Glucocorticoid receptor mutants: man-made tools for functional research

Glucocorticoid receptor mutants:man-made tools for functional researchIlse M. Beck1, Karolien De Bosscher2,3 and Guy Haegeman3

Laboratory of Eukaryotic Gene Expression and Signal Transduction (LEGEST), Department of Physiology, Ghent University, K.L.

Ledeganckstraat 35, B-9000 Gent, Belgium

Review

The glucocorticoid receptor (GR) is a ligand-dependenttranscription factor that can bind to glucocorticoids(GCs). Upon ligand binding, GR sheds its cytoplasmicchaperoning complex and translocates to the nucleus,where it can act as a ligand-dependent transcriptionfactor, transactivating or transrepressing specific genepromoters. Often, GR interacts with specific cofactors toimplement a variety of gene promoter effects. GR activi-ty and function is further modulated by post-translation-al modifications. To assess the diverse aspects of GRmechanisms of activation and gene regulation, research-ers continue to use a range of artificial GR mutants. Inthis review we analyze the characteristics of GR mutantswith the aim of assisting the design and interpretation ofGR mutant-based experiments.

Glucocorticoid receptor activation and functionThe glucocorticoid receptor (GR), NR3C1, belongs to thenuclear receptor family and can bind glucocorticoids (GCs).These naturally occurring steroids play a role in the fight-or-flight stress response. Their immunomodulatory effectsmake these steroids the preferred anti-inflammatory drugof today. However, the multitude of diverse steroid-medi-ated effects generates an unfavorable side-effect profile inchronic GC-based therapy [1,2] (Box 1).

GR domains

Full-length GR was cloned in 1985 [3], after which the GRwas subjected to a range of mutagenesis studies to assessdomain functionality (Figure 1a). The N-terminal domaincomprises a transactivation function AF-1 and containsmost residues subject to post-translational modifications.The C-terminal domain (CTD) harbors a second transacti-vation domain AF-2, the ligand-binding domain (LBD),nuclear localization signals (NLSs) and protein-bindingsites. The DNA-binding domain (DBD) plays importantroles in both GR homodimerization and DNA binding [4].

GR-mediated activation and transcription regulation

Upon ligand binding, the cytoplasmic GR changes confor-mation, dissociates from its chaperoning complex andtranslocates to the nucleus where it gives rise to positive

Corresponding author: Beck, I.M. ([email protected])1 Current address: Laboratory of Experimental Cancer Research, Department of

Radiation Oncology and Experimental Cancer Research, Ghent University Hospital,Ghent University, De Pintelaan 185, B-9000, Belgium.

2 Current address: Cytokine Receptor Laboratory, Department of Medical ProteinResearch, VIB-UGent, Albert Baertsoenkaai 3, B-9000 Gent, Belgium.

3 Shared senior authorship.

1043-2760/$ – see front matter � 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tem.2011.

and negative transcriptional effects on specific gene pro-moters via diverse mechanisms (Figure 1b) [4–6]. GR canalso modulate gene expression by affecting several signal-ing cascades, such as p38, ERK and JNK MAPKs, mitogen-and stress-activated protein kinase MSK1, the cyclin-de-pendent kinase complex pTEFb and TANK-binding kinaseTBK1 [5]. Furthermore, GR function and signalingstrength are regulated via post-translational modulation[5] and its dynamic interaction with cofactors in multi-subunit coregulatory complexes [7].

To refine our knowledge of GR structure–function rela-tionships researchers have used GR mutants. Some GRmutants in which large peptide sequences are ablated orreplaced are currently still used, whereas new mutants,especially point mutations affecting single amino acids(AAs), are still being developed (Box 2). Concerns regard-ing the interpretation of GR mutant-based results arediscussed in Box 3. Some of these mutants were con-structed de novo, but others occur naturally and displayparticular characteristics [4,8]. Exemplary in this respectare the GC-resistant GR mutants derived from murinelymphoma S49 cell lines [9]. In this review we focus on thecharacteristics displayed by new GR mutants with partic-ular attention to mutations that have helped to define keyknowledge on GR function over the past decade (Table 1).

GR mutants in GR-mediated transcription regulationGR isoforms

Although GR is ubiquitously expressed and transcribedfrom a single gene, its cellular distribution cannot beconsidered to be homogenous. The discovery of isoformsoriginating from alternative translation start sitesemerged from a mutational study of the GR start codonsM1 and M27 [10]. The isoforms are generated from acombination of alternative splicing and differential utili-zation of transcription and translation start sites, and areassociated with distinct responses to GC treatment [4,8].

GR ligand binding

GRa is the most widely expressed and researched isoform[8]. Because its LBD is necessary for ligand binding, pointmutations in the ligand-binding pocket obviously affectthis process [11,12]. For instance, mouse GR (mGR)C644G (in the LBD) enhances GR ligand-binding affinity[13], whereas mGR Y770N (at the end of the CTD) does not[14]. The single point mutation in the human GR (hGR),M604L (mGR M610L), which alters the H3–H5 interactionimportant for coactivator binding, displays increased GC

03.009 Trends in Endocrinology and Metabolism, August 2011, Vol. 22, No. 8 295

mailto:[email protected]

http://dx.doi.org/10.1016/j.tem.2011.03.009

Box 1. GC-associated side-effects

GC treatment is associated with a detrimental side-effect profile

[1,2], in which prolonged therapy and dosage are central risk factors.

Mechanistically, these side-effects predominantly arise from GR

transactivation, although undesired adverse effect can also originate

via GR transrepression or both GR transrepression and GR

transactivation [1,2,104] (Figure I). Although unwanted in most GC

therapies, these adverse side-effects stem from overstimulation of

normal physiological GC/GR actions.

GRTransactivation

GR Transactivation& GR Transrepression

GRTransrepression

CataractGlaucoma

Skin atrophyHypertension

Muscle wastingDiabetes mellitus

Thymus weight reduction

Increased susceptibilityto infections

Suppression of HPA axisFatty liver development

OsteoporosisGrowth retardation

Cushingoid appearance

TRENDS in Endocrinology & Metabolism

Figure I. GC-associated side-effects: GR transactivation versus GR

transrepression. Other reported GC-associated side-effects involve the PI3K-

and SGK1-mediated increase in gastric acid secretion, and the concomitant risk

of peptic ulcer development and gastrointestinal bleeding [105,106]; and also

GC-induced emotional lability, insomnia, with enhanced appetite and weight

gain, for which the mechanisms appear more complex and have not been fully

elucidated [1,2].

Review Trends in Endocrinology and Metabolism August 2011, Vol. 22, No. 8

affinity and GR transactivation [15]. This mutant wasfurther examined in a gain-of-function GR M610Lknock-in mouse [16], which confirmed the in vitro resultsand therefore could represent a candidate model to studythe effects of increased GC sensitivity [16]. A deletion of 14AAs of the C-terminal end of the mGR b strand results inan inactive GR unable to bind GCs [14], most probablybecause the active conformation of helix 12 of GR, that isknown to cap the ligand-binding site and is differentiallytilted by agonist versus antagonist binding, is destabilized[17]. Interestingly, X-ray structure superposition of dexa-methasone (DEX)-bound and deacylcortivazol (DAC)-bound rat GR (rGR) led to the identification of a residuethat, upon mutation (rGR R629Y), generates a rGR thatcan only accommodate DAC- but not DEX-induced GRtransactivation [12], suggesting that GR modificationcan affect ligand-specific activity.

Initial screening for novel GR ligands made use of aGal4(DBD)–GR chimera [18]. The ability of a potentialligand to activate a Gal4-responsive reporter gene viabinding to the Gal4(DBD)–GR-LBD chimera indicates di-rect binding to the ligand-binding pocket and transforma-tion of the LBD into the active conformation. Although this

296

strategy allows for rapid screening, Gal4(DBD)–GR-LBD-based experiments have a bias towards ligands whichsupport GR transactivation mechanisms. Another pitfallis that GR modulators affecting GR without actually bind-ing to its ligand-binding pocket are also missed.

Currently, GR research focusing on improved therapiesfor inflammatory diseases have sought more selective GRligands/modulators (SGRMs) or ‘dissociative’ ligands thatpermit GR transrepression of proinflammatory genes butthat do not induce GR activation of glucose metabolism-associated genes [1,19]. For this, GR deletion mutants havebeen useful in detecting which GR domains are essentialfor GR/SGRM action [20]. Drug screens for selective estro-gen receptor (ER) modulators have exploited a GR chimerain which the GR LBD is swapped for the ER LBD, whichgives a more potent response [21]. This chimera approach,utilizing the GR N-terminal domain and DBD as a scaffoldfor the LBD of a different nuclear receptor LBD, haspermitted screening for ER ligands. However, chimera-based results should be interpreted with caution andshould always be controlled for by comparing chimeraeffects to those induced by full-length GR or ER on theirrespective reporter gene vectors, to avoid false negativesand positives.

GR subcellular translocation

Although GR is classically thought to reside in the cyto-plasm prior to ligand binding, in fact GR continuallyshuttles between the nucleus and cytoplasm, constantlyscanning the cellular environment [22–24]; even within thenucleus the mobility of GR is ligand-dependent [25]. Thesubcellular location of GRa is controlled by both the importand export rates of the receptor through the nuclear porecomplexes.

Import of GR via importina/b-based mechanisms iscontrolled via the NL1 and NL2 domains [26] (Figure 1a).Deletion studies showed that NL1 supports rapid and hor-mone-independent translocation of GR, whereas NL2 facil-itates slower and hormone-dependent nuclear import[22,27–29].

GR export appears to be more complex. Pharmacologicalblockade of exportin1/CRM1-mediated export abrogatesthe translocation of GR to the cytosol [22,30]. In thisrespect, a leucine- and isoleucine-rich nuclear export se-quence (NES) was identified in the GR LBD on the basis ofsequence homology [31], but awaits experimental confir-mation via mutation analysis. It is equally likely that GRexportin1/CRM1-mediated export can be mediated by aNES-containing bridging partner. The NES-containingcoactivators SRC-1 [32] and 14-3-3s [33] are candidatesfor this piggyback strategy. In support, the GC-mediatedassociation of 14-3-3s with the GR LBD enhances nuclearexport of GR and inhibits GR transactivation potential, allof which takes place in a 14-3-3s NES-dependent manner[33]. Moreover, exportin1/CRM1 appears to account onlyfor the export of unliganded GR because export of GC-bound GR is suggested to be mediated via a Ca2+-depen-dent calreticulin-based mechanism [34,35]. However, an-other study contested this calreticulin-dependent export[36]. Notably, fusing the nuclear-export signal (NES) of IkBto the N-terminal tail of GR not only results in accelerated

Box 2. GR mutation strategies

The glucocorticoid receptor can be mutated via several strategies,

listed below, ranging from coarse to refined.

� Deletion mutants: large or small stretches of oligopeptides are

removed from the translated region of GR, for example rGR N525

in which all amino acids after position 525 were deleted[49]. Such

mutants were often used in initial GR screening to map receptor

domains.

� Chimeric mutants

� Added sequences: a sequence of the translated region of a non-

GR heterologous origin is added to the GR sequence, predomi-

nantly at the C- and/or N-termini, for example NES–GR[37].

� Swapped sequences: a sequence of the translated region of a

particular part of GR is replaced by a sequence of non-GR origin,

for example GRSV122–139 [24]. In some cases the inserted sequence

is derived from other nuclear receptors, as in the ER–GR chimera

[21].

� Point mutants: single amino acids within the GR are changed, for

example mGR S212A. These point mutations are sometimes also

combined to obtain a GR variant with multiple point mutations, for

example mGR S220A/S234A[73].

Of course, these strategies can be combined to generate a more

complex GR mutant, for example NES–GST–GRhinge,LE507–508AA–

GFP–NLS[24]. However, the physiological relevance of experimental

results obtained with highly artificial mutants should be confirmed

and additionally demonstrated by experiments using these muta-

tions in the full-length GR sequence.


export of NES–GR chimeras, but also in increased steroid-dependent downregulation of GR protein by cytoplasmicproteasomes [37]. Therefore, the interpretation of translo-cation-altering mutants should also take GR expressionlevel changes into account.

Following an elegant mutational study of GR, Carriganand coworkers identified a nuclear-retention signal (NRS)in GR. This NRS, which overlaps closely with the NL1 inthe hinge region of GR, can actively counteract the redis-tribution of GR to the cytoplasm after steroid withdrawal.Although specific point mutations could abrogate NRSfunction in a recombinant GR hinge mutant, in the contextof full-length GR these same point mutations still allow forretarded redistribution to the cytoplasm after hormonewithdrawal and are insufficient to abolish NRS function[24]. These results thus warn us not to overinterpretresults of recombinant proteins carrying only small GRpeptide sequences. Nevertheless, research using greenfluorescent protein (GFP)–GR chimeras or diverse GRmutants harboring alternative translocation characteris-tics have provided insights into GR translocation mechan-isms and GR functionality in different subcellularcompartments [25,33].

GR chaperone complex

The GR chaperone complex has an important function inassisting the proper protein folding of GR, but also sup-ports GR stability and ligand binding. The CTD of GR isthought to be the main interaction platform for chaperon-ing heat-shock proteins (Hsp). Hsp90 plays an importantrole in maintaining the ligand accessibility of the ligand-binding pocket in the hydrophobic core of the LBD [38]. Theinteraction of Hsp90 to rGR was mapped to AAs 547–553 inhelix 1 of the GR LBD, containing a LxxLL motif [38,39].

Interestingly, double mutations of the L residues in theLxxLL motif of this helix 1 do not affect the hsp90:GRassociation, but can affect ligand binding and thus GRtransactivation [38,40]. Because this LxxLL motif is notpart of the ligand-binding pocket, the decline in ligandbinding was suggested to take place via a conformationchange [40].

GR-mediated transcription activation

Structurally, GR transactivation is regulated via twotransactivation domains. The activation function AF-1 issituated in the NTD and functions ligand-independently,whereas the ligand-dependent AF-2 is located in the LBD.Gene targets of GR can be differentially affected whenablating either GR AF-1 or AF-2 [41]. Nonetheless, despitethe C-terminal location of both AF-2 and LDB, mutationsimpairing AF-2 function do not affect GR ligand binding,suggesting that the two functions use different amino acidsor structures [12,42]. Another study using a GR AF-2 pointmutant (mGR C644G) suggested that AF-2 function super-seded AF-1-mediated activation in a mouse mammarytumor virus (MMTV) nucleosomal array [13].

The NTD AF-1 contributes to the interaction of GR withcofactors, chromatin-remodeling enzymes, RNA Pol II, theTAT-binding protein and TBP-associated proteins(TAFIIs). However, the CTD of GR can also accommodatecoactivator binding to the C-terminal AF-2 domain [43].These GR-bound multisubunit coregulator complexes canconsist of p300 or CBP, p/CAF, steroid receptor coactiva-tors SRC1, SRC2 and/or SRC3, all of which possess histoneacetyl-ransferase (HAT) activities, and also PGC-1a, whichcan recruit HAT activity-containing co-factors, such asSRC-1, p300 or DRIP/TRAP [7]. The GR-bound enhanceo-some of promoters governed by glucocorticoid responseelements (GREs) could also contain the ATP-dependentchromatin-remodeling complex SWI/SNF and/or elementsof the DRIP/TRAP complex [7].

GR mutations confirmed that the two zinc-finger motifsin the DBD of the GR are crucial for GR binding to itsrespective GREs in targeted gene promoters [44,45]. With-in the evolutionarily conserved DBD, GR–ER mutationalswap analyses and point mutations have suggested thatthe P-box and particularly rGR K461 play a role in GRErecognition, whereas the D-box in the second zinc fingerplays a role in GR dimerization [44,45]. In that respect,mutation of mGR A465T in this second zinc finger rendersthis GR dimerization-defective, hence its name GRdim,and is therefore unable to bind or drive classic GRE-regulated transcription [46]. However, GRdim retainsthe capability to transrepress NF-kB- and AP-1-regulatedgenes in vivo [47]. The initial results using GRdim sparkedthe belief that the therapeutically exploited anti-inflam-matory effects of GR are mainly due to GR-mediatedtransrepression of AP-1- and NF-kB-regulated, inflamma-tion-associated genes, whereas GC-associated side-effectsare predominantly mediated by GR transactivationmechanisms [1,47].

Ligands that mimic this selectivity in GR function arestill highly sought-after because their enhanced selectivitycould hold great therapeutic promise [19]. However, not allGC-mediated promoter activation is abrogated by the

297

TRENDS in Endocrinology & Metabolism

DNA binding

hGR 777aa

LBDDBDNTDNH2

T8

PP P P P P P P P P

S45

S11

3S

134

S14

1

S20

3S

211

S22

6S

234

S26

7

Ub

K41

9

Ac

K48

0A

c K

494

Ac

K49

5A

c K

492

SU

MO K

277

SU

MO K

293

SU

MO K

703

S40

4

HR COOH

Transcription factor binding

LocalizationDimerization

Extracellular

Intracellular

Glucocorticoid

Cytoplasm

Nucleus

Tethering

Tethering

nGREComposite GRE

Competitive GRE Sequestration

Composite GRE

Simple GRE

GR

GR

(b)

(a)

TF

TF

TF TFGR GR

GR GR

GR

GR

GR

TF

GR

TFGR

GREGRE

Chaperones

TransactivationCofactor binding

Chaperone binding

Ligand binding

AF1

NL2

AF2NL1/NRS

Figure 1. (a) Structural properties of the human glucocorticoid receptor. The GR consists of a N-terminal domain, a DNA-binding domain, a hinge region and a C-terminal

ligand-binding domain. Above the diagram, possible post-translational modifications of the human GR are shown. Below the diagram, the designated functions of GR

domains are indicated. (b) Glucocorticoid receptor-mediated transcriptional regulation. Glucocorticoids, which posses a steroidal structure, can diffuse freely across the

plasma membrane. In the cytoplasm, a NLS-masking chaperoning complex retains the unactivated and unliganded GR in a ligand-receptive state. Upon ligand binding, GR

changes conformation, dissociates from its chaperoning complex and then translocates into the nucleus. However, liganded and unliganded GR actually shuttle between

cytoplasm and nucleus. In the nucleus, liganded GR can exert positive and negative transcriptional effects via different mechanisms, as displayed. The prototypical

mechanism of GR transactivation features binding of a GR homodimer to a palindromic glucocorticoid response element (GRE), whereas GR-mediated transrepression

mechanism most commonly deploys a tethering mechanism in which GR interacts with a DNA-bound transcription factor such as NF-kB or AP-1. Nevertheless, GR can also

activate and repress gene promoters via other mechanisms. Abbreviations: Ac, acetylation; HR, hinge region; h, human; NTD, N-terminal domain; P, phosphorylation;

SUMO, sumoylation; TF, transcription factor; Ub, ubiquitination.


298

Box 3. Concerns of GR mutant-based experiments

One must always tread carefully when interpreting results from GR

mutant-based experiments due to various concerns. Although the GR

protein sequence shows 94% homology between human and mouse,

92% homology between human and rat, and 97% homology between

mouse and rat, minor sequence differences do exist and structural

features could be species-specific. For this reason GR mutants could

also display species-specific effects; for example, the enhanced ability

of mGR in comparison to hGR to activate a TAT–GRE reporter gene,

which has been mapped to a single AA acid difference[99]. GR

mutants could also have cell type-specific characteristics [72] because

various cells differ in their cofactor, transcription factor, kinase and

other regulatory protein concentrations, associations and post-

translational modulations. Promoter-specific effects [76,77,98] are

also relevant; the level of GR-mediated promoter activation can be co-

determined by the number of GRE elements in the promoter, the GR-

binding sequence and its allosteric control over the GR conformation,

and the composition of the promoter-bound enhanceosome. How-

ever, cautions in extrapolating from promoter-specific, cell type-

specific and species-specific results are not only restricted to GR

mutant-based results.

More specifically for GR mutants, concerns regarding whether the

results can be extrapolated to physiological settings could depend on

how the GR mutants are expressed. Transient transfection is used

most often in cell lines with a background of no or very low levels of

endogenous GR. However, the less-used method of stable transfec-

tion of the GR mutant expression vector entails better stabilization of

expression and thus protein levels. Nevertheless, both methods

express excessive and non-physiological levels of GR, possibly

causing artefactual results; furthermore, expression level differences

between cell lines expressing different GR mutants cannot be

avoided. Preferably the GR mutant of interest should be inserted into

the genome as a single copy and placed under the control of its

endogenous promoter in an in vivo system, such as in mice. However,

this in vivo strategy dismisses the possible benefit of swiftly scanning

large numbers of GR mutants. Thus far, this approach has only been

used for researching GRdim mutant characteristics [46,47,64,66] and

recently those of mGR M610L [16] and rGR LS7 [65].

Moreover, sequence deletions, swaps, and single point mutations

could inadvertently change GR conformation [14,17]. These undesired

structural changes might then unveil previously unexposed GR

oligopeptides, and thus unintentionally alter GR interactions,

GR-driven mechanisms and even overall GR function. Control

experiments analyzing GR mutant ligand-binding affinity, GR inter-

action dynamics with its chaperones and coregulators, and GR

subcellular translocation could indicate potential effects of the

mutation on proper GR folding.


GRdim mutant [41], indicating that there are additionalmechanisms for GR-mediated transcription activation(Figure 1b). Indeed, monomeric GRs enhance transcriptionvia tethering mechanisms in which GR binds to a DNA-bound transcription factor (and not to DNA itself), forexample GR tethers to STAT for the stimulation of b-caseingene expression, or via a composite GRE in which DNA-bound GR combines forces with another DNA-bound tran-scription factor to enhance gene transcription, for exampleGR collaborates with AP-1 to stimulate proliferin expres-sion [6] (Figure 1b).

GR-mediated transcription repression

GR-mediated transrepression most commonly features atethering mechanism in which non DNA-bound GR associ-ates with and thus inhibits the function of a DNA-boundtranscription factor, for example NF-kB p65 or AP-1(Figure 1b) [5,6]. Mutational analyses have demonstratedthat diverse GR domains are involved, with the second zincfinger of the GR DBD affecting binding to NF-kB [48–50].Deletions or point mutations in the second GR zinc fingerresults in loss of IL6 repression. However, a GR mutantwith deletion or point mutations in the first zinc finger canactivate IL6 and other gene promoters without specific

Table 1. Characteristics of GR mutants

GR ligand binding

Effect Mutant

Mut>wt hGR M604L

mGR C644G

Mut<wt rGR L550S, rGR L550S/L553S

GST–547C/L550S, GST–520C/L550S, GST–520C/L553

rGR P548A/T549A/V551A

hGR 532 LxxAA, hGR 718 LxxAA

mGR L550A, mGR D555A

rGR E558A, rGR L584D/Q588K, rGR R629Y

rGR L584D

rGR L584S

rGR L671S, rGR E706K

DNA binding [50]. Notably, GR transrepression ofSmad3-mediated gene expression requires the GR LBDand not the GR DBD or AF-1 [51]. Therefore, each specificassociation appears to require a particular subset of GRdomains and AAs.

GR can act as a monomer to repress NF-kB and AP-1,evidenced by GR dimerization-deficient mutants that stilltransrepress [47,52]. The GR:transcription factor associa-tion can be located by ChIP to NF-kB- or AP-1-binding sitesin promoter regions of inflammatory genes [20,49,53]. Inthis respect, the fact that a NLS-deficient GR mutant canstill repress NF-kB-mediated gene transcription could pos-sibly be explained via cytoplasmic crosstalk [54]. Recently,the LIM domain-containing protein thyroid receptor-inter-acting protein 6 (Trip6) was proposed to be a necessarymediator in the association between GR and AP-1(Fos) orNF-kB to allow GR-mediated transrepression, but also toallow reciprocal repression [53,55,56]. Proteins interactingwith the CTD of GR appear not to be involved in GRtransrepression because the hGR E755A C-terminal pointmutation, which abolishes the association of GR withLxxLL-containing cofactors, shows clear GR-mediatedtransrepression. Because this mutation, as expected,severely impaired GRE-regulated transcription [57], these

Exp Ref.

COS-7 (TT) [15]

COS-7 (TT) [13]

COS-7 (TT) [38]

S, GST–520C/L550,553Sa COS-7 (TT) [38]

COS-7 (TT) [39]

COS-1 (TT) [40]

In vitro [96]

COS-7 (TT) [12]

COS-7 (TT) [12]

COS-7 (TT) [11]

COS-7 (TT) [11]

299

Table 1 (Continued )

GR ligand binding

Effect Mutant Exp Ref.

Mut�wt mGR E548A, mGR S561A In vitro [96]

rGR E773T nm [41]

GR localization in the absence of agonist


C>Nb YFP–hGRa COS-1 (TT) [25]

rGRNL1- COS-7 (TT) [22]

GFP–rGRNL1- COS-7 (TT) [24]

GFP–rGRN525NL1- COS-7 (TT) [22]

GFP–SV40–rGRNL1-c COS-7 (TT) [24]

c-abl–rGRNL1-d COS-7 (TT) [23]

mGR S212A/S234A, mGR S220A/S234A, mGR A3 (S212/220/234A), mGR A4 (S150/212/220/

234A), mGR A5 (S150/212/220/234A,T159A), mGR A5+412A (S150/212/220/234/412A,T159A),

mGR A7 (S150/212/220/234/315/412A,T159A), mGR A8 (S122/150/212/220/234/315/412A,T159A)

COS-1 (TT) [73]

hGR S203/211/226A COS-1 (TT) [77]

hGR S226A COS-7 (TT) [81]

hGR S404D U2OS-hGR S404D (ST) [82]

hGR Y598F/Y663F COS-1 (TT) [58]

rGR N768, rGR N694, rGR N508, rGR N464, rGR N237.b, rGR N237.540C COS-7 (TT) [26]

Z.4C (rGR4–795), Z.4–445, Z.407C, Z.540C, N794.Z, 407–794.Z, 407–768.Z,407–740.Z, 440–493.Ze COS-7 (TT) [26]

N>C c-abl–rGRd COS-7 (TT) [23]

GFP–rGRN525 COS-7 (TT) [22]

rGR N615, rGR N508 COS-7 (TT) [26]

407–615.Z, 407–545.Z, 440–545.Z, 497–524.Ze COS-7 (TT) [26]

N�C NES–rGRf COS-1 (TT) [37]

hGR S404A U2OS-hGR S404A (ST) [82]

hGR K419A COS-1 (TT) [89]

GR localization in the presence of agonist (e.g. DEX)

Effect Mutant Exp Remarks Ref.

C>N rGR N768, rGR237.b, rGR N237.540C COS-7 (TT) [26]

N>C YFP–hGRa COS-1 (TT) [25]

c-abl–rGR d COS-7 (TT) [23]

NES–rGRf COS-1 (TT) [37]

GFP–SV40–rGRNL1-c COS-7 (TT) Accelerated Redistribution to the

cytoplasm after steroid withdrawal

[24]

rGRSV122–139g COS-7 (TT) [24]

mGR S212A/S234A, mGR S220A/

S234A, mGR A3 (S212/220/234A),

mGR A4 (S150/212/220/234A), mGR A5

(S150/212/220/234A,T159A), mGR

A5+412A (S150/212/220/234/

412A,T159A), mGR A7 (S150/212/220/

234/315/412A,T159A), mGR A8 (S122/

150/212/220/234/315/412A,T159A)

COS-1 (TT) [73]

hGR S226A COS-7 (TT) Delayed re-export after UV exposure

or DEX withdrawal

[81]




rGR L507A/E508A COS-7 (TT) [24]

hGR Y598F/Y663F COS-1 (TT) [58]

N�C rGRNL1- COS-7 (TT) Slower import rate than wt GR [22]

GFP–rGRNL1- COS-7 (TT) Accelerated redistribution to the

cytoplasm after steroid withdrawal in

comparison to wt

[24]

c-abl–rGRNL1-d COS-7 (TT) [23]

hGR S203/211/226A COS-1 (TT) [77]

GR transrepression

Effect Mutant Exp Read-out; remarks Ref.

Mut>wt hGR D589–697 CV-1 (TT) Rec NF-kB reporter (TT) [54]

hGR S404A U2OS-hGR S404A (ST) Rec NF-kB reporter (TT) [82]

hGR S404A U2OS-hGR S404A (ST) IL1b (End) [82]


300


GR transrepression


hGR D4X (N454D/A458T/R460D/D462C),

hGR N454D/A458T

CV-1 (TT) Rec TRE reporter (TT) [52]

hGR K494A/K495A, hGR K494N/K495N A549 (TT) GM-CSF (End) [91]

Mut<wt rGR N525 CV-1 (TT) Rec NF-kB reporter [49]

hGR D77-262/D532-647, hGR D450-487 HEK-293T (TT) Rec NF-kB reporter (TT) [20]

hGR C421G, hGR D420-451, hGR C457G,

hGR D450-487, hGR D428-490

HeLa (TT) IL6 reporter (TT) [50]

hGR GtttG, hGR GgttG, hGR GggtG h COS-1 (TT) Rec NF-kB reporter (TT) [48]

hGR DGM, hGR MGM, hGR MGG, hGR

GMM, hGR MMGi


hGR S211A, hGR S226A COS-1 (TT) Rec NF-kB reporter, rec AP-1 reporter

(TT); ligand-dependent effects

[97]

hGR S211A U2OS-hGR S211A (ST) cMyc, ephrin, Fra, JunB, Smad7 (End) [76]

hGR S226A U2OS-hGR S226A (ST) Ephrin, Smad7, cMyc, JunB (End) [76]

hGR S404D U2OS-hGR S404D (ST) Rec NF-kB reporter (TT) [82]

hGR N454D/A458T, hGR C476W/R479Q MM1.R (TT) Rec NF-kB reporter (TT) [63]

hGR S425G, hGR L436V, hGR S425G/

L436V, hGR C476W/R479Q, hGR

R477S, hGR R479G hGR Y478L/R479G


hGR S425G COS-7 (TT) Rec NF-kB reporter (TT) [61]

hGR S425G MM1.R (TT) Rec NF-kB reporter (TT) [63]

hGR D4X (N454D/A458T/R460D/D462C),

hGRmH12DAF1(E755Q/D77–262)

COS-7 (TT) Urokinase-type plasminogen activator

(uPA) reporter (TT)

[55]

hGRmDBD(C476W/R479Q) COS-7 (TT) Collagenase reporter (TT) [53]

rGR K461A CV-1 (TT) Rec NF-kB reporter (TT); stimulation [49]

rGR K461A COS-7 (TT) Rec AP-1 reporter (TT) and rec

proliferin reporter (TT); stimulation

[62]

rGR K461A F9 (TT) Coll reporter (TT); stimulation [98]

rGR K461T, rGR K461Q F9 (TT) Coll reporter (TT); stimulation [98]

rGR R488Q CV-1 (TT), HEK 293 (ST) Rec NF-kB, ICAM1 and COX2 reporters

(TT); COX2, JunB, IL6R (End)

[60]

rGR R488Q COS-1 (TT) Rec NF-kB reporter (TT) [48]

rGR K490E COS-1 (TT) Rec NF-kB reporter (TT) [48]

rGR E558A, rGR L584D, rGR L584D/

Q588K, rGR R629Y

U2OS (TT) Coll3 (End) [12]

mGR S561A, hGR D488-532, hGR

D532–697, hGR 1–726

Hep3B (TT) Rec TRS reporter (TT) [51]

hGR P625A CV-1 (TT) MCP1 reporter (TT) [17]

Mut�wt hGR D77–262 HEK-293T (TT) Rec NF-kB reporter (TT) [20]

hGR GMG i CV-1 (TT) Rec TRE reporter (TT) [52]

hGR GgtgG, hGR GttgGh COS-1 (TT) Rec NF-kB reporter (TT) [48]

rGR T171A U2OS (TT) Rec AP-1 reporter (TT) [83]

hGR S211A U2OS-hGR S211A (ST) c-Jun (End) [76]

hGR S226A U2OS-hGR S226A (ST) Fra, c-Jun (End) [76]

hGR S404D U2OS-hGR S404A (ST) Bcl3 (End) [82]

rGR 407C CV-1 (TT) Rec NF-kB reporter [49]

rGR R488Q CV-1 (TT), HEK 293 (ST) Rec AP-1 and MMP1 reporter (TT),

MMP1, ActivinA, GADD45B (End)

[60]

rGR N491A COS-1 (TT) Rec NF-kB reporter (TT) [48]

rGR LS7 (P493R/A494S) HEK 293 (TT) Rec NF-kB reporter (TT), MMP1

reporter (TT)

[60]

rGR LS7 (P493R/A494S) COS-1 (TT) Rec NF-kB reporter (TT) [48]

rGR LS7 (P493R/A494S) Keratinocyte cell line PB (TT) Rec NF-kB reporter(TT) [65]

rGR LS7 (P493R/A494S) In vivo (skin,K5-GR-TR in

C57Bl/6J x DBA/2J mice)

IL1b, MMP-3 (End) [65]

rGR R576N, rGR R576D COS-7 (TT) Urokinase-type plasminogen activator

(uPA) reporter (TT)

[55]

hGR C442G HeLa (TT) IL6 reporter (TT) [50]

hGR D4X (N454D/A458T/R460D/

D462C)

COS-1 (TT) Rec NF-kB reporter (TT) [48]

hGR S425G, hGR L436V, hGR N454D/

A458T

COS-7 (TT) Rec AP-1 reporter (TT) [61]


301


GR transrepression


hGR L436V, hGR N454D/A458T COS-7 (TT) Rec AP-1 reporter (TT) [61]

hGR L436V MM1.R (TT) Rec AP-1 reporter (TT) [63]

hGR dim A458T CV-1 (TT) Rec TRE reporter (TT) [52]

mGR dim A465T Peritoneal macrophages

(GRdim mice)

IL6, COX2, TNF, IL1b, IFNgamma,IL2,

(End)

[47]

mGR dim A465T MEFs from GRdim mice Coll3, gelatinase B (End) [46]

mGR dim A465T Primary osteoblasts from

GRdim mice

IL11, LIF, NGF2, IL6 (End) [64]

rGR K461A HOS D4 (TT), COS-7 (TT) Osteocalcin reporter (TT) [62]

hGR F602S, hGR I628A CV-1 (TT) MCP1 reporter (TT) [17]

hGR E755A HeLa (TT), CV-1 (TT), HT-29 (TT) Gal4-p65/Gal4-reporter (TT) [57]

hGRmH12(E755Q), hGRmH12DAF1(E755Q/D77–262) COS-7 (TT) Collagenase reporter (TT) [53]

GR transactivation


Mut>wt rGR T171A U2OS (TT) Rec GRE (TT) [83]

hGR S203A, hGR S211A, hGR S226A,

hGR S203A/S211A, hGR S203A/S211A/

S226A, hGR S45A/S203A/S211A/S226A

HCT116 (TT) MMTV (TT) [72]

hGR S226A COS-1 (TT) Rec TAT–GRE (TT) [97]

hGR S226A U2OS-hGR S226A (ST) MMTV (TT), IGFBP1, IRF8, GILZ, LAD1

(End)

[76]

rGR S246A, rGR DM (K297R/K313R),

rGR TM (S246A/K297R/K313R)

COS-7 (TT) Rec TAT3 GRE (TT) [87]

hGR K277R, hGR K293R, hGR K277R/

K293R), hGR (K277R/K293R/K703R)

HeLa (TT) Rec GRE (TT) [86]

hGR S404D U2OS-hGR S404D (ST) Rec GRE (TT) [82]

hGR S404A U2OS-hGR S404A (ST) Kip2 (End) [82]

mGR V437G COS-1 (TT), E8.2 (TT) MMTV (TT) [99]

rGR G449V, hGR G430V COS-1 (TT) TAT2 reporter (TT) [99]

rGR D481R, rGR R479D, rGR K297R/

K313R

CV-1 (TT) Rec composite TAT–GRE (TT) [85]

rGRdim A477T, rGR R479D, rGR D481R CV-1 (TT) PNMT reporter (TT) [100]

hGR N454D/A458T MM1.R (TT) Rec GRE (TT) [63]

hGR L436V MM1.R (TT) Rec GRE (TT) [63]

hGR S425G, hGR L436V, hGR S425G/

L436V, hGR R477S

CV-1 (TT) MMTV (TT) [52]

rGR K461A F9 (TT) Rec composite GRE (TT) [98]

mGR K426A COS-1 (TT) Rec GRE (TT) [88]

hGR K419A COS-1 (TT) Rec GRE, MMTV (TT) [89]

rGR L550S, rGR L553S CV-1 (TT) Rec GRE (TT) [38]

hGR M604L COS-7 (TT) MMTV (TT) [15]

rGR C656G H4IIE (TT) PEPCK reporter (TT) [43]

rGR M622T, rGR M622L F9 (TT) Rec TAT–GRE (TT) [101]

Mut<wt hGR I696, hGR I626, hGR I599, hGR

I582, hGR I559, hGR I550, hGR I532,

hGR I532, hGR I500j


rGR C440R, rGR S444P, rGR C460Y

rGR F463Y, rGR R466K, rGR C482Y,

rGR N491S, rGR C492R


rGR 407C, rGR 407–525, rGR N525,

rGR D107–318, rGR EX525

F9 (TT) TAT–GRE reporter (TT) [98]

hGR D428–490, hGR D420–451, hGR

D450–487, hGR D77–262, hGR D9–385

COS-1 (TT) Rec GRE (TT) [59]

hGR D491–516; hGR D488–533, hGR

D491–551, hGR D515–551, hGR

D515–583, hGR D515–627, hGR

D532–590, hGR D532–697, hGR

D550–600, hGR D550–627, hGR

D589–697k


rGRNL1- COS-7 (TT) MMTV (TT) [22]

mGR A5 (S150/212/220/234A,T159A) COS-1 (TT) MMTV (TT) [73]


302


GR transactivation


mGR S212A, mGR S220A, mGR S212A/

S234A, mGR S220A/S234A, mGR A3

(S212/220/234A), mGR A4 (S150/212/

220/234A), mGR A5 (S150/212/220/234A,

T159A), mGR A5+412A (S150/212/220/234/

412A,T159A), mGR A7 (S150/212/220/234/

315/412A,T159A), mGR A8 (S122/150/212/

220/234/315/412A,T159A)


hGR 500 S211A CV-1 (TT) Rec GRE (TT) [75]

hGR S211A COS-1 (TT) Rec TAT–GRE (TT) [97]

hGR S211A U2OS-hGR S211A (ST) IGFBP1, IRF8 (End) [76]

hGR S203/211/226A COS-1 (TT) MMTV, TAT1 reporter (TT); GILZ (End) [77]

hGR S404A U2OS-hGR S404A (ST) STAT4, IL4 (End) [82]

rGR D481R, rGR R479D, rGR K297R/K313R CV-1 (TT) Rec simple TAT-GRE (TT) [85]

hGR K277R/K293R), hGR (K277R/K293R/K703R) HeLa (TT) MMTV (TT) [86]

hGR K494A/K495A, hGR K494N/K495N,

hGR K494Q/K495Q,

A549 (TT) SLPI (End) [91]

mGR V437G COS-1 (TT), E8.2 (TT) TAT2 reporter (TT) [99]

rGR G449V, hGR G430V COS-1 (TT) MMTV reporter (TT) [99]

hGR dim A458T CV-1 (TT) Rec GRE, MMTV reporter (TT) [52]

mGR dim A465T MEFs from GRdim mice MMTV, rec GRE (TT); TAT (End) [46]

mGR dim A465T Primary osteoblasts from

GRdim mice

GILZ, Per1, KLF15 (End) [64]

rGRdim A477T, rGR R479D, rGR D481R CV-1 (TT) Rec TAT–GRE (TT) [100]

rGR E219K/F220L/W234R (AF1mut), rGR E773

(AF2mut), rGR dim A477T

U2OS-rGRmut (ST) Target -selective effects (End) [41]

hGR C476W/R479Q MM1.R (TT) Rec GRE (TT) [63]

rGR RK461A, rGR V462A, rGR R466A F9 (TT) Rec simple GRE (TT) [98]

hGR D4X (N454D/A458T/R460D/D462C),

rGR K490E, rGR N491A, rGR LS7 (P493R/A494S)


rGR LS7 (P493R/A494S) Keratinocyte cell line PB (TT) MMTV (TT) [65]

rGR R488Q HEK 293 (ST) Rec GRE (TT) [60]

rGR R488Q COS-1 (TT) Rec GRE (TT) [48]

hGR C421G, hGR D420–451 HeLa (TT) MMTV (TT) [50]

hGR C476W/R479Q, hGR R479G CV-1 (TT) MMTV (TT) [52]

hGR C476W/R479Q MM1.R (TT) Rec GRE (TT) [63]

hGR K579A, hGR E755A Huh-7 (TT) GR–VP16 fusion–Gal4 reporter (TT),

rec GRE reporter (TT)

[57]

rGR E558A, rGR L584D, rGR L584D/Q588K CV-1 (TT), U2OS (TT) Rec GRE (TT), MMTV (TT), LAD1 (End) [12]

rGR P548A/T549A/V551A CV-1 (TT) Rec GRE (TT) [39]

rGR L550S/L553S CV-1 (TT) Rec GRE (TT) [38]

hGR 532 LxxAA, hGR 718 LxxAA COS-1 (TT) Rec GRE (TT) [40]

mGR L550A, mGR D560A, mGR S561A CV-1 (TT) MMTV (TT) [96]

hGR Y598F/Y663F COS-1 (TT) MMTV (TT) [58]

hGR P625A, hGR I628A CV-1 (TT) MMTV (TT) [17]

hGR LBD E755A, hGR LBD E755R,

hGR LBD R585A/D590A

CV-1 (TT) Fusion Gal4(DBD)–GR(LBD) with Gal 4

reporter (TT)

[17]

rGR Y616N F9 (TT) Rec TAT-GRE (TT) [101]

rGR C656G/E773A H4IIE (TT) PEPCK reporter (TT) [43]

rGR R629Y CV-1 (TT), U2OS (TT) Rec GRE, MMTV (TT); LAD1 (End);

ligand-selective

[12]

mGR C644G 1471.1(TT) MMTV (ST) [13]

mGR M785A/L759A, mGR E761A,

mGR I762A/I763A

NIH3T3 (TT) MMTV (TT) [42]

Gal4–mGR M785A/L759A, Gal4–mGR E761A,

Gal4–mGR I762A/I763A

COS-1 (TT) Gal4 reporter (TT) [42]

hGRmH12(E755Q), hGRmH12DAF1(E755Q/

D77–262), hGRmDBD(C476W/R479Q)

COS-7 (TT) MMTV (TT) [53]

Mut�wt NES-rGRf COS-1 (TT) MMTV (TT) [37]

hGR S45A, hGR S395A HCT116 (TT) CV-1 (TT) [72]

hGR S211A U2OS-hGR S211A (ST) GILZ, LAD1 (End) [76]


303


GR transactivation


hGR S203/211/226A COS-1 (TT) Rec GRE (TT) [77]

mGR S212A, mGR S220A, mGR

S234A, mGR S212A/S234A,

mGR S220A/S234A, mGR A3

(S212/220/234A), mGR A4 (S150/

212/220/234A), mGR A5+412A

(S150/212/220/234/412A,T159A),

mGR A7 (S150/212/220/234/315/

412A,T159A), mGR A8 (S122/150/

212/220/234/315/412A,T159A)

COS-1 (TT) MMTV (TT) [73]

mGR S234A COS-1 (TT) Rec GRE (TT) [73]

hGR S226A COS-7 (TT) Rec GRE (TT) [81]

hGR S404A U2OS-hGR S404A (ST) Rec GRE (TT), FOXO4 (End) [82]

rGR D481R/R479D CV-1 (TT) Rec simple and composite TAT–GRE

(TT)

[85]

rGR T547A, rGR P548A, rGR V551A,

rGR S552A, rGR T547A/T549A/S552A

CV-1 (TT) Rec GRE (TT) [39]

hGR S425G MM1.R (TT) Rec GRE (TT) [63]

rGR V462A, rGR R466A F9 (TT) Rec composite GRE (TT) [98]

hGR C442G HeLa (TT) MMTV (TT) [50]

hGR Y478L/R479G CV-1 (TT) MMTV (TT) [52]

rGR G453E, rGR R489K CV-1 (TT) MMTV (TT) [45]

rGR M770I F9 (TT) Rec TAT–GRE (TT) [101]

hGR F602S CV-1 (TT) CV-1 (TT) [17]

hGR K703R HeLa (TT) Rec GRE (TT) [86]

GR DNA binding


Mut<wt rGRN556 C440R, rGRN556 S444P,

rGRN556 C457Y, rGRN556 C460Y,

rGRN556 F436S, rGRN556 R466K,

rGRN556 C482R, rGRN556 C482Y,

rGRN556 R489K, rGRN556C492R,

rGRN556 C495Y, rGRN556 C500R,

rGRN556 L501P, rGRN556 M505I,

rGRN556 M505Tl

In vitro EMSA, GRE binding [45]

hGR C421G, hGR D420–451 HeLa (TT) EMSA, MMTV binding [50]

hGR440–525 GSA (V462A), hGR440-

525 EGV (G458Q/S459G), hGR440–525

GSA (G458Q/S459G/V462A)m

In vitro EMSA, GRE binding [44]

hGR S203/211/226A COS-1 (TT) ChIP, MMTV (TT) binding [77]

hGRdim A458T COS-7 (TT) EMSA, GRE binding [52]

mGRdim A465T Liver nuclear extracts

from GRdim mice

EMSA, GRE binding [46]

Mut�wt rGRN556 R488Q, rGRN556 N491Sl In vitro EMSA, GRE binding [45]

hGR S203/211/226A COS-1 (TT) ChIP, GILZ (End) binding [77]

rGR K461A COS-7 (TT) EMSA, GRE binding [62]

rGR dim A477T, GR D481R In vitro EMSA, PNMT binding [100]

GR interactions

Effect Mutant Exp Interacting partner Ref.

Mut>wt hGR S203/211A Yeast TSG101 [84]

hGR S211D Yeast MED14 [76]

hGR S404A U2OS-hGR S404A (ST) NF-kB p65 [82]

mGR S561A Hep3B (TT) SRC2 [51]

rGR F620S, rGR M770 In vitro (GST) SRC2 [101]

Mut<wt mGR delta574–632 mGR D575–605 COS-7 (TT) Hsp90 [103]

rGR P548A/T549A/V551A COS-7 (TT) Hsp90 [39]

hGR 30IIB (E198A/F220A/W213A) Yeast TSG101 [84]

hGR 30IIB (E198A/F220A/W213A) Yeast MED14 [76]

hGR S211A Yeast MED14 [76]

hGR S203/211/226A COS-1 (TT) SRC2 [77]

hGR AF1 S211A In vitro (HeLa extracts) TBP, CBP, SRC1 [79]


304


GR interactions

Effect Mutant Exp Interacting partner Ref.

hGR 500 S211A CV-1 (TT) TBP, CBP, SRC1 [79]

rGR S246A COS-7 (TT) SUMO 2/3 [87]

hGR S404A U2OS-hGR S404A (ST) p300 [82]

hGR D532–697, hGR 1–726 Hep3B (TT) SRC2 [51]

rGR C656G Yeast SRC1 [43]

hGR Y598F/Y663F COS-1 (TT) PI3K (p85a) [58]

hGR R485A/D590A Huh-7 (TT) SRC1, SRC2, SRC3 [57]

hGR E755A Huh-7 (TT) SRC1, SRC2, SRC3, MED1, PGC1a,

PGC1b, RIP140, ASC2

[57]

Mut�wt GST–520C/L550S, GST–520C/L553S,

GST–520C/L550,553S (a)

COS-7 (TT) Hsp90 [38]

hGR 532 LxxAA, hGR 718 LxxAA COS-1 (TT) Hsp90 [40]

mGR D603–633 COS-7 (TT) Hsp90 [103]

rGR 407C, rGR N577(D108–317) In vitro (GST) NF-kB p65 [49]

rGR R488Q NF-kB p65 [60]

hGR D488-532 Hep3B (TT) SRC2 [51]

rGR Y616N, rGR M622T In vitro (GST) SRC2 [101]

hGR S404A U2OS-hGR S404A (ST) SRC2 [82]

mGR S561A, hGR D488–532, hGR

D532–697, hGR 1–726

Hep3B (TT) SRC1 [51]

hGR K494A/K495A, hGR K494N/K495N A549 (TT) HDAC2 [91]

GR downregulation


Mut>wt NES–rGRh COS-1 (TT) [37]


Mut<wt mGR A3 (S212/220/234A), mGR A4 (S150/212/220/234A), mGR A5 (S150/212/220/234A,T159A),

mGR A5+412A (S150/212/220/234/412A,T159A), mGR A7 (S150/212/220/234/315/412A,T159A),

mGR A8 (S122/150/212/220/234/315/412A,T159A)

COS-1 (TT) [73]


mGR K426A COS-1 (TT) [88]


hGR 718 LxxAA COS-1 (TT) [40]

Mut�wt hGR S211A, hGR S226A COS-1 (TT) [97]

mGR S212A, mGR S220A, mGR S234A, mGR S212A/S234A, mGR S220A/S234A COS-1 (TT) [73]

hGR S203/211/226A COS-1 (TT) [77]

GR post-translational modulation

Modulation Mutant Exp Effect Ref.

Phoshorylation (ph) hGR S03A U2OS-hGR S203A (ST) Increase S226 ph [67]

hGR S211A U2OS-hGR S211A (ST) Increase S226 ph [67]

hGR S226A U2OS-hGR S226A (ST) Increase hGR S203 and 211 ph [67]

Sumoylation rGRS246A In vitro Decrease GR SUMOylation [87]

End, endogenous; exp, experimental system; h, human; m, mouse; mut, mutant; nm, not mentioned; r, rat; rec, recombinant; ST, stably transfected; TT, transiently

transfected; wt, wild-type.

aFusion of GST to the N-terminus of truncated rGR.

bC, cytoplasm; N, nucleus.

cAdded SV40 NLS.

dc-Abl DBD does not carry a NLS function.

erGR chimeras fused to LacZ (b-galactosidase–receptor fusion) whose localization is unaffected by DEX.

fFusion of the IkB NES to the N-terminus of rGR.

gGR NL1/NRS (506–523) was replaced by SV40NLS.

hC-terminally truncated rGR with point mutations.

iChimera between hGR (G) and hMR (M): N-terminal domain, DBD, LBD.

jhGR truncation mutations.

khGR internal deletion mutations in the hinge and LBD.

lC-terminally truncated rGR with point mutations.

mER-specific substitutions in GR DBD.


305


results suggest that cofactors regulating GR transactiva-tion and GR transrepression processes might require dis-tinct GR interaction platforms.

Activated GR also inhibits gene transcription by dis-rupting different kinase signaling cascades targetingdownstream transcription factors such as NF-kB or AP-1[5]. Interestingly, GCs and inflammatory stimuli can cost-imulate expression of the pattern-recognition receptorTLR2, and GR plays a role in proinflammatory cytokine-inducing TLR2 signaling. Recent progress mapped theinteraction of GR with the TLR2-activated PI3K to twoconsensus YxxM binding motifs (Y598 and Y663) in thehGR LBD and indicated that this PI3K:GR interaction isnecessary in the regulation of TLR2-stimulated TNFa

production [58]. Currently, the role of this PI3K:GR inter-action needs to be addressed further in vivo. Typically,transcription factors targeted by GR display reciprocalrepression of GRE-mediated gene transcription, a processinvolving multiple GR domains. For instance, stimulationor overexpression of NF-kB p65 also affects GC-inducedGRE-regulated promoter activity [6,48,59].

In contrast to GRdim, which mediates GR transrepres-sion mechanisms but not classic GRE-mediated transacti-vation [46,52], other dissociative GR mutants selectivelymediate particular GR actions. For instance, the doubleDBD point mutant rGR P493R/A494S, commonly knownas GR LS7, does not induce GR transactivation, but doesrepress NF-kB- and AP-1-mediated gene transcription[48,60]. At the level of transrepression, the rGR R488Qmutant selectively represses AP-1-mediated gene tran-scription whereas NF-kB-regulated promoters are unaf-fected [60]. Conversely, the hGR S425G mutation retainsits transactivation capabilities and, similar to rGR R488Q,inhibits AP-1-mediated gene expression but not NF-kB-regulated transcription [61]. Alternatively, rGR K461 pos-sibly distinguishes between GR-repressing mechanismsbased on either tethering or GR DNA binding [62]. BecauseGR DBD point mutants hardly affect the overall secondarystructure of the GR DBD, it is likely that the selectivetransrepression characteristics of such GR mutants origi-nate from specific AA alterations in the functional interac-tion surface or by minute but decisive local conformationchanges, thus modulating distinct protein–protein inter-actions [61]. The mechanistic basis of these dissociativemutations requires additional research. Ultimately, thesespecific mutants will allow us to discern the role of GRtransactivation and transrepression actions for specificGR-regulated processes not only in vitro [63], but mostimportantly in vivo [64–66]. Recent examples include theexploration of the GC-mediated mechanisms in vivo inkeratinocyte proliferation [65], skin barrier competence[66] and suppression of bone formation [64].

GR mutants and post-translational modulation of GR

GR phosphorylation Phosphorylation of GR is ameticulously regulated mechanism becausephosphorylation of some sites can prevent and/or favorphosphorylation at specific other sites [67]. Althoughsome sites can be phosphorylated in unactivated GR, GRbecomes hyperphosphorylated upon ligand binding.Phosphorylation of GR takes place in the variable

306

N-terminal domain of GR. For hGR, residues S113,S141, S203, S211, S226 and S404 have been confirmedto be phosphorylation sites [5,68] (Figure 1a). These areconserved in mGR as S122, S150, S212, S220, S234 andS412. Additional identified mGR phosphorylation sites areS315 and T159 [5,68]. Recently, mass spectrometrysuggested additional phosphorylation at hGR T8, S45,S134, S234 and S267 [69]. However, mutational studiesto confirm and analyze the possible role of these recentlydiscovered phosphorylations are lacking.

Diverse kinases play a role in GR phosphorylation.Cdk2/CyclinA kinase complexes and Cdk5 are involvedin phosphorylation of hGR S203 and S211, whereasCdk2/CyclinE affects hGR S203 phosphorylation [70–72].Phosphorylation of these residues increases GR transacti-vation [70,73–77], albeit in a promoter-selective manner[76,77]. However, the nature of the tissue also plays a role,for example central nervous system (CNS)-specific Cdk5-mediated GR phosphorylation is not linked to an increasebut to a decrease in transcription activation by GR [72].Alternatively, hGR S211 was suggested to be phosphory-lated by p38 MAPK [75,78,79]. The c-Jun N-terminalkinase (JNK) phosphorylates rGR S246 (conserved ashGR S226) and inhibits GR transactivation mechanisms[80,81] and is thought to be involved in GR nuclear export[81]. Lastly, glycogen synthase kinase 3b (GSK3b) phos-phorylates hGR S404, and this enhances GR nuclear ex-port and downregulation, and diminishes GRtransactivation and transrepression [82]. Also, GSK3 phos-phorylates rGR T171. Although this rGR T171 phosphory-lation site is conserved in mGR at T159, hGR harbors analanine residue at the corresponding hGR A150. However,when this corresponding hGR A150 is mutated to a Tresidue, the GSK3-regulated decrease in GR transactiva-tion, as observed in rGR experiments, can be reinstatedalso for hGR [83]. This case warns us that not all GRphosphorylation sites are conserved across species. Tofurther complicate the system, GR phosphorylation iscoregulated by phosphatases PP1, PP2A and PP5 [5,67].The Hsp90-binding PP5 has been shown to be implicated inhGR S203, S211 and S226 dephosphorylation [67].

The effects of phosphorylation are crucially dependenton the phosphorylation site. Gene-specific receptor recruit-ment can be matched to the phosphorylation profile of GRand the concomitant GR interaction profile [73,74,76,82].Recent mutational analyses of hGR S211 phosphorylationsuggested that this site-specific phosphorylation stronglycontributes to secondary and tertiary structural GR foldingof the GR AF1 and its association with cofactors such asSRC1, CBP, TBP and MED14 [76,79]. Other studies basedon mutations of hGR S203, S211 and S226 have demon-strated that phosphorylation of these residues plays a rolein the GR:SRC2 interaction and SRC2 occupancy of GRE-regulated promoters, and also affect GR recruitment in apromoter-selective manner [77]. Lastly, unphosphorylatedhGR S203A/S211A shows an enhanced association withTSG101, an interaction which stabilizes the hypopho-sphorylated, unliganded GR [84]. Notably, the link be-tween hGR S211 phosphorylation and enhanced GRtransactivation is dependent on the promoter and theamount of activated receptor [76]. Furthermore, mutations


of hGR S404 suggest that phosphorylation of this residueaffects GR association with NF-kB p65 and p300 [82].Overall, phosphorylation of GR plays a role in the regula-tion of GR ligand binding, conformation, localization, half-life, DNA and protein binding, and receptor activity [5].However, the field still lacks in-depth assessments ofGR phosphorylation-defective and -mimicking mutantsin vivo.

Other GR post-translational modifications

Other post-translational modifications of GR have beenidentified and confirmed using GR mutants. SUMOylationof hGR K703, and particularly K293 and K277, has beenimplicated in the control of GR transactivation synergismon multiple GREs [85,86]. These post-translational mod-ifications can additionally be controlled by GR phosphory-lation because SUMOylation of the N-terminal residues inrGR can be enhanced by JNK-mediated phosphorylation ofrGR S246 [87].

Furthermore, ubiquitination at mGR K426 appears toplay a role in ligand-dependent downregulation of GRbecause mutated mGR K426A displays reduced GR down-modulation and enhanced GR transactivation [88]. Thesefindings were recently confirmed via the human counter-part hGR K419A [89]. The hGR K419A shows similarsubcellular trafficking to wild-type GR [89]. Pharmacolog-ical inhibition of proteasomal degradation (MG132) se-verely impedes GR intranuclear mobility [90], althoughthe cause for this diminished mobility remains unclear.Just as SUMOylation can be regulated by phosphoryla-tion, ubiquitination of GR was also found to be a phos-phorylation-directed post-translational modulationbecause a GR mutant of which all known phospho-acceptorsites are mutated to A, resists ligand-induced degradation[73].

Lastly, HDAC2-mediated deacetylation of the hGRK494 and K495 acetylation sites in the hinge region ofGR is necessary for GR:NF-kB p65 interaction and ulti-mately GR transrepression of GM-CSF gene expression[91]. Nevertheless, and in seeming contradiction, mutationof hGR K494 and/or K495 does not appear to affect GR:NF-kB p65 association [91]. Further, it was suggested that,unlike wild-type hGR, overexpression of hGR K494/495Aand hGR K494/495N did not enhance the induction of theGRE-regulated SLPI gene expression levels, and experi-ments with hGR K494/495A mutants also indicate thatnon-acetylated GR can contribute to GR-mediated repres-sion of GM-CSF gene expression [91]. However, becausethese overexpression experiments were performed in A549cells containing wild-type GR, confirmation is still war-ranted. Although the experiments described in A549 cellssuggest that high GR acetylation levels contribute to GRtransactivation and that deacetylation of GR could contrib-ute to GR transrepression [91], overexpression and knock-down experiments focusing on the acetyl-transferaseheterodimer CLOCK:BMAL1 in HCT116, HepG2 and Helacells suggested that elevated acetylation actually countersthe GR transactivation potential, assumedly via decreasedGR:GRE binding, and assists in GR transrepression of NF-kB-regulated gene expression via an unknown mechanism[92]. In support, mutation of hGR acetylation sites

abolished the transactivation-modulating effect ofCLOCK:BMAL1 overexpression. However, mutations ofonly K494 and K495 were insufficient to achieve this effect,and additional mutations of acetylation target residuesK480 and K492 were required [92]. Confirmation of theseacetylation sites via mass spectrometry and/or acetylation-specific antibodies is still warranted. Notably, deletionmapping suggests that hGR interacts with CLOCK, ofwhich the AA sequence and structure highly resembleSRC-3, via the hGR hinge/LBD and irrespective of GRacetylation [92]. Currently, the apparent cell type-depen-dency of the effects of these mutations on general GRtransactivation or transrepression mechanisms is notcompletely clear and requires additional research. Becausemutation of the corresponding hinge region acetylationsites in the progesterone receptor, androgen receptorand/or ERa, most probably via p300, can affect theirphosphorylation, nuclear translocation dynamics, transac-tivation magnitude and kinetics [93–95], it could proveinteresting to perform a similar in-depth analysis of therange of acetylation-mediated effects on GR functionality.

ConclusionsIn addition to the cell- or tissue-specific knock-out of GR,the introduction of mutated GR proteins provides an in-teresting and validated tool for exploring GR structure–

function relationships, GR protein-interacting domainsand the mechanisms underlying GR-mediated cellulareffects. Future studies on new or alternatively modulatedGR post-translational modifications via mass spectrome-try, identifying new modulated peptide sequences or AAs,could launch a new wave of research into novel GRmechanisms; the generation of corresponding GR mutantswould provide an interesting strategy for obtaining com-plementary data. However, the validity and impact of GRmutant research would be strengthened by confirmation invivo. In conclusion, the plethora of newly generated GRmutants, which are in general freely distributed among theGR community, continues to fuel current research.

AcknowledgmentsThis work was financially supported by Interuniversity Attraction Poles(IAP) 6/18. I.M.B. and K.D.B. are postdoctoral fellows of the ResearchFoundation – Flanders (FWO-Vlaanderen).

References1 Schacke, H. et al. (2004) Dissociation of transactivation from

transrepression by a selective glucocorticoid receptor agonist leadsto separation of therapeutic effects from side effects. Proc. Natl. Acad.Sci. U.S.A. 101, 227–232

2 McDonough, A.K. et al. (2008) The epidemiology of glucocorticoid-associated adverse events. Curr. Opin. Rheumatol. 20, 131–137

3 Hollenberg, S.M. et al. (1985) Primary structure and expression of afunctional human glucocorticoid receptor cDNA. Nature 318, 635–641

4 Nicolaides, N.C. et al. (2010) The human glucocorticoid receptor:molecular basis of biologic function. Steroids 75, 1–12

5 Beck, I.M. et al. (2009) Crosstalk in inflammation: the interplay ofglucocorticoid receptor-based mechanisms and kinases andphosphatases. Endocr. Rev. 30, 830–882

6 Kassel, O. and Herrlich, P. (2007) Crosstalk between theglucocorticoid receptor and other transcription factors: molecularaspects. Mol. Cell. Endocrinol. 275, 13–29

7 Rosenfeld, M.G. et al. (2006) Sensors and signals: a coactivator/corepressor/epigenetic code for integrating signal-dependentprograms of transcriptional response. Genes Dev. 20, 1405–1428

307


8 Gross, K.L. and Cidlowski, J.A. (2008) Tissue-specific glucocorticoidaction: a family affair. Trends Endocrinol. Metab. 19, 331–339

9 Danielsen, M. et al. (1989) Mutational analysis of the mouseglucocorticoid receptor. Cancer Res. 49, 2286s–2291s

10 Yudt, M.R. and Cidlowski, J.A. (2001) Molecular identification andcharacterization of a and b forms of the glucocorticoid receptor. Mol.Endocrinol. 15, 1093–1103

11 Garabedian, M.J. and Yamamoto, K.R. (1992) Genetic dissection ofthe signaling domain of a mammalian steroid receptor in yeast. Mol.Biol. Cell. 3, 1245–1257

12 Tao, Y.G. et al. (2008) Mutations of glucocorticoid receptordifferentially affect AF2 domain activity in a steroid-selectivemanner to alter the potency and efficacy of gene induction andrepression. Biochemistry 47, 7648–7662

13 Sheldon, L.A. et al. (1999) A ligand binding domain mutation in themouse glucocorticoid receptor functionally links chromatinremodeling and transcription initiation. Mol. Cell. Biol. 19, 8146–8157

14 Zhang, S. et al. (1996) Role of the C terminus of the glucocorticoidreceptor in hormone binding and agonist/antagonist discrimination.Mol. Endocrinol. 10, 24–34

15 Zhang, J. et al. (2005) A critical role of helix 3–helix 5 interaction insteroid hormone receptor function. Proc. Natl. Acad. Sci. U.S.A. 102,2707–2712

16 Zhang, J. et al. (2009) Characterization of a novel gain of functionglucocorticoid receptor knock-in mouse. J. Biol. Chem. 284, 6249–6259

17 Bledsoe, R.K. et al. (2002) Crystal structure of the glucocorticoidreceptor ligand binding domain reveals a novel mode of receptordimerization and coactivator recognition. Cell 110, 93–105

18 Matthews, L. et al. (2009) Thiazolidinediones are partial agonists forthe glucocorticoid receptor. Endocrinology 150, 75–86

19 De Bosscher, K. et al. (2010) Classic glucocorticoids versus non-steroidal glucocorticoid receptor modulators: survival of the fittestregulator of the immune system? Brain Behav. Immun. 24, 1035–

104220 De Bosscher, K. et al. (2005) A fully dissociated compound of plant

origin for inflammatory gene repression. Proc. Natl. Acad. Sci. U.S.A.102, 15827–15832

21 Maru, B.S. et al. (2009) Potential use of an estrogen-glucocorticoidreceptor chimera as a drug screen for tissue selective estrogenicactivity. Bone 44, 102–112

22 Savory, J.G. et al. (1999) Discrimination between NL1- and NL2-mediated nuclear localization of the glucocorticoid receptor. Mol. Cell.Biol. 19, 1025–1037

23 Hache, R.J. et al. (1999) Nucleocytoplasmic trafficking of steroid-freeglucocorticoid receptor. J. Biol. Chem. 274, 1432–1439

24 Carrigan, A. et al. (2007) An active nuclear retention signal in theglucocorticoid receptor functions as a strong inducer of transcriptionalactivation. J. Biol. Chem. 282, 10963–10971

25 Schaaf, M.J. et al. (2005) Ligand-selective targeting of theglucocorticoid receptor to nuclear subdomains is associated withdecreased receptor mobility. Mol. Endocrinol. 19, 1501–1515

26 Picard, D. and Yamamoto, K.R. (1987) Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBOJ. 6, 3333–3340

27 Freedman, N.D. and Yamamoto, K.R. (2004) Importin 7 and importinalpha/importin beta are nuclear import receptors for theglucocorticoid receptor. Mol. Biol. Cell. 15, 2276–2286

28 Tao, T. et al. (2006) Importin 13 regulates nuclear import of theglucocorticoid receptor in airway epithelial cells. Am. J. Respir.Cell Mol. Biol. 35, 668–680

29 Echeverria, P.C. et al. (2009) Nuclear import of the glucocorticoidreceptor-hsp90 complex through the nuclear pore complex is mediatedby its interaction with Nup62 and importin beta. Mol. Cell. Biol. 29,4788–4797

30 Beck, I.M. et al. (2008) Altered subcellular distribution of MSK1induced by glucocorticoids contributes to NF-kappaB inhibition.EMBO J. 27, 1682–1693

31 Lombardi, M. et al. (2008) Hormone-dependent nuclear export ofestradiol receptor and DNA synthesis in breast cancer cells. J. CellBiol. 182, 327–340

32 Amazit, L. et al. (2003) Subcellular localization and mechanisms ofnucleocytoplasmic trafficking of steroid receptor coactivator-1. J. Biol.Chem. 278, 32195–32203

308

33 Kino, T. et al. (2003) Protein 14-3-3sigma interacts with and favorscytoplasmic subcellular localization of the glucocorticoid receptor,acting as a negative regulator of the glucocorticoid signalingpathway. J. Biol. Chem. 278, 25651–25656

34 Kumar, S. et al. (2004) Shuttling components of nuclear importmachinery involved in nuclear translocation of steroid receptorsexit nucleus via exportin-1/CRM-1 independent pathway. Biochim.Biophys. Acta 1691, 73–77

35 Holaska, J.M. et al. (2002) Ca2+-dependent nuclear export mediatedby calreticulin. Mol. Cell. Biol. 22, 6286–6297

36 Walther, R.F. et al. (2003) Nuclear export of the glucocorticoidreceptor is accelerated by cell fusion-dependent release ofcalreticulin. J. Biol. Chem. 278, 37858–37864

37 Liu, J. and DeFranco, D.B. (2000) Protracted nuclear export ofglucocorticoid receptor limits its turnover and does not require theexportin 1/CRM1-directed nuclear export pathway. Mol. Endocrinol.14, 40–51

38 Giannoukos, G. et al. (1999) The seven amino acids (547-553) of ratglucocorticoid receptor required for steroid and hsp90 binding containa functionally independent LXXLL motif that is critical for steroidbinding. J. Biol. Chem. 274, 36527–36536

39 Kaul, S. et al. (2002) Mutations at positions 547-553 of ratglucocorticoid receptors reveal that hsp90 binding requires thepresence, but not defined composition, of a seven-amino acidsequence at the amino terminus of the ligand binding domain. J.Biol. Chem. 277, 36223–36232

40 Dong, D.D. et al. (2006) Functional analysis of the LXXLL motifs of thehuman glucocorticoid receptor: association with altered ligandaffinity. J. Steroid Biochem. Mol. Biol. 101, 106–117

41 Rogatsky, I. et al. (2003) Target-specific utilization of transcriptionalregulatory surfaces by the glucocorticoid receptor. Proc. Natl. Acad.Sci. U.S.A. 100, 13845–13850

42 Danielian, P.S. et al. (1992) Identification of a conserved regionrequired for hormone dependent transcriptional activation bysteroid hormone receptors. EMBO J. 11, 1025–1033

43 Kucera, T. et al. (2002) A point mutation of the AF2 transactivationdomain of the glucocorticoid receptor disrupts its interaction withsteroid receptor coactivator 1. J. Biol. Chem. 277, 26098–26102

44 Alroy, I. and Freedman, L.P. (1992) DNA binding analysis ofglucocorticoid receptor specificity mutants. Nucleic Acids Res. 20,1045–1052

45 Schena, M. et al. (1989) Mutations in the glucocorticoid receptor zincfinger region that distinguish interdigitated DNA binding andtranscriptional enhancement activities. Genes Dev. 3, 1590–1601

46 Reichardt, H.M. et al. (1998) DNA binding of the glucocorticoidreceptor is not essential for survival. Cell 93, 531–541

47 Reichardt, H.M. et al. (2001) Repression of inflammatory responses inthe absence of DNA binding by the glucocorticoid receptor. EMBO J.20, 7168–7173

48 Liden, J. et al. (1997) A new function for the C-terminal zinc finger ofthe glucocorticoid receptor Repression of RelA transactivation. J. Biol.Chem. 272, 21467–21472

49 Nissen, R.M. and Yamamoto, K.R. (2000) The glucocorticoid receptorinhibits NFkappaB by interfering with serine-2 phosphorylation ofthe RNA polymerase II carboxy-terminal domain. Genes Dev. 14,2314–2329

50 Ray, A. et al. (1991) Repressor to activator switch by mutations in thefirst Zn finger of the glucocorticoid receptor: is direct DNA bindingnecessary? Proc. Natl. Acad. Sci. U.S.A. 88, 7086–7090

51 Li, G. et al. (2006) Role of steroid receptor coactivators inglucocorticoid and transforming growth factor beta regulation ofplasminogen activator inhibitor gene expression. Mol. Endocrinol.20, 1025–1034

52 Heck, S. et al. (1994) A distinct modulating domain in glucocorticoidreceptor monomers in the repression of activity of the transcriptionfactor AP-1. EMBO J. 13, 4087–4095

53 Kassel, O. et al. (2004) A nuclear isoform of the focal adhesion LIM-domain protein Trip6 integrates activating and repressing signals atAP-1- and NF-kappaB-regulated promoters. Genes Dev. 18, 2518–

252854 Doucas, V. et al. (2000) Cytoplasmic catalytic subunit of protein kinase

A mediates cross-repression by NF-kappa B and the glucocorticoidreceptor. Proc. Natl. Acad. Sci. U.S.A. 97, 11893–11898


55 Diefenbacher, M. et al. (2008) Restriction to Fos family members ofTrip6-dependent coactivation and glucocorticoid receptor-dependenttrans-repression of activator protein-1. Mol. Endocrinol. 22, 1767–

178056 Diefenbacher, M.E. et al. (2010) The nuclear isoform of the LIM

domain protein Trip6 integrates activating and repressing signalsat the promoter-bound glucocorticoid receptor. Mol. Cell. Endocrinol.320, 58–66

57 Wu, J. et al. (2004) Repression of p65 transcriptional activation by theglucocorticoid receptor in the absence of receptor-coactivatorinteractions. Mol. Endocrinol. 18, 53–62

58 Arancibia, S. et al. (2011) Phosphatidylinositol 3-kinase interacts withthe glucocorticoid receptor upon TLR2 activation. J. Cell. Mol. Med.15, 339–349

59 McKay, L.I. and Cidlowski, J.A. (1998) Cross-talk between nuclearfactor-kappa B and the steroid hormone receptors: mechanisms ofmutual antagonism. Mol. Endocrinol. 12, 45–56

60 Bladh, L.G. et al. (2005) Identification of endogenous glucocorticoidrepressed genes differentially regulated by a glucocorticoid receptormutant able to separate between nuclear factor-kappaB and activatorprotein-1 repression. Mol. Pharmacol. 67, 815–826

61 Tao, Y. et al. (2001) Mapping of glucocorticoid receptor DNA bindingdomain surfaces contributing to transrepression of NF-kappa B andinduction of apoptosis. J. Biol. Chem. 276, 2329–2332

62 Meyer, T. et al. (1997) The rat glucocorticoid receptor mutant K461Adifferentiates between two different mechanisms of transrepression.J. Biol. Chem. 272, 21090–21095

63 Sharma, S. and Lichtenstein, A. (2008) Dexamethasone-inducedapoptotic mechanisms in myeloma cells investigated by analysis ofmutant glucocorticoid receptors. Blood 112, 1338–1345

64 Rauch, A. et al. (2010) Glucocorticoids suppress bone formation byattenuating osteoblast differentiation via the monomericglucocorticoid receptor. Cell Metab. 11, 517–531

65 Donet, E. et al. (2008) Transrepression function of the glucocorticoidreceptor regulates eyelid development and keratinocyte proliferationbut is not sufficient to prevent skin chronic inflammation. Mol.Endocrinol. 22, 799–812

66 Bayo, P. et al. (2008) Glucocorticoid receptor is required for skinbarrier competence. Endocrinology 149, 1377–1388

67 Wang, Z. et al. (2007) Modulation of glucocorticoid receptorphosphorylation and transcriptional activity by a C-terminal-associated protein phosphatase. Mol. Endocrinol. 21, 625–634

68 Galliher-Beckley, A.J. and Cidlowski, J.A. (2009) Emerging roles ofglucocorticoid receptor phosphorylation in modulating glucocorticoidhormone action in health and disease. IUBMB Life 61, 979–986

69 Dephoure, N. et al. (2008) A quantitative atlas of mitoticphosphorylation. Proc. Natl. Acad. Sci. U.S.A. 105, 10762–10767

70 Krstic, M.D. et al. (1997) Mitogen-activated and cyclin-dependentprotein kinases selectively and differentially modulatetranscriptional enhancement by the glucocorticoid receptor. Mol.Cell. Biol. 17, 3947–3954

71 Wang, Z. and Garabedian, M.J. (2003) Modulation of glucocorticoidreceptor transcriptional activation, phosphorylation, and growthinhibition by p27Kip1. J. Biol. Chem. 278, 50897–50901

72 Kino, T. et al. (2007) Cyclin-dependent kinase 5 differentiallyregulates the transcriptional activity of the glucocorticoid receptorthrough phosphorylation: clinical implications for the nervous systemresponse to glucocorticoids and stress. Mol. Endocrinol. 21, 1552–

156873 Webster, J.C. et al. (1997) Mouse glucocorticoid receptor

phosphorylation status influences multiple functions of the receptorprotein. J. Biol. Chem. 272, 9287–9293

74 Blind, R.D. and Garabedian, M.J. (2008) Differential recruitment ofglucocorticoid receptor phospho-isoforms to glucocorticoid-inducedgenes. J. Steroid Biochem. Mol. Biol. 109, 150–157

75 Miller, A.L. et al. (2005) p38 Mitogen-activated protein kinase(MAPK) is a key mediator in glucocorticoid-induced apoptosis oflymphoid cells: correlation between p38 MAPK activation and site-specific phosphorylation of the human glucocorticoid receptor atserine 211. Mol. Endocrinol. 19, 1569–1583

76 Chen, W. et al. (2008) Glucocorticoid receptor phosphorylationdifferentially affects target gene expression. Mol. Endocrinol. 22,1754–1766

77 Avenant, C. et al. (2010) Glucocorticoid receptor phosphorylationmodulates transcription efficacy through GRIP-1 recruitment.Biochemistry 49, 972–985

78 Nader, N. et al. (2010) AMPK regulates metabolic actions ofglucocorticoids by phosphorylating the glucocorticoid receptorthrough p38 MAPK. Mol. Endocrinol. 24, 1748–1764

79 Garza, A.M. et al. (2010) Site-specific phosphorylation inducesfunctionally active conformation in the intrinsically disordered N-terminal activation function (AF1) domain of the glucocorticoidreceptor. Mol. Cell. Biol. 30, 220–230

80 Rogatsky, I. et al. (1998) Antagonism of glucocorticoid receptortranscriptional activation by the c-Jun N-terminal kinase. Proc.Natl. Acad. Sci. U.S.A. 95, 2050–2055

81 Itoh, M. et al. (2002) Nuclear export of glucocorticoid receptor isenhanced by c-Jun N-terminal kinase-mediated phosphorylation.Mol. Endocrinol. 16, 2382–2392

82 Galliher-Beckley, A.J. et al. (2008) Glycogen synthase kinase 3beta-mediated serine phosphorylation of the human glucocorticoid receptorredirects gene expression profiles. Mol. Cell. Biol. 28, 7309–7322

83 Rogatsky, I. et al. (1998) Phosphorylation and inhibition of ratglucocorticoid receptor transcriptional activation by glycogensynthase kinase-3 (GSK-3) Species-specific differences betweenhuman and rat glucocorticoid receptor signaling as revealedthrough GSK-3 phosphorylation. J. Biol. Chem. 273, 14315–14321

84 Ismaili, N. et al. (2005) Stabilization of the unliganded glucocorticoidreceptor by TSG101. J. Biol. Chem. 280, 11120–11126

85 Holmstrom, S.R. et al. (2008) SUMO-mediated inhibition ofglucocorticoid receptor synergistic activity depends on stableassembly at the promoter but not on DAXX. Mol. Endocrinol. 22,2061–2075

86 Tian, S. et al. (2002) Small ubiquitin-related modifier-1 (SUMO-1)modification of the glucocorticoid receptor. Biochem. J. 367, 907–911

87 Davies, L. et al. (2008) Cross talk of signaling pathways in theregulation of the glucocorticoid receptor function. Mol. Endocrinol.22, 1331–1344

88 Wallace, A.D. and Cidlowski, J.A. (2001) Proteasome-mediatedglucocorticoid receptor degradation restricts transcriptionalsignaling by glucocorticoids. J. Biol. Chem. 276, 42714–42721

89 Wallace, A.D. et al. (2010) Lysine 419 targets human glucocorticoidreceptor for proteasomal degradation. Steroids 75, 1016–1023

90 Deroo, B.J. et al. (2002) Proteasomal inhibition enhancesglucocorticoid receptor transactivation and alters its subnucleartrafficking. Mol. Cell. Biol. 22, 4113–4123

91 Ito, K. et al. (2006) Histone deacetylase 2-mediated deacetylation ofthe glucocorticoid receptor enables NF-kappaB suppression. J. Exp.Med. 203, 7–13

92 Nader, N. et al. (2009) Circadian rhythm transcription factor CLOCKregulates the transcriptional activity of the glucocorticoid receptor byacetylating its hinge region lysine cluster: potential physiologicalimplications. FASEB J. 23, 1572–1583

93 Daniel, A.R. et al. (2010) The progesterone receptor hinge regionregulates the kinetics of transcriptional responses throughacetylation, phosphorylation, and nuclear retention. Mol.Endocrinol. 24, 2126–2138

94 Fu, M. et al. (2000) p300 and p300/cAMP-response element-bindingprotein-associated factor acetylate the androgen receptor at sitesgoverning hormone-dependent transactivation. J. Biol. Chem. 275,20853–20860

95 Wang, C. et al. (2001) Direct acetylation of the estrogen receptor alphahinge region by p300 regulates transactivation and hormonesensitivity. J. Biol. Chem. 276, 18375–18383

96 Milhon, J. et al. (1997) Identification of amino acids in the tau 2-regionof the mouse glucocorticoid receptor that contribute to hormonebinding and transcriptional activation. Mol. Endocrinol. 11, 1795–

180597 Avenant, C. et al. (2010) Role of ligand-dependent GR phosphorylation

and half-life in determination of ligand-specific transcriptionalactivity. Mol. Cell. Endocrinol. 327, 72–88

98 Starr, D.B. et al. (1996) Intracellular receptors use a commonmechanism to interpret signaling information at responseelements. Genes Dev. 10, 1271–1283

99 Necela, B.M. and Cidlowski, J.A. (2004) A single amino acid change inthe first zinc finger of the DNA binding domain of the glucocorticoid

309


receptor regulates differential promoter selectivity. J. Biol. Chem.279, 39279–39288

100 Adams, M. et al. (2003) Homodimerization of the glucocorticoidreceptor is not essential for response element binding: activation ofthe phenylethanolamine N-methyltransferase gene by dimerization-defective mutants. Mol. Endocrinol. 17, 2583–2592

101 Ricketson, D. et al. (2007) A conformational switch in the ligand-binding domain regulates the dependence of the glucocorticoidreceptor on Hsp90. J. Mol. Biol. 368, 729–741

102 Hollenberg, S.M. et al. (1989) Identification of two regions of thehuman glucocorticoid receptor hormone binding domain that blockactivation. Cancer Res. 49, 2292s–2294s

310

103 Housley, P.R. et al. (1990) Evidence that the conserved region in thesteroid binding domain of the glucocorticoid receptor is required forboth optimal binding of hsp90 and protection from proteolyticcleavage A two-site model for hsp90 binding to the steroid bindingdomain. J. Biol. Chem. 265, 12778–12781

104 Lemke, U. et al. (2008) The glucocorticoid receptor controls hepaticdyslipidemia through Hes1. Cell Metab. 8, 212–223

105 Lang, P.A. et al. (2007) PI3 kinase dependent stimulation of gastricacid secretion by dexamethasone. Cell. Physiol. Biochem. 20, 527–534

106 Sandu, C. et al. (2007) Role of the serum and glucocorticoid induciblekinase SGK1 in glucocorticoid stimulation of gastric acid secretion.Pflugers Arch. 455, 493–503

Glucocorticoid receptor mutants: man-made tools for functional research

Documents

Transcript of Glucocorticoid receptor mutants: man-made tools for functional research