Glucocorticoids, steroid hormones secreted from the adrenal cortex, play essential roles in the maintenance of internal homeostasis by influencing virtually all organs and tissues. These diverse actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), which belongs to the nuclear receptor superfamily and functions as a hormone-dependent transcription factor. The human GR protein is produced from 9 exons of the GR gene and is composed of 3 major subdomains, each of which has specific structural and functional properties. Since glucocorticoids are essential for life, other cellular signaling pathways strongly regulate GR actions in many different ways, such as physical interaction via their effector transcription factors and epigenetic modifications including phosphorylation and acetylation. Further, the GR gene expresses a splicing variant GRbeta, which does not bind glucocorticoids but functions as a dominant negative isoform for GRalpha. In this chapter, recent progress in the field investigating the GR actions is discussed.
Glucocorticoids are steroid hormones secreted from the zona fasciculata of the adrenal cortex, and play numerous important roles in the maintenance of internal homeostasis by influencing activities of virtually all organs and tissues, such as the brain, liver, skeletal muscles and the immune-related organs and cells . In addition to their effects on a basal state, these hormones are essential for responding properly to stress, the environmental changes that occur internally and externally . Thus, organisms cannot survive in the absence of glucocorticoid action, as observed in the mouse model in which the glucocorticoid receptor (GR) gene is genetically deleted . In pharmacologic or stress-related doses, glucocorticoids act as potent immunosuppressive agents, and as a result, are broadly used in the management of many inflammatory, autoimmune and proliferative diseases . Since glucocorticoids are indispensable for life and essential in the response to stress, a complex but organized feedback system, called the hypothalamic-pituitary adrenal (HPA) axis, regulates the production of glucocorticoids in the adrenal cortex to meet with timely requirement of glucocorticoid actions in their target tissues . This regulatory system consists of three components: the hypothalamic paraventricular nucleus (PVN), the anterior lobe of the pituitary gland and the adrenal cortex. These components respectively secrete corticotropin-releasing hormone, adrenocorticotropic hormone and glucocorticoids that regulate downstream components as well as to suppress upper centers, ultimately creating a closed regulatory loop . The HPA axis, in the absence of stress, is under the strong influence of the upper regulatory center suprachiasmatic nucleus (SCN) of the hypothalamus that functions as a core for organizing the body’s circadian activity [2, 5]. Thus, levels of circulating cortisol in humans create a typical oscillation fluctuation that completes in a 24-hour cycle, with the zenith occurring in the early morning and the nadir at midnight [2, 5].
Glucocorticoids influence a variety of biologic processes and exert profound influences on many physiologic functions by affecting mRNA abundance of up to ~20% of the human genome [6, 7]. Glucocorticoids do this by binding to their intracellular receptor molecule, the glucocorticoid receptor (GR), which functions as a hormone-activated transcription factor for changing expression of glucocorticoid-responsive genes positively and negatively [8, 9]. The GR is ubiquitously expressed in almost all human tissues and organs . The human (h) GR, a single polypeptide chain of 777 amino acid residues, belongs to the steroid/sterol/thyroid/retinoid/orphan receptor superfamily, which consists of over 130 members currently cloned and characterized across species . This chapter will describe the structure and action of the GR, focusing on recent progress in this field.
Biological actions of circulating glucocorticoids, such as cortisol in humans and corticosterone in rodents, are transduced into cells by the ubiquitously expressed intracellular receptor, the GR . The GR cDNA was isolated by expression cloning in 1985 . The GR, also known as nuclear receptor superfamily 3, group C, member 1 (NR3C1), belongs to the nuclear receptor superfamily, which are functional and preserved from the early metazoans to humans [8, 9]. The GR forms the steroid hormone receptor subfamily together with the mineralocorticoid (MR), progesterone (PR), estrogen (ER) and androgen (AR) receptors . The GR and its phylogenetically closest receptor MR are thought to have evolved approximately 450 million years ago from an ancestral corticosteroid receptor (CR), which originated from the ancestral estrogen receptor after two genome duplication events that occurred early in chordate evolution [13-15].
The human GR gene, located in the short arm of chromosome 5 (5q31.3), consists of 9 exons, and its expression is regulated by at least three different promoters A, B and C, with promoter A alternatively used with three unique promoter fragments 1A1, 1A2 and 1A3 . Thus, the GR gene can produce five different transcripts from different promoters that encode the same GR proteins. In addition to alternative transcripts using the 5’ different promoters, the GR gene generates two 3’ splicing variant transcripts with alternative use of exon 9alpha and 9beta (Figure 1). Thus, the GR gene generates 10 different transcripts that encode two protein molecules, GRalpha and GRbeta. Recently, it became evident that the GRalpha variant mRNA is translated from at least 8 initiation sites into multiple GRalpha isoforms termed A through D (A, B, C1-C3 and D1-D3), producing different amino terminal isoforms with distinct transcriptional activities on glucocorticoid-responsive genes  (Figure 1). These GR molecules are also differentially expressed in several different cell lines and tissues . Given that GRalpha and GRbeta share a common mRNA domain that contains the same translation initiation sites , it appears that the GRbeta variant mRNA is also translated through the same initiation sites to a similar host of 8 beta isoforms  (Figure 1).
Figure 1. Genomic and complementary DNA and protein structures of the human GR isoforms. The human GR gene consists of 9 exons. Exon 1 is an untranslated region, exon 2 codes for NTD, exons 3 and 4 for DBD, and exons 5-9 for the hinge region (HR) and LBD. The GR gene contains two terminal exons 9 (9a and 9b) alternatively spliced to produce the classic GRa (GRalpha-A) and the nonligand-binding GRb-A, which exerts dominant negative effects upon GRalpha (GRa-A). C-terminal gray colored domains in GRalpha-A and GRb-A show their specific portions. GRalpha N-terminal translational isoforms expressed from a single GRa transcript are shown in the middle of the Figure. The GRb transcript may also produce similar N-terminal isoforms from the same start sites. (Modified from Reference 9). Abbreviations: AF-1: activation function-1; DBD: DNA-binding domain; HD: hinge region; GR: glucocorticoid receptor; LBD: ligand-binding domain; NTD: N-terminal domain.
The classic receptor GRa and its molecular structure
GRalpha, the classic glucocorticoid receptor, is ubiquitously expressed and mediates most of the known actions of glucocorticoids [1, 9]. The human GRalpha consists of 777 amino acids and has 3 major distinct functional domains, the N-terminal or immunogenic domain (NTD), the DNA-binding domain (DBD) and the ligand-binding domain (LBD)  (Figure 2).
Figure 2. Functional distribution of the human GRa in its linearized molecule. The human GRalpha consists of 3 subdomains, NTD, DBD and LBD, each of which contains several functional molecular structures, such as the nuclear localization signals and transactivation domains. The human GRalpha also contains several amino acid residues for phosphorylation (serines) or acetylation (lysines), as indicated. Abbreviations: AF-1 and -2: activation function-1 and -2; DBD: DNA-binding domain; GR: glucocorticoid receptor; HR: hinge region; HSPs: heat shock proteins; LBD: ligand-binding domain; NL-1 and -2: nuclear localization signal 1 and 2; NTD: N-terminal domain.
Between the DBD and LBD, there is a small portion called the “hinge region” that influences specificity of the receptor to binding to its cognate DNA sequences, the glucocorticoid response elements (GREs) . The LBD of GRalpha, corresponding to amino acids 520-777, consists of 12 alpha-helices and 4 beta-sheets, among which helices 3, 4, 11 and 12 form the ligand-binding pocket for binding to glucocorticoids [20-22]. The DBD of GRalpha corresponds to amino acids 420-480 and contains two zinc finger motifs through which GRalpha binds to GREs [23, 24]. The DBD has two similar zinc finger modules, each nucleated by a Zn ion coordination center held by four cysteine residues and followed by an alpha-helix . The N-terminal’s first alpha helix lies in the major groove of the double-stranded DNA, while the C-terminal part of each module is positioned over the minor groove . In contrast to LBD and DBD, the structure of the NTD has not yet been well elucidated .
Nucleocytoplasmic shuttling of GRalpha
GRalpha is located primarily in the cytoplasm in the absence of a glucocorticoid ligand, as part of hetero-oligomeric complexes containing heat shock proteins (HSPs) 90, 70, 50, 20, and possibly, other proteins as well [9, 18] (Figure 3). After binding to its agonist ligand, GRalpha undergoes conformational changes, dissociates from the heat shock proteins (HSPs), homo-dimerizes, and translocates into the nucleus through the nuclear pore, via an active ATP-dependent process mediated by its nuclear localization signals (NL)-1 and -2 [8, 27]. NL-1 is located in the junction of the DBD and the hinge region, while NL-2 spans the entire LBD  (Figure 2). The function of NL-1 is mediated by the classic importin alpha/beta-nuclear pore complex, while that of the NL-2 has not been well elucidated . In the absence of a ligand, binding of HSPs with the LBD of GRalpha masks/inactivates NL-1 and NL-2, thereby maintaining GRalpha in the cytoplasm. Several mechanisms have been postulated for the regulation of GRalpha nuclear export . The CRM1/exportin and the classic nuclear export signal (NES)-mediated nuclear export machinery is not functional in GRalpha, based on evidence that GRalpha is insensitive to leptomycin B, an inhibitor of this export system, and does not contain the classic NES(s) [27-29]. Rather, the Ca2+-binding protein calreticulin plays a role in the nuclear export of GRalpha, directly binding to the DBD of this receptor [29-31].
Regulation of GRalpha transcriptional activity
Inside the nucleus, the ligand-activated GRalpha directly interacts as a dimer with GREs located in the promoter region of target genes  (Figure 3).
Figure 3. Circulation of the GRa between the cytoplasm and the nucleus, and its transactivating or transrepressive activities. Upon binding to ligand, GRalpha dissociates from HSPs and translocates into the nucleus, where it regulates the transcriptional activity of glucocorticoid-responsive genes positively and negatively either by binding to GREs located in the promoter region of target genes or by physically interacting with other transcription factors. After completion of changing the transcriptional activity of glucocorticoid-responsive genes, the receptor is exported into the cytoplasm and is incorporated into the complex with HSPs. Abbreviations: GR: glucocorticoid receptor; GRE: glucocorticoid responsive element; HSPs: heat shock proteins; TF: transcription factor; TFREs: transcription factor responsive elements.
The optimal recognition site of GREs is an inverted hexameric palindrome separated by 3 base pairs, PuGNACANNNTGTNCPy, with each GRalpha molecule binding to one of the palindromes . The interaction of GRalpha with GREs is dynamic, with the GRalpha binding to and dissociating from GREs in the order of seconds . GRalpha contains two transactivation domains, activation function (AF)-1 and -2, located at its NTD and LBD, respectively. Through these domains, promoter-bound GRalpha interacts with many proteins and protein complexes, such as the nuclear receptor histone acetyltransferase coactivator [p160, p300/CREB-binding protein (CBP) and p300/CBP-associated factor (p/CAF)] complexes and the SWI/SNF and vitamin D receptor-interacting protein/thyroid hormone receptor-associated protein (DRIP/TRAP) chromatin-remodeling complexes . By attraction of these cofactor complexes, GRalpha changes the chromatin structure, influences the activity of RNA polymerase II and its ancillary factors, and eventually alters the transcription rates of glucocorticoid-responsive genes [9, 18, 34] (Figure 2 and 3). GRalpha also interacts with the nuclear receptor corepressor (NCoR) and its homolog the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), which are macromolecular docking platforms for nuclear receptors and many transcription factors, and repress the transcriptional activity of the GRalpha by attracting HDAC/Sin3 complexes . The p160 type coactivators and the NCoR/SMRT type corepressors establish equilibrium in their interaction with the GRalpha to respectively facilitate or block its transcriptional activity . Accumulation of coactivators and corepressors on the promoter-bound GRalpha is dependent on the kind of ligands bound to the GRalpha: agonist glucocorticoids attract the coactivator complexes to the promoter-bound GRalpha, while antagonists, like RU 486, accumulate the corepressor complexes .
In addition to transactivation of the glucocorticoid-responsive genes explained above, GRalpha modulates other signal transduction cascades through mutual protein-protein interactions with specific transcription factors, by influencing their ability to stimulate or inhibit the transcription rates of their respective target genes (Figure 3). This activity may be more important than the GRE-mediated one, granted that mice harboring a mutant GRalpha, which is active in terms of protein-protein interactions but inactive in terms of transactivation via DNA GREs, survive and procreate. In contrast, mice with a deletion of the entire GR gene die immediately after birth from severe respiratory distress syndrome [37, 38].
The protein-protein interactions of GRalpha with other transcription factors may take place on promoters that do not contain GREs, as well as on promoters that have both GRE(s) and responsive element(s) of transcription factors that interact with GRalpha . Suppression of transactivation of other transcription factors through protein-protein interactions may be particularly important in the suppression of immune function and inflammation by glucocorticoids [37, 40]. A substantial part of the effects of glucocorticoids on the immune system may be explained by the interaction between GRalpha with the nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and probably the signal transducers and activators of transcription (STATs) [41-44]. In addition to these transcription factors, GRalpha interacts physically with many other transcription factors including the cAMP response element-binding protein (CREB), transforming growth factor (TGF) beta-downstream Smad6, the CAAT/Enhancer-binding proteins (C/EBPs), the chicken ovalbumin promoter-upstream transcription factor II (COUP-TFII), hepatocyte nuclear factor (HNF)-6, Nur77, p53, GATA-1, Oct-1 and -2, nuclear factor-1 and Sp-1. Through these transcription factors, GRalpha develops specific biologic effects, such as those influencing intermediary metabolism, adipocyte differentiation and regulation of the CRH expression in the hypothalamus [1, 45-50]. Importantly, these GRalpha-interacting transcriptional factors also influence the transcriptional activity of the GRalpha on glucocorticoid-responsive genes, indicating that the effect of their protein-protein interaction is mutual.
Epigenetic modulation of GRalpha transcriptional activity
In addition to co-regulators and other transcription factors that modulate GRalpha-induced transcriptional activity, several distinct signaling pathways regulate the transcriptional activity of the GRalpha via post-translational modifications of the receptor protein . These include methylation, acetylation, nitrosylation, sumoylation, ubiquitination and phosphorylation, the last of which has been studied the most. For example, yeast cyclin-dependent kinase p34CDC28 phosphorylates rat GRalpha at serines 224 and 232, which are orthologous to serines 203 and 211 of the human GRalpha, with the resultant phosphorylation enhancing rat GRalpha transcriptional activity in the yeast . These residues are also phosphorylated after binding of the GRalpha with agonists or antagonists and the phosphorylated receptor shows reduced translocation into the nucleus and/or altered subcellular localization in mammalian cells [52, 53]. The p38 mitogen-activated protein kinase (MAPK) phosphorylates serine 211 of the human GRalpha, enhances its transcriptional activity and mediates GR-dependent apoptosis . p38 MAPK and the c-Jun N-terminal kinase (JNK) also phosphorylate serine 226 of the human GRalpha and suppress its transcriptional activity by enhancing nuclear export of the receptor . We recently found that adenosine 5’ monophosphate-activated protein kinase (AMPK), a central regulator of energy homeostasis that plays a major role in appetite-modulation and energy expenditure, indirectly phosphorylates human GRalpha at serine 211 and enhances its transcriptional activity through phosphorylation/activation of p38 MAPK . The central nervous system (CNS)-specific cyclin-dependent kinase 5 (CDK5) also phosphorylates the human GRalpha at multiple serines including those at 203 and 211, and modulates GR-induced transcriptional activity by physically interacting with the receptor through its activator component p35, and by changing accumulation of transcriptional cofactors on GRE-bound GRalpha . Indeed, the majority of phosphorylated sites are located in the AF-1 domain of the NTD (Figure 2), thus phosphorylation of some or all of them modulates GRalpha-induced transcriptional activity mainly through alteration of co-regulator attraction to the promoter region of glucocorticoid-responsive genes, possibly by changing their affinity to the AF-1 domain of GR . CDK5 and p35 are expressed mainly in neuronal cells and play important roles in embryonic brain development. Aberrant activation of CDK5 in the central nervous system also plays a significant role in the pathogenesis of neurodegenerative disorders, such as Alzheimer’s disease and amyotrophic lateral sclerosis .
Acetylation: Implication to modulation of glucocorticoid action under circadian fluctuation of circulating glucocorticoids
The human GRalpha is acetylated at several lysine residues under various physiologic and pathologic conditions. For example, the human GRalpha is acetylated at lysines located at amino acid positions 494 and 495 of the hinge region in response to glucocorticoids, and acetylation of these amino acids reduces the transrepressive action of the receptor to NF-kappaB-induced transcriptional activity . Interestingly, HDAC2 deacetylates these lysine residues and restores the repressive effect of glucocorticoids on the NF-kappaB signaling pathway . We recently found that the circadian rhythm transcription factor Clock acetylates the human GRalpha at a multiple lysine cluster located in its hinge region, and represses GR-induced transcriptional activity of several glucocorticoid-responsive genes . Clock, the “circadian locomotor output cycle kaput”, and its heterodimer partner “brain-muscle-arnt-like protein 1” (Bmal1) belong to the basic helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) superfamily of transcription factors [59, 60], and plays an essential role in the formation of the circadian oscillation rhythm of both hypothalamic SCN-located master CLOCK and peripheral tissue-harboring slave CLOCKs that function as internal circadian time keepers . Clock physically interacts with the LBD of the GR through its nuclear receptor-interacting domain and acetylates lysine residues located at amino acid positions 480, 492, 494 and 495 of the human GRalpha in its hinge region  (Figure 2). Acetylation of GRalpha further suppresses binding of this receptor to GREs, underlining the mechanism of its repressive effect . It is likely that acetylation-mediated repression of GRalpha transcriptional activity by Clock functions as a local counter regulatory mechanism to diurnally oscillating circulating glucocorticoids, which is created by the strong influence of the central master CLOCK located in hypothalamic SCN .
The splicing variant GRbeta isoform
The GRbeta isoform, which is expressed from the GR gene through alternative use of its specific exon 9beta, is expressed ubiquitously in most tissues similar to the classic human GRalpha. This isoform was identified in both the zebrafish and humans, and was recently reported in mice [11, 62, 63]. The human (h) GRbeta contains 742 amino acids and shares the first 727 amino acids from the N-terminus with hGRalpha [11, 18] (Figure 1). hGRbeta encodes an additional 15 nonhomologous amino acids in the C-terminus, while hGRalpha possesses an additional 50 amino acids forming a 777 amino acid protein [11, 18] (Figure 1). Therefore, hGRbeta shares the same NTD and DBD with hGRalpha, but has a unique “LBD”. Since the divergence point (amino acid 727) is located at the C-terminal end of helix 10 in the hGRalpha LBD, the hGRbeta “LBD” does not have the helices 11 and 12 of the hGRalpha. As these helices are important for forming the ligand-binding pocket and for the creation of the AF-2 surface upon ligand binding , GRbeta cannot form an active ligand-binding pocket, does not bind glucocorticoids, and so, does not directly regulate GRE-containing, glucocorticoid-responsive gene promoters. In the absence of the hGRbeta “LBD”, the truncated hGR consisting of NTD and DBD is transcriptionally active on GRE-containing promoters , thus the hGRbeta “LBD” somehow attenuates the transcriptional activity of the other subdomains of the molecule on GRE-driven promoters. Recently, the human GRbeta was shown to possess the intrinsic transcriptional activity independent to its dominant negative effect on the GRalpha-induced transcriptional activity, while physiologic role(s) of this activity remain(s) to be examined [65-67]. Inside the cells, hGRbeta can localize both in the cytoplasm and in the nucleus [68, 69].
Similar to the human GR gene, the zebrafish (z) GR gene consists of 9 exons and produces the zGRalpha and zGRbeta proteins, which contain 746 and 737 amino acids, respectively  (Figure 4).
Figure 4. Genomic and complementary DNA and protein isoforms of the zebrafish GR The zebrafish (z) GR gene consists of 9 exons. The zGR gene expresses zGRa and zGRb splicing variants through intron retention . C-terminal gray colored and shaded domains in zGRalpha and zGRbeta show their specific portions. They are respectively encoded by exon 9 and the 3’ portion of exon 8, which are also shown in the same labeling. (From Reference 65). Abbreviations: DBD: DNA-binding domain; GR: glucocorticoid receptor; LBD: Ligand-binding domain; NTD: N-terminal domain; UTR: untranslated region.
zGRalpha and zGRbeta share the N-terminal 697 amino acids, whereas they have specific C-terminal portions, which contain 47 and 40 amino acids, respectively. In contrast to hGRalpha and hGRbeta, which are produced through alternative use of specific exon 9alpha and 9beta, zGRalpha and zGRbeta are formed as a result of intron retention . zGRalpha and zGRbeta use exon 1 to exon 8 for their common N-terminal 697 amino acids. zGRalpha uses exon 9 for its specific C-terminal portion, while zGRbeta continuously employs the rest of exon 8 and uses a stop codon located at the 3’ portion of this exon to express its specific C-terminal peptide . Protein alignment comparison of hGRbeta and zGRbeta indicated that these two molecules employ exactly the same divergence point, while their beta isoform-specific C-terminal peptides show little sequence homology . These pieces of molecular information indicate that hGRbeta and zGRbeta evolved independently. Mouse (m) GRbeta is also produced in the same fashion as zGRbeta, indicating that intron retention appears to be a general mechanism for expressing this receptor isoform in organisms, while splicing-mediated expression employed by the human GRbeta is rather unique . Nevertheless, zebrafish and mouse GRbeta demonstrated the same functional properties as those of the hGRbeta, namely, the inability to bind glucocorticoids, a dominant negative activity on zGRalpha- and mGRalpha-induced transactivation a of GRE-drive promoters, and strikingly similar tissue distribution as hGRbeta [62, 70]. Thus, hGRbeta, mGRbeta and zGRbeta were produced through convergent evolution, most likely developed through strong requirement of this type of GR isoform in a physiologic situation.
The presence of nonligand-binding C-terminal variants is not unique to the GR. Similar to the human, mouse and zebrafish GR, several other human steroid and nuclear receptors, e.g. estrogen receptor beta (ERbeta), thyroid hormone receptor alpha (TRalpha), vitamin D receptor, constitutive androstane receptor (CAR), dosage-sensitive sex reversal-1 (DAX-1), nuclear receptor related-2 (NURR-2), neuron-derived orphan receptor-2 (NOR-2), peroxisome proliferators-activated receptor alpha (PPARalpha), and PPARgamma, all have C-terminally truncated receptor isoforms that are defective in binding to cognate ligands and have dominant negative activity on their corresponding classic receptors [71-80]. This suggests that evolution has allowed the development and retention of such alternative nuclear receptors, probably because they play useful biologic roles.
The GR transduces diverse actions of glucocorticoids found in most organs and tissues, by strongly modulating transcriptional activity of numerous glucocorticoid-responsive genes. To support such extensive activities of glucocorticoids, the GR proteins are differentially expressed in a tissue-specific fashion through alternative use of 5 distinct promoters, terminal exon 9alpha/beta and 8 translation initiation sites, and have multiple domains with specific functional motifs in their single polypeptide chain that spans ~800 amino acids. These include 3 sub-domains, NTD, DBD and LBD, which contain the structures for ligand- or DNA-binding, nuclear localization, transactivation and transrepression, and the portions for interacting with other transcription factors. Further, other cellular signaling cascades regulate GR activities through chemical modifications including phosphorylation and acetylation. Importantly, these numerous different activities of GR are regulated by binding of the receptor to a glucocorticoid ligand, indicating that GR is a highly sophisticated intracellular molecular switch, possibly developed under the strong influence of long lasting evolutional pressure and biological needs.
Literary work of this article was funded by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD.
T Kino – Unit on Molecular Hormone Action, Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1109, USA
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