of dexamethasone.Bars represent mean  SEM of at least five independent experiments.Asterisks indicate statistically significant differences. C and D,Subcellular localization and nuclear translocation studies of the wild-type and mutant receptors. GFP-hGR  V575G demonstrates delayednuclear translocation compared with the GFP-hGR   after exposure todexamethasone. HeLa cells transiently expressing GFP-hGR   (C) orGFP-hGR  V575G (D) were treated with 10  6 M of dexamethasone.Images of the same cells were obtained at the indicated time points. E904  Nicolaides et al Novel hGR Mutation in Glucocorticoid Resistance J Clin Endocrinol Metab, May 2014, 99(5):E902–E907 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 06 May 2014. at 13:52 For personal use only. No other uses without permission. . All rights reserved.  hGR  V575G demonstrates delayed nucleartranslocation compared with the wild-type hGR  In the absence of dexamethasone, both the wild-typeand the mutant receptors were localized in the cytoplasmof cells. Addition of dexamethasone (10  6 M) resulted intranslocationofhGR  andhGR  V575Gintothenucleuswithin 15 minutes (mean  SE, 12.6  0.4 min) and 36minutes (mean  SE, 34.8  1.4 min;  P  .001), respec-tively (Figure 1, C and D), suggesting a 2.6-fold delay innuclear translocation of the hGR  V575G. hGR  V575G preserves its ability to bind to DNA ChIP assays showed no significant differences in theability of hGR   and hGR  V575G to bind to glucocorti-coid response elements of the endogenous  G6Pase  and GILZ  genes. hGR  V575G demonstrates impaired interactionwith the GRIP1 coactivator due to defectiveactivation function (AF)-2 domain The hGR  V575G demonstrated decreased ability tointeract with the nuclear receptor binding domain of GRIP1 and the full-length GRIP1, whereas its interactiontothecarboxyl-terminalfragmentofthiscoactivatorwaspreserved. Therefore, the hGR  V575G displays an im-pairedinteractionwiththeGRIP1coactivatorthroughitsdefective AF-2 surface. hGR  V575G displays impaired interaction with theLXXLL motif in computer-based structuralsimulation Inthe3-dimensionalcrystallographicstructuralmodelof the hGR   LBD bound to dexamethasone, V575 is lo-catedintheC-terminalportionofhelix5.Valhasalarge,hydrophobic side chain, which protrudes into the AF-2surfaceandcreatesanoncovalentinteractionwiththesec-ondandthirdleucineoftheLXXLLmotif(Figure2  ).Inthe LBD of hGR  V575G, this Val residue is replaced byGly. In our molecular simulation for hGR  V575G LBD,two noncovalent bonds observed between V575 and theleucines of the LXXLL motif were completely lost, sug-gesting that hGR  V575G displays impaired interactionwiththeLXXLLmotif(Figure2B).Wefurthercalculatedtherootmeansquaredeviationforthehelicalresiduecon-taining the LXXLL motif (amino acids 743–750) of thehuman GRIP1 against the AF-2 surface. As expected, thehGR  V575G LBD demonstrated a 2-fold increase in thenumber of distance between LXXLL motif and AF-2 sur-face (Figure 2C); hence, this mutant LBD has reduced af-finityfortheLXXLLmotif.WealsodemonstratedthatthehGR  V575GLBDhadasignificantlywiderAF-2surface(Figure 2D). Taken together, these results provide a mo- L745L748L749V575 WT L745L748G575L749 V575G BAC WTV575G    R   M   S   D   (    Å   ) 43210 **** D WTV575G    B  u  r   i  e   d   S  u  r   f  a  c  e   A  r  e  a   (    Å    2    ) 1210987 ** 11 AF-2 Surface AreaDistance of LXXLL to AF-2 Figure 2.  Three-dimensional crystallographic structural model of the LBDof the wild-type (WT) hGR  bound to dexamethasone. In the wild-typereceptor (A), the V575 creates a noncovalent interaction with the secondand third leucine (L) of the LXXLL motif of the GRIP1 coactivator, therebyfacilitating their interaction through the AF-2 domain of the hGR  . In theLBD of hGR  V575G (B), the replacement of Val by Gly resulted in a loss ofthe two noncovalent bonds and a reduced interaction of the mutantreceptor with the LXXLL motif through an increase in the distancebetween the AF-2 domain and the LXXLL motif (C). Estimation of the areaof the AF-2 surface of the WT and mutant receptors (D) indicated that theLBD of the mutant receptor had significantly wider AF-2 surface than thatof the WT receptor. RMSD, root mean square deviation.doi: 10.1210/jc.2013-3005 jcem.endojournals.org  E905 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 06 May 2014. at 13:52 For personal use only. No other uses without permission. . All rights reserved.  lecular explanation for the reduced transcriptional activ-ity of hGR  V575G and its defective interaction with theNRB domain of the GRIP1 coactivator. Discussion In the present study, we identified a novel heterozygouspoint mutation in exon 5 of the  hGR  gene in three newcases of primary generalized glucocorticoid resistance,whichresultedinsubstitutionofValtoGlyataminoacidposition 575 in the LBD of hGR  . We systematically in-vestigated the molecular mechanisms through whichhGR  V575G affects normal glucocorticoid signal trans-duction.ThemutantreceptorhGR  V575Gdemonstrateda significant reduction in its ability to transactivate glu-cocorticoid-responsive genes, had a 50% lower affinityfor the ligand, and a 2.5-fold delay in nuclear transloca-tion, it preserved its ability to bind to DNA and demon-strated an impaired interaction with the GRIP1 coactiva-torduetoitsdefectiveAF-2domain.Ourfindingssuggestthat hGR  V575G affects multiple steps in the glucocor-ticoid signaling pathway.The reduced affinity of the mutant receptor for the li-gand is most likely due to the location of the mutationwithin the LBD of the receptor, given that substitution of Val to Gly at amino acid position 575 in helix 5 mightinduce a conformational change of this domain. In addi-tion, hGR  V575G showed a 2.5-fold delay in nucleartranslocation, indicating that the V575G mutation altersthe nucleocytoplasmic shuttling of hGR  . This defectmightbecausedbyimpairmentofthenuclearlocalizationsequence 1 and/or nuclear localization sequence 2 func-tions or occurs secondary to the reduced affinity for theligand (8, 10–17).InourmolecularsimulationforhGR  V575GLBD,wefound that the V575 of hGR   is part of the AF-2 surfaceand that the hydrophobic side chain of this amino acidcontributeddirectlytotheattractionoftheLXXLLmotif by forming a noncovalent bond with its second and thirdleucine.TheG575ofthemutantreceptorlostthesebondstotheLXXLLmotif;therefore,itinteractedwiththeNRBdomainofGRIP1lessefficientlythanthewild-typerecep-tor.TheseresultsconcurwithourpreviousstudiesofotherhGR mutations located in the LBD of the receptor; how-ever, the hGR  V575G is the first mutant receptor thataffects directly a residue within the AF-2 surface (8,10–17).Interestingly, the transrepression assays showed thathGR  V575G displayed enhanced ability to transrepressthe NF-    signaling pathway. Although the underlyingmechanism is not known, a stronger interaction of themutant receptor hGR  V575G with the p65 subunit of NF-    heterodimer and/or reduced induction of gluco-corticoid-responsiveI  Bbytheactivatedmutantreceptormight play a role.The insensitivity to glucocorticoids in patients withpathological hGR   mutations accounts for the hyperac-tivity of HPA axis and consequent alterations in stressresponse(18,19).TheelevatedCRHconcentrationsmaybe further accentuated by activation of the mineralocor-ticoid receptors in the hippocampus by glucocorticoids(20).In summary, we identified and functionally character-ized a novel point mutation in the  hGR  gene causing pri-mary generalized glucocorticoid resistance. ThehGR  V575G impairs multiple steps in the pathway of glucocorticoid signal transduction, including the affinityfortheligand,thetimerequiredfornucleartranslocation,and the interaction with the GRIP1 coactivator, and ex-pressessignificantdissociationbetweenitstransactivatingand transrepressive activities. Acknowledgments Address all correspondence and requests for reprints to:Evangelia Charmandari, MD, MSc, PhD, MRCP(UK),CCST(UK), Division of Endocrinology, Metabolism, and Dia-betes,FirstDepartmentofPediatrics,UniversityofAthensMed-icalSchool,’AghiaSophia’Children’sHospital,ThivonandPa-padiamantopoulou Street, Athens 11527, Greece. E-mail:evangelia.charmandari@googlemail.com.ThisworkwassupportedbytheEuropeanUnion(EuropeanSocialFund)andGreeknationalfundsthroughtheOperationalProgram “Education and Lifelong Learning” of the NationalStrategic Reference Framework, Research Funding Program,THALIS, University of Athens, Athens, Greece; the intramuralprogram of the  Eunice Kennedy Shriver  National Institute of Child Health and Human Development, National Institutes of Health,Bethesda,Maryland20892;andtheNationalInstituteof Allergy and Infectious Diseases, National Institutes of Health,Bethesda, Maryland.Disclosure Summary: The authors have nothing to disclose. References 1.  Chrousos GP, Vingerhoeds A, Brandon D, et al.  Primary cortisolresistanceinman.Aglucocorticoidreceptor-mediateddisease.  JClinInvest  . 1982;69(6):1261–1269.2.  Chrousos GP, Detera-Wadleigh SD, Karl M.  Syndromes of gluco-corticoid resistance.  Ann Intern Med  . 1993;119(11):1113–1124.3.  Charmandari E, Kino T, Ichijo T, Chrousos GP.  Generalized glu-cocorticoid resistance: clinical aspects, molecular mechanisms, andimplications of a rare genetic disorder.  J Clin Endocrinol Metab .2008;93(5):1563–1572.4.  Charmandari E, Kino T.  Chrousos syndrome: a seminal report, aphylogenetic enigma and the clinical implications of glucocorticoidsignalling changes.  Eur J Clin Invest  . 2010;40(10):932–942. E906  Nicolaides et al Novel hGR Mutation in Glucocorticoid Resistance J Clin Endocrinol Metab, May 2014, 99(5):E902–E907 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 06 May 2014. at 13:52 For personal use only. No other uses without permission. . All rights reserved.
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