Gene regulation by the glucocorticoid receptor: Structure:function relationship

Journal of Steroid Biochemistry & Molecular Biology 94 (2005) 383–394

Gene regulation by the glucocorticoid receptor: Structure:function relationship Raj Kumar, E. Brad Thompson ∗ Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1068, USA Received 2 November 2004; accepted 30 December 2004

Abstract The glucocorticoid receptor (GR) belongs to the superfamily of ligand-activated transcription factors, the nuclear hormone receptors. Like other members of the family, the GR possesses a modular structure consisting of three major domains—the N-terminal (NTD), DNA binding (DBD), and ligand binding (LBD). Although the structures of independently expressed GR DBD and LBD are known, the structures of the NTD and of full-length GR are lacking. Both DBD and LBD possess overall globular structures. Not much is known about the structure of the NTD, which contains the powerful AF1/tau1/enh2 transactivation region. Several studies have shown that AF1 region is mostly unstructured and that it can acquire folded functional conformation under certain potentially physiological conditions, namely in the presence of osmolytes, when the GR DBD is bound to glucocorticoid response element (GRE), and when AF1 binds other transcription factor proteins. These conditions are discussed here. The functions of the GR will be fully understood only when its working three-dimensional structure is known. Based on the available data, we propose a model to explain data which are not adequately accounted for in the classical models of GR action. In this review, we summarize and discuss current information on the structure of the GR in the context of its functional aspects, such as protein:DNA and protein:protein interactions. Because of the close similarities in modular organization among the members of the nuclear hormone receptors, the principles discussed here for the GR should be applicable to many other receptors in the family as well. © 2005 Elsevier Ltd. All rights reserved. Keywords: Gene regulation; Glucocorticoid receptor; Structure:function relationship; Activation domain; Protein folding

1. Introduction Many classic effects of glucocorticoids and other steroid hormones occur through the regulation of gene transcription. Excluding the steroid effects that occur within seconds to a few minutes after the hormone reaches its target cell, regulation through the glucocorticoid receptor (GR), a ligandactivated transcription factor, appears to be the primary way in which these steroids modulate body processes. Transcriptional regulation can be divided into primary and secondary regulations. By primary regulation is meant the alteration in gene transcription that occurs as a direct result of the activated receptor interacting in some fashion with the regulatory regions of the affected genes. Secondary gene regulation refers ∗

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to instances in which the RNA or protein products from genes regulated at the primary level affect the transcription of additional genes. These latter mechanisms do not concern us here, and this review will focus on primary regulation. After entry into the cell the steroid finds the receptor as part of a large heteromeric complex. This complex has been studied extensively and best estimates indicate that it consists of several proteins including HSP90, HSP70, Immunophilins, FKBPs, CyP-40, P23, and possibly few others [1–3]. A key part of the complex is the receptor:HSP90 interaction, which seems to keep the ligand binding pocket of the receptor in its optimal, high-affinity configuration. Once steroid is bound, the complex disassembles and the “activated” receptor, with the steroidal ligand occupying its site in ligand binding domain (LBD) of the receptor enters the nucleus, where it interacts with critical regulatory sites on the relevant genes. Our concepts of what defines these sites have changed significantly

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in recent years. Once in the nucleus, the “activated” GR now interacts at the regulatory regions of its primary targets to either enhance (upregulate, induce), transcription or repress (down regulate, deinduce). This sequence of events is termed primary regulation. For the most part, genes that are under primary regulation show changes in transcriptional output in a few hours [4]. The sequence from steroid entry and binding to the receptor, to receptor reaching the nucleus takes only a few minutes. In the nucleus, the onset of changes in transcription require a few more minutes, and in order to accumulate enough messenger RNA, depending on the size of the gene and the time required for post-transcriptional processing, a few more minutes are necessary. Thus, in very rapidly responding genes, increases in messenger RNA have been detected as early as 15 min after the addition of steroid [5]. In other cases of primary regulation an interval as long as a few hours has been necessary. The traditional models proposed that all such regulations required the receptor to weakly bind and release at nonspecific DNA sites until it finds the sequence-specific sites termed “response elements” (RE), to which receptor binds tightly as a homodimer [6]. This is no longer sufficient to fit the accumulated data. It has become apparent that the receptor also can cause primary gene regulation by interacting with other regulatory proteins, which are bound to DNA at their own high-affinity binding sites [7]. The steroid receptor (SR) in such circumstances may be simply “tethered” indirectly to the DNA or may also make secondary DNA contacts as well. Due to the fact that such tethering sites were discovered on genes in which addition of steroids to cells caused down regulation, it was believed that all receptor-tethering events would cause gene repression. By several suggested mechanisms, the GR interaction with the heterologous transcription factor resulted in diminution of transcription of that particular gene. More recent data however, has shown that upregulation is also possible at receptor-tethering sites, depending on the particular DNA binding site involved for the heterologous transcription factor and/or the factor interaction with receptor [7,8]. While not documented as yet, in principle, such factor:receptor interaction also could occur through intermediary proteins by a bridging mechanism. In addition, our examination of the promoters of a number of genes found to be induced by glucocorticoids in CEM leukemic lymphoid cells suggests that induction of a significant number of genes may proceed from regulatory regions lacking classic glucocorticoid response elements (GRE) [9,10]. The data suggest that GR, tethered at genes through heterologous factors, can induce as well as repress genes in a significant number of cases. One important implication of this broader model is that the surfaces of the GR must be employed in various ways in order to allow interaction with a variety of other macromolecules, and thus change transcription [7]. How can this be? Results from a variety of systems have made it clear that transcription factors are platforms on which large multiprotein complexes are built [11–13]. These multifunctional

Fig. 1. A model showing possible factors that may alter conformation(s) of the GR. Under the influence of these factors, the GR displays surface(s), which are suitable for cofactor binding. Each conformation allows the GR to efficiently interact with specific coactivators/corepressors, and may also facilitate a direct or indirect cross talk between AF1 and AF2.

complexes “remodel” chromatin and make protein:protein contacts that by several mechanisms enhance or reduce transcription. The members of the complexes can vary, and the ultimate composition of the complex determines the outcome. As transcription factors, receptors such as the GR, appear to exhibit structural plasticity; and this property affects the surfaces of the receptors [7]. We and others propose that this property allows differential “selection” of a variety of ubiquitous and cell-specific cofactors. More precisely, modulations in the surfaces that the receptor presents to the solvent result in altered affinities for various cofactors. In a particular cell, the recruitment of various cofactors depends on their availability at the gene locus to which the receptor binds. Modulation of the receptor can come through the influence of specific ligands, by post-translational modifications, from the specific DNA sequence of a response element, through the induced fit engendered by binding particular proteins, and possibly by the effects of intracellular osmolytes (Fig. 1). In recent years, through structural biological and molecular genetics approaches, a great deal of progress has been made on the question of how the GR transmits the transcriptional signal from ligand to its specific target genes. Since the modulated structures of the receptor seem to lie at the center of this problem, focus has been on these structures. In this review article, we will discuss certain of these important structure function aspects of the GR.

2. Overview of the modular organization of the GR The organization of the GR into its three major functional domains, N-terminal (NTD), DNA binding (DBD), and ligand-binding (LBD) is well characterized. In terms of both size and sequence homology the NTD represents the most variable domain among the nuclear hormone receptors (NHRs) and even between different species of the GR [14,15]. Yet the NTD contains a powerful transcriptional activation region (AF1/tau1/enh2) required for maximal transcriptional enhancement [16–19]. The AF1 can act constitutively in the

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absence of the LBD, and is quite active in stimulating transcription from simple promoters containing cognate GR binding sites (response elements) [18]. This ability to increase gene transcription appears to be cell/coactivator-dependent [7,20,21]. The AF1 is rich in acidic amino acid residues, similar to the “acidic activation domains” of several non-receptor transcription factors [22,23]. The NTD is highly immunogenic and contains the major known sites of phosphorylation in the GR [24–26]. The DBD, located centrally in the amino acid sequence of the GR, is the most conserved region among NHRs. It is composed of two highly conserved “zinc fingers”, though they do not form classic three-dimensional zinc finger structures as in the Xenopus protein and other true zinc finger proteins. The DBD contains amino acids that contact specific bases in GRE sequences to provide site specificity for GR:DNA binding. These amino acids are primarily located in the first zinc finger, where the “P box” comprised of three amino acids is responsible for RE discrimination [27,28]. The second zinc finger region stabilizes DBD:GRE interactions, and five amino acids in the second zinc finger, termed “D box”, play an important role in homodimerization at the GRE [27,28]. The DBD also interacts with certain heterologous proteins, c-Jun for example. The C-terminal LBD is responsible for recognition and binding of steroid hormone ligands, chaperones and other proteins [14]. In addition, the LBD contains a small but important ligand-dependent activation function (AF2) subdomain located towards its C-terminal end [19,29]. The AF2 functions in a ligand-dependent manner to fold so as to complete binding surfaces for other proteins, e.g. coactivators and corepressors. 2.1. The N-terminal domain Within the NTD, the importance of the AF1 domain as the major activation region was established long ago by molecular mapping and structure:function techniques, showing that much of the GR’s transcriptional activity depends on this domain [16,18,19]. Though quantitatively important, the GR AF1 is not necessary for life. Transgenic mice have been created that express only a GR form lacking the AF1 [30]. The three-dimensional structure of the entire NTD or (where present) its AF1 is not known for any NHR member. The major obstacle in determining the structure of this domain has been the unstructured nature of the AF1 peptide. When expressed independently as a recombinant peptide, AF1 seems to exist as an ensemble of conformers without stable secondary/tertiary structures [31,32]. When AF1 is present in a two-domain fragment of the GR containing its entire NTD and DBD, the GR AF1 component displays slightly more structure, but clearly is not fully folded [33]. Similar results have been shown for the progesterone and androgen receptor AF1s [34–36]. It is known that AF1 interacts with several other transcription factors, and the available data strongly suggest that conditional folding of AF1 is the key for these interactions and subsequent transcriptional activity [37–41].

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How AF1 folds and what kind of functional folded conformation it adopts are open questions, actively being pursued. The flexible AF1 domain is ideally suited to provide modulated surfaces in the GR. In recent years, it has become clear that in many proteins, significant regions are coded by amino acid sequences that do not automatically fold into their fully condensed, functional structures, even in the presence of chaperone proteins [42]. However, most proteins must have structure to carry out their proper functions. In the case of GR AF1, application of predictive algorithms for secondary structure suggests that it may contain some helical structure. Consistent with this prediction, in the presence of the helixpromoting alcohol trifluoroethanol (TFE), some helical structure was formed in AF1 [31]. Independent experiments have shown that substitution of a helix-breaking amino acid (Proline) in the putative helical segments interfered with AF1 functions [31]. However, TFE-induced conformation cannot be presumed under in vivo conditions. Taking the lead from these findings, we have found several conditions that cause the GR AF1 to acquire a folded conformation. 2.1.1. Osmolytes induce AF1 to fold into a native-like functional conformation Osmolytes are small molecules used by many organisms to promote or maintain protein folding during metabolic or environmental extremes. The large literature on osmolytes suggests that osmolyte-induced structure is in fact physiological [43–50]. Human kidneys, for example, contain several osmolytes. It has been calculated that osmolyte concentrations in whole tissues often reach 400 mmol/kg of cell water, meaning that in certain compartments, the concentrations are almost surely much higher. Some tissues have higher concentrations of osmolytes than do others. The proteins of these tissues do not appear to differ in structure or function from those found elsewhere. Osmolytes induce folding not by affecting the amino acid side chains, but as a result of solvophobic effects on the peptide backbone [48,50]. In this way, osmolytes can provide an additional force for protein folding, while allowing natural hydrophobic forces that drive protein folding to occur unhindered. Because the protein backbone comprises the most numerous functional groups of proteins, osmolyte-induced conformations result in native folded functional species [43,44]. In a recent study, oral administration of the osmolyte, trehalose has been used in a transgenic mouse model of Huntington’s disease to attenuate protein aggregation [51], a major pathogenic component of the disease. Our data indicate that in vitro the osmolyte trimethylamine-N-oxide (TMAO) causes recombinant GR AF1 to fold into a form that selectively binds certain relevant proteins. When recombinant AF1 is incubated in increasing concentrations of TMAO, the protein cooperatively folds into a compact, monomeric structure [41]. Such cooperativity is a hallmark of spontaneous protein folding [43]. When thus folded, AF1 selectively binds TATA box-binding protein (TBP), CREB-binding protein (CBP) and a member of

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the steroid receptor coactivator-1 (SRC-1) family of proteins [41]. While the concentrations of TMAO required in vitro to produce AF1 folding are unlikely to be relevant in vivo, these results demonstrate the principle that when folded, AF1 can function to bind strongly to known coactivators and a member of the TFIID complex. Similar studies have also been done with the androgen receptor (AR) AF1 [36]. 2.1.2. Folding is induced in AF1 when the GR’s DBD binds a palindromic GRE When a two-domain form of the GR containing the entire N-terminal region through the complete DBD, but no LBD, was bound stoichiometrically to a DNA oligomer containing a complete palindromic GRE, secondary and tertiary structure, monitored by circular dichroism (CD) and fluorescence emission spectroscopies, was induced in the NTD [33]. This implies an intramolecular flow of information from the DBD to the N-terminal region. Data from several groups has suggested that the binding of steroid receptors’ DBD’s to their respective REs provides more than passive localization of receptor to gene regulatory region. The nucleotide sequence of the RE can influence selection of genes to be regulated as well as the quantitative aspect of that regulation [7]. It seems appropriate to think of the DNA sequence of an RE as an allosteric ligand [52]. Our data provide direct, physical evidence that DBD:GRE binding results in acquisition of tertiary structure in the NTD/AF1 of the GR. We propose that this allosteric effect results in a partially folded, “cocked” AF1 conformation, such that it can readily recognize certain other proteins important for transcriptional regulation. Indeed, we have shown that this AF1/NTD binds with great efficiency to TBP and CBP (unpublished data), the same proteins to which the osmolyte-folded AF1 shows enhanced affinity. These conditional structural changes in the N-terminal region of the GR following DNA binding via the DBD to its cognate GRE thus may play an important role in triggering gene regulation. Rogatsky et al. have demonstrated that alterations in GRE nucleotide sequence can result in conversion of a corepressor to a coactivator [53]. The supposed corepressor GRIP demonstrated coactivator activity when bound to a GR located on a particular GRE. This model of the RE as allosteric ligand in no way rules out the possibility of further structural modulations in AF1 (or the entire GR) as a result of protein:protein interactions. In a study with a similar twodomain fragment of the progesterone receptor, it is shown that binding of this fragment protein to its cognate RE also stabilizes AF1 structure [34]. 2.1.3. Binding of a coregulatory protein induces more helical structure in AF1 Coregulatory proteins influence or modulate the transcriptional activity of the GR by multiple mechanisms. Once bound to GR, coregulators can act to modify chromatin, influence RNA polymerase II phosphorylation, and bind other transcription factors, mediators and proteins of the basal tran-

scription complex. But how is the choice of coregulator:AF1 interaction made? We have just indicated that specific sequences in the nucleotides of RE’s may be one mechanism. Another may be the GR structural alteration imposed by protein:protein interactions, i.e. induced fit. There are many examples in which the unfolded or partially folded regions of a protein take full shape upon interaction with particular binding partner(s) [15]. Applied to the GR AF1 for example, this induced-fit model of folding hypothesizes that AF1 is not fully structured in vivo until it binds one or another key partner molecule. This induced-conformation or limited set of conformations in AF1 is needed in order for it to interact with specific sets of other necessary factors. The initial fold in AF1 could derive from the tethering of the GR to a heterologous transcription factor bound to DNA at its own site. The fold in GR could also be driven by a locally high concentration of some particular coregulator. The actual inducedfit change in GR could occur by initial non-specific interactions between the unfolded AF1 domain and the binding partner. In this version of the model, when such interactions occur, the proximity of the two proteins leads to rapid acquisition of the proper structure in AF1. Alternatively, there could initially be more specific interactions of binding partner(s) with a partially folded AF1, or even with a tiny pool of fully folded AF1 molecules, creating a kinetic “sink” into which the general population falls. Physical proof of inducedfit alterations in steroid receptor AF1s comes from studies of AR AF1:TFIIF binding, the result of which is a significant increase in ␣ helical content of the AF1, with corresponding reduction in ␤ sheet [36]. In analogous experiments, GR AF1:TBP protein:protein interactions cause increased helix in AF1 [54]. This direct physical evidence for induced-fit changes in the AF1s from two different SRs suggests that the assembly of AF1:binding partner complexes is an essential step in realizing AF1’s properly folded, functioning structure. It remains to be determined what kind of functional structure(s) AF1 adopts. An induced-fit mechanism has also been reported for the activation domains of several transcription factors, including those of cMyc and the estrogen receptor NTD, via interactions with TBP [55,56]. These limited data indicate that the activation domains of each of these transcription factors may be adopting a folded functional conformation under physiological conditions through interaction with coregulatory proteins of the transcriptional machinery complex. We hypothesize that in the holoreceptor, under physiological conditions, AF1 exists as a partially folded structure due to inter-domain influences. Each receptor’s interaction with the DNA of its RE, or with a tethering transcription factor leads to further acquisition of AF1 structure, with specific variations in DNA sequence or heterologous transcription factor providing different signals. The resulting structurally modified forms of AF1 suit it for its varied interactions with other critical coregulatory proteins, and possibly additional modulations in receptor structure essential for gene regulation by the receptor. These interactions give the final folded structure

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to AF1 and form the basis for the multi-protein assemblies involved in GR-mediated regulation of transcription. 2.2. The DNA-binding domain In structural terms, the DBD is an extensively studied domain of the GR. The DBD consists of highly conserved amino acid sequences, which include two highly conserved groups of four Cys residues, each group coordinately binding a single Zn atom. The resulting tertiary structure contains helices that interact specifically with DNA [27,28]. The zinc atoms serve a structural role, each capping the N-terminus of an amphipathic ␣-helix and supporting a peptide loop. The first stabilized helix contains the few amino acids critical for DNA sequence-specific binding in the major groove of the GRE. Two more helices are found in the second zinc finger. These bind DNA non-specifically in the minor groove, and this contribution is important for the cooperative, highaffinity binding of GR dimers at classic palindromic GREs. The central feature of the secondary structure elements within the DBD is three helical regions. Helices I & III are oriented perpendicular to each other and form the base of a hydrophobic core. NMR studies indicate that helices I & III are both regular ␣-helices, whereas helix II is somewhat distorted [27]. Comparison of the crystal structure of the DBD complexed with DNA with that in the absence of DNA in solution reveals a small difference in one region of secondary structure. The crystal structure of the GR DBD:GRE complex in which DBD is shown to bind to GRE as a dimer shows that the helix I, which includes the region between the distal two cysteines of the first zinc finger, fits into the major groove of the DNA helix. This arrangement provides critical contacts between three amino acids in the helix and certain bases in the major groove of the DNA [28]. The second zinc finger region appears to be less well defined in solution as compared to the crystal structure, suggesting that this region is stabilized upon formation of the (DBD)2 :GRE complex [28]. NMR relaxation measurements, and molecular dynamics simulations of the GR DBD revealed a uniform and limited mobility along the backbone [57]. Concerted motions in and between the sub-domains could facilitate structural rearrangements that lead to the cooperativity of DNA binding [57]. Molecular dynamics simulation and free energy perturbation studies suggest that the binding of the DBD to the GRE distorted conformations for bases at positions 5 and 6, propagation of which through the DNA may facilitate cooperative binding of another monomer at half-site [58]. In sum, DBD:GRE binding induces slight, but definite changes in DBD structure. The DBD also is known to participate in interactions with heterologous proteins, such as c-Jun and GT198 [59,60]. 2.3. The ligand-binding domain Analogous approaches to the study of independently expressed recombinant LBDs from over a dozen receptors, in-

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cluding the GR, led to solution of their crystal structures. In each case this domain consisted of about 12 helices that fold overall into a globular structure consisting of three sets of helices that form the sides and top of the globule, making a central pocket, the ligand-binding site [61]. Crystal structure of the GR ligand-binding domain reveals a novel mode of receptor dimerization and coactivator recognition [61]. Molecular genetics had shown that near the C-terminus of the LBD of several of these hormone-binding receptors is a short helical amino acid sequence (usually helix-12 important for activating gene transcription [61,62]). The surface formed by this sub-domain (AF2) binds a number of coactivator and corepressor proteins via their LXXLL motifs [63–68], and these proteins often are histone acetylases as well as acting as molecular bridges between the receptor and the fundamental transcription initiation complex of proteins, tethered through TBP to the TATA box. The LBD crystal structures provide a presumptive physical explanation for these data. The most C-terminal helix (usually helix-12) contains the sequence for the important AF2 function, the site upon which coactivator/corepressor binding depends. Helix-12 changes position upon ligand binding, flipping from an “open” position to one closed over the bound ligand [61]. The result is that the LBD now presents a surface favorable for binding the coactivators [61]. At least some ligands that act as antagonists appear to do so by causing helix-12 to close in a position that creates an unfavorable surface for coactivator binding [61]. The crystal structure of the GR LBD bound to dexamethasone and a peptidecoactivator motif derived from the transcriptional intermediary factor-2 (TIF-2) has been published [61]. The overall folding pattern of folding is shown to be similar to other NHR LBDs, i.e. three antiparallel sandwiches of helices. However, some interesting unique features are seen in the GR LBD structure. It adopts a dimer configuration involving formation of an inter-molecular ␤-sheet. This dimer interface is different from the helix-10 dimer interface observed in the estrogen receptor LBD structure [62]. Mutations in this dimer interface that disrupt LBD dimerization resulted in loss of GR activation function without affecting transrepression activity. These results are similar to those found with GR containing a defective DBD dimerization interface [69]. Unlike other NHRs, the GR LBD structure revealed an additional charge clamp that determines the binding selectivity of a coactivator and a distinct ligand-binding pocket that explains its selectivity for endogenous ligands. Another distinctive ligand-binding pocket feature seen in the GR LBD is an additional branch [61,62]. While the either/or structural model of helix-12 folding with ligand antagonists and agonists explains some data, still unexplained is the fact that some “antagonists” act as agonists in specific tissues. This has been most extensively explored for the estrogen receptor. To explain this behavior, a more dynamic view [70,71] applied to the structure of the NHRs will be necessary.

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3. Role of phosphorylation in modulating the action of the GR Phosphorylation is a frequent post-translational modification that modulates protein function, often by altering protein structure [72–75]. There is increasing evidence that phosphorylation of many transcription factors regulates their ability to affect gene expression. Phosphorylation by cellular kinases plays a prominent role in the regulation of the NHRs [25,76]. Ligand binding, nuclear translocation, modulation of binding to REs, receptor dimerization, and interaction with general transcription factors have all been linked to phosphorylation [77]. It has been reported that kinases enhance the ligand-dependent transactivation activity of several SRs [78,79]. In some cases kinases can cause NHRs to function in a ligand-independent manner [80]. Transcription mediated by the estrogen receptor AF1 domain is strongly modulated by cell-specific phosphorylation [80]. There are also reports that phosphorylation plays a major role in the recruitment of cofactors for Vitamin D receptor, and steroidogenic factor-1 [81,82]. The GR is a phosphoprotein, and regulated phosphorylation is an obvious possibility for modulating its activity, for example by influencing its interactions with other transcription factors. The GR is partially phosphorylated in the absence of glucocorticoid, with increased phosphorylation occurring when the GR is bound to agonist, but not antagonist steroids [83]. Some reports suggest that phosphorylation affects GR stability [84]. All seven phosphorylation sites (6 Ser plus 1 Thr) identified in mouse GR are found in the NTD, in or near the AF1 domain [26]. Five of these are well conserved in mouse, rat and human GRs. Individual mutations of these sites do not alter receptor activity in an MMTV-driven promoter–reporter construct, but a GR lacking all five conserved sites shows significantly decreased activity [24]. It was suggested that this result was due to the increased acidity of the AF1 domain [24]. In the human GR AF1, the phosphorylated residues are S203, S211 and S226. At least two of these (S211 and S226) are thought to be important for transcriptional activity of the GR [85,86]. A recent study has shown that site-specific (S211) phosphorylation of the GR AF1 enhances its interaction with a protein from the DRIP/TRAP coactivator complex [85,86]. These observations clearly suggest that site-specific phosphorylation in GR AF1 may be affecting GR function. The widespread use of phosphorylation in nature to regulate protein activity makes the phosphorylation of the GR protein an attractive possibility for controlling some aspects of their function. Some recent studies have shown that GR phosphorylation, by affecting GR interaction with some corepressors, plays an important role in GR’s immunosuppressive action [87,88]. The pattern of phosphorylation in the GR is variable among species, suggesting that species-specific differences in phosphorylation may influence functions of the GR [89]. Despite these tantalizing observations, the exact role of phosphorylation in the action of the GR is lacking.

4. Protein:DNA interactions Many hormonally regulated target genes contain one or more REs. These have been found within the promoter close to the transcriptional start site, up to several kilobases upstream of the start site, or occasionally within the intronic sequences of the regulated gene itself. The GRE situated in the long terminal repeat of the mouse mammary tumor virus (MMTV) was the first RE described [90] and established the pattern for all NHR REs. In their complete form, all are composed of two 5–6 nucleotide sequences, laid head to head or head to tail, and separated by 0 or up to 6 nucleotides of no particular sequence. The inclusion of two specified sequences in each complete RE is key, for it provides the basis for cooperative, high-affinity binding of receptor dimers [6]. As do many other DNA binding proteins, in their interaction with their response elements, the NHR: bind to the DNA double helix essentially through interactions within the major groove in which they recognize individual base pairs. Analysis of REs suggests that essentially all the members of the steroid receptor subfamily recognize one of the two hexameric DNA sequences (half-sites) of the GRE or of the estrogen response element (ERE). The targets of the nuclear subgroup of the receptors represented by the thyroid, retinoic acids, and Vitamin D receptors generally share binding sites based on the ERE, but in varied orientations and spacing, whereas the GRE can function virtually as effectively as an RE for glucocorticoid, progesterone, androgen and mineralocorticoid receptors. Thus, the basic building block of these REs is the specific 6-nucleotide sequence that comprises a half-site. The classic RE is composed of a pair of these hexamers, and the spacing between them, plus their orientation to one another, makes for a considerable degree of receptor-binding specificity. High affinity, however, requires a second half-site to allow cooperativity of the receptor dimers. Additional modifications in receptor binding specificity and affinity can be provided by variations in the use of certain nucleotides, differently from the consensus hexameric sequences, and by nucleotides in nearby DNA “flanking” sequences. The classical model of the GR action considers GR:GRE interaction as a simple tethering of macromolecular GR homodimer to DNA. However, our data suggest otherwise, that binding of GRE to GR DBD results in the formation of structure in the relatively unstructured AF1 domain [33]. This would mean that considerable binding energy is diverted to cause structure in the AF1 domain through intramolecular rearrangements. Similar results have been demonstrated with several other transcription factors, including SRs [34]. Earlier calculations suggested that there were such thermodynamic consequences for recombinant DBD:GRE interactions, consistent with the relatively small structural adjustments within the DBD alone [58,91]. Whether the LBD also achieves structural arrangements due to site-specific DNA-binding is not yet clear. However, there is evidence to indicate that this could be the case with some NHRs [92,93]. At this point, it may be

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intellectually satisfying to suggest that in the case of GR:GRE interaction, much of the energy from DBD:DNA binding is spent in structuring the NTD due to its relatively unstructured nature compared with other domains. We have already mentioned the fact that the DBD in the DBD:GRE crystal structure has some structural differences from its unbound state in solution. These considerations lead us to re-evaluate the classical model of GR:GRE binding. It appears that one reason why sequence-specific DNA binding has such a profound effect on GR AF1 structure (and possibly other SRs) is that AF1 can adopt a conformational surface such that it can physically interact with critical cofactors. Therefore the specific variations in RE sequence to which each SR binds may influence the functional conformation of the receptor, especially in AF1, so that the ancillary proteins that bind to the receptor are excluded or included in the ultimate complex by virtue of the availability of the conformed surface after DNA binding [52,53,65,94]. This theory is consistent with recent studies showing that the particular nucleotide sequence of the RE hexamer can influence transcriptional potency and gene selectivity [95–97].

5. Protein:protein interactions From the foregoing it is clear that protein:protein interactions can influence GR structure, particularly that of its AF1 region. In this section, we present an overview of the types of protein interactions in which the GR is known to participate, to emphasize the possibilities for influences on receptor structure and function. The GR can interact with coregulatory proteins, other types of transcription factors, and may also show intramolecular domain–domain interactions. Each of these will be considered in turn. 5.1. Interactions with coregulators 5.1.1. Coactivators and corepressors These are proteins that interact with DNA-bound receptors and participate in their transcriptional activation function [64]. The general model of an SR:coactivator or coactivator complex suggests that the ligand-bound receptor is able to recruit one or more of these proteins, which subsequently results in the recruitment of additional proteins to an assembly [65,68]. The histone acetylation, methylation and other activities of various constituents of the complex facilitate alterations in the chromatin architecture at the target gene promoter, thereby affecting transcriptional activation [68]. It is likely that several additional cofactors are involved. Among the many coregulatory proteins, some are ubiquitous, while others are cell-specific, and different SRs may recruit different components of the complex. The specific combination of SR and coactivators results in the specific control of particular genes [65]. Two classes of coregulatory complexes are recruited, either simultaneously or combinatorially. One class promotes

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the nucleosomal remodeling required for transcriptional activation, while the other forms a direct bridge to the transcriptional machinery. Most of the best-studied coregulatory proteins bind to the AF2 sub-domain through their LXXLL motif [63–68]. The AF1 domain is known to play an important role in many coregulator:SR interactions, by binding to regions of the coactivator remote from the LXXLL motif [38,39]. For example, CBP, SRC-1, AdA2, and several other co-regulators have been shown to bind to the GR AF1 [40]. The topic of SR:coregulator interactions has been reviewed extensively elsewhere [64]. We give this extremely brief description only to point out the wide potential for modulations of structure to occur and to affect outcomes of receptor control of transcription. 5.1.2. Interactions with basal transcription machinery Several SRs have been shown capable of directly binding with certain general transcription factors [15,55,64]. This raises the possibility that such contacts occur and influence transcription in vivo. In light of the extensive data indicating large coregulatory complexes, the role of direct SR interactions with general factors remains to be explained. It is striking, however, that GR:DBD binding to a GRE causes a fold of the GR AF1 which greatly enhances AF1:TBP interaction. Furthermore, TBP added to recombinant GR AF1 causes induced-fit folding of AF1 [54]. The AR AF1, whose primary amino acid sequence does not resemble that of the GR AF1, can bind the RAP74 component of the basal factor complex TFIIF, and RAP74:AR AF1 causes a shift in the composition of the AF1 from ␤ sheet to ␣ helix [36]. These data suggest that by directly binding to certain of its basal transcription component proteins, the GR, the AR (and by extension, other SRs) directly influence transcription. On at least some genes, the GR shows another mechanism by blocking transcription through interfering with the phosphorylation of the C-terminal portion of RNA Pol II [8]. 5.1.3. Interactions with other transcription factors The GR is known to be capable of binding with several other important transcription factors, each of which has its DNA sequence-specific binding site. It is believed that in two general ways, such interactions can alter the regulation of transcription by either or both factors. First, GR:heterologous transcription factor binding may lead to sequestration of the two away from their respective binding sites. This mechanism need not concern us here. Second, the GR may be tethered to genes at the heterologous factor’s DNA site. At such sites, the GR need not necessarily interact with the DNA at all, though it may do so in some situations, particularly by binding at an adjacent 1/2 GRE. Since the protein heterodimers that form to create these tethering situations may result in induced-fit or allosteric alterations in GR confirmation, they afford a range of possibilities for altering the surfaces available for additional coregulators to bind, building multi-protein complexes.

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While this has not yet been proven directly, two types of experiments attest to its likelihood. First, differing surfaces of the GR are important for regulation of various genes, and the makeup of regulatory complexes can vary with site [7,30]. Second, our proof that in two cases, protein:protein interactions result in physical induced-fit alterations in GR and AR structure suggest that other protein interactions, i.e. with transcription factors, do so as well. 5.1.4. Intrareceptor/domain interactions Though in many cases either AF1 or AF2 is to some extent capable of regulating transcription alone, full transcriptional activation by SRs requires functional synergy between the two AFs. This might be achieved through a physical intramolecular association between the NTD and LBD. This possibility is supported by studies showing a direct interaction between the N-terminal and C-terminal domains of the progesterone receptor-B [98]. The interaction takes place only when an agonist is bound, and antagonist binding prevents it, suggesting a specific conformational change in the agonist-bound LBD. Furthermore, coactivators such as SRC1 and CBP/p300 are not required for this physical association, though they are capable of enhancing a functionally productive interaction. Direct interactions between the NTD and LBD of the AR have also been reported. These may occur by a dimeric head to tail interaction [99]. There are suggestions that agonist-dependent interaction of the AR NTD and AF2 contributes to stabilization of helix12 to slow down ligand dissociation [100]. The AR AFs’ interactions are also suggested to be a prerequisite for the efficient recruitment of coactivators and for AR’s transcriptional activity [101]. A second possibility is that AF1 and AF2 interact via mutual binding of other proteins, such as coactivators. The synergy between the ER AF1 and AF2 is reported to be due to cooperative recruitment of members of p160 coactivators [102,103]. SRC-1 seems to be able to interact with both the AF1 and AF2 regions of several steroid receptors. This type of interaction between AF1 and AF2 regions via bridge proteins appears to be assisted by other cofactor proteins as well, such as TIF2 [104]. The TRAP/DRIP coactivator complex is reported to interact with both AF1 and AF2 regions in several SRs, including the GR [105]. Similar synergy between AF1 and AF2 is also observed involving CBP/p300 and estrogen receptor [106].

6. Summary and future perspectives The structural knowledge of individual domains of the GR and several other members of the NHR family has provided some initial outline of their structure, which has been helpful in answering several important questions related to the actions of the GR; however, this overall structural view has also raised certain critical questions. What is the structure of the holoreceptor, and how is structure influenced, by the binding of GRE, ligands, and other cofactor proteins? Recent stud-

ies provided some clues, suggesting that in fact these factors may be influencing the conformation of the GR so that it conforms to the surfaces for the interaction with the appropriate coactivators/corepressors. In addition, studies with the GR and other NHRs indicate that the structure and functions of the receptor are influenced by cross communication between receptor domains. Also remaining to be determined are the precise assemblies of cofactors on the receptor under various circumstances, and how these mediate communication between the GR and transcription initiation machinery. The ability of the GR AF1 to interact both with a component of the general transcriptional machinery (TBP) and with coregulator proteins provides a challenge to understand the overall mechanism. If the GR AF1 contacts TBP (or any member of the TBP–TAF complex) directly, how is this to be reconciled with the current models, which emphasize chromatin remodeling and complex protein “bridges” between GR and basal transcription machinery? An additional important problem yet to be solved is the means by which AF1 and AF2 synergize with each other. Recent studies have hinted that both the sequence and final components in the assembly of the receptor:cofactors may be the basis for these effects. A major obstacle to solving these problems is the limited knowledge of AF1 structure and functions. Of course obtaining a threedimensional structure of the full-length receptor is vital, but it must be recognized that the dynamic structure of the GR and like proteins means that a single structure will only give a starting point, albeit an important one. Finally, the rapid

Fig. 2. Model showing gene regulation by two alternative schemes are shown. In scheme (1), ligand-bound GR interacts with a GRE site. This protein:DNA interaction opens surfaces on the GR in such a conformation that allows both AF1 and AF2 regions to recruit certain specific cofactors. Alternatively, in scheme (2) GR binds to another transcription factor (TF) in the proximate region of the promoter. This GR:TF binding allows specific cofactors to be recruited either at GR, TF or both. A bridge is formed between AF1 and AF2 through these and/or other cofactor(s). AF1 and AF2 may also interact directly. In both (1) and (2), the eventual complex alters local chromatin structure, catalyzes histone acetylation or deacetylation, and affects the stabilization of the transcription pre-initiation complex. Other regulatory effects on transcription also may occur. The receptor complex, bound to a GRE (1), or TF (2) thus recruits and regulates Pol II via direct associations with specific subunits of the mediator complex, which makes a bridge between the receptor and Pol II. The receptor:cofactor assembly may also interact directly with the basal transcription machinery at the TBP/TATA box to regulate transcription. By affecting GR structure, factors shown in Fig. 1 thus influence the GR:cofactor complex in a cell type- and promoter-specific manner.

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progress being made in the determination of the assembly of the GR with cofactor proteins is paving the way to ultimately solving the mystery of how the signals from a specific ligand are passed through the GR to regulate specific sets of genes. The general GR:coactivator complex model suggests that the ligand-bound receptor is able to recruit one or more coactivators, which subsequently results in the recruitment of additional coactivators to the assembly. The histone acetylation and methylation activities of various constituents of the coactivator complex facilitate the relaxation of the chromatin architecture at the target gene promoter, thereby enhancing transcriptional activation (Fig. 2). It is likely that additional cofactors are involved, and that different NHRs may recruit different components of the complex, thus achieving a level of specificity among NHRs and coactivators. Analogous arguments can be made for receptor:corepressor complexes. Yet to be understood is how the GR functions in the transcription regulation when tethered to DNA via a heterologous transcription factor. From the wealth of new findings, briefly outlined here, a concept of the GR (and by analogy, other NHRs) as a highly dynamic protein emerges. GR functions, based on a structure that is highly malleable, can be modulated differentially by specific steroid ligands, post-transcriptional modifications, DNA sequence of the GRE, and protein:protein interactions.

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