Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
Address all correspondence and requests for reprints to: Dr. John Cidlowski, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T. W. Alexander Drive, NC MDF307, Research Triangle Park, North Carolina 27709-2233.
ABSTRACT
The ability of natural and synthetic glucocorticoids to elicit numerous and diverse physiological responses is remarkable. How the product of a single gene can participate in such a myriad of cell- and tissue-specific pathways has remained largely unknown. The last several years have seen increased description of glucocorticoid receptor (GR) protein isoforms. Here we review the current state of knowledge regarding naturally occurring GR isoforms and discuss how this array of receptor species generates the diversity associated with the glucocorticoid response. We propose that the multiplicity of receptor forms have unique tissue- specific actions on the downstream biology providing a mechanism to create GR signaling networks.
GLUCOCORTICOIDS ARE A vital class of steroid hormones that mediate profound and diverse physiological effects in vertebrates from fish to man. Produced and secreted by the adrenal cortex, levels of circulating glucocorticoids are regulated by ACTH largely under the control of the hypothalmic-pituitary-adrenal axis (HPA). Although named for their role in glucose homeostasis, glucocorticoids are also eminently important throughout physiology, with regulatory roles in development, metabolism, neurobiology, programmed cell death, and many other functions. In addition to these far-reaching physiological roles, corticosteroids are among the most widely prescribed class of drugs in the world. The pharmacological benefits of glucocorticoids are primarily antiinflammatory and immunosuppressive. They also find widespread use in chemotherapeutic regimes in patients with leukemias, lymphomas, and other cancers due to their critical role in the induction of apoptosis.
The physiological response and sensitivity to glucocorticoids varies among species, individuals, tissues, cell types, and even during the cell cycle (1, 2). Additionally, several pathological conditions lead to, or are a result, of glucocorticoid resistance or hypersensitivity (3). The molecular basis of glucocorticoid resistance varies widely and is not completely understood. Generalized inherited glucocorticoid resistance is classified by a failure of synthetic glucocorticoids to elicit a normal physiological response. Although abnormalities in glucocorticoid responsiveness can be attributed in many cases to specific inherited mutations, epigenetic factors are also involved, because in many diseases long-term glucocorticoid treatment can result in glucocorticoid resistance (4). Several reviews on glucocorticoid resistance discuss the complexity of the syndrome and investigations of its origins (5, 6, 7).
Natural glucocorticoids and their synthetic derivatives work through the glucocorticoid receptor (GR). The GR is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. Like all members of this superfamily, the dimeric GR mediates transactivation of target genes by binding sequence-specific recognition elements (glucocorticoid response elements) in their promoter region. The homologous structural organization of the nuclear receptors is well known. A centrally located DNA binding domain (6070 amino acids) is flanked on the carboxy terminal by an approximately 250-amino acid ligand binding domain (LBD), and by a variably sized, nonhomologous amino-terminal domain. Biophysical and structural studies over the last half decade, most notably with the estrogen receptor, have led to an understanding of steroid hormone action and antagonism at the atomic scale (8, 9, 10). Structure models predict both subtle and drastic changes in ligand-induced receptor conformations can direct or impede specific protein interactions responsible for downstream biological effects.
The ligand-activated GR also interacts with a multitude of transcription factors such as c-jun (11), nuclear factor-B (NF-
B) (12), the TFIID complex (13), STAT5 (14), as well as a host of coactivators (15) where they are known to act on the function of these signaling molecules. In addition, the GR interacts with numerous cytosolic proteins including chaperones, kinases, phosphatases, nuclear shuttling proteins, and the proteasome. Clearly, nuclear hormone receptors like the GR are much more than simple molecular switches turning on and off genes in response to hormonal stimuli. They are part of a dynamic and intricate network, linking and integrating signaling pathways and regulatory processes in cells, tissues, and whole organisms. Even before the genomic explosion led to the new era of proteomics, it was apparent that the level of complexity in hormone action arose from the diversity of receptor isoforms themselves. Whether a product of distinct genes, splice variants of a primary message, or posttranslational alteration, various steroid receptor isoforms have been identified that appear to have a role in normal homeostasis as well as disease pathology. We hypothesize that nature, lacking natural selective hormone modulators, favored the evolution of different receptor isoforms that provide more finely tuned stimuli or cell or tissue specific signaling networks. Thus, the paradox of how one receptor can elicit such enormous variety of cell and tissue specific responses is at least partially explained by the diversity of receptor isoforms. Here we summarize recent discoveries of how cells produce multiple forms of GRs from a single gene that in turn provides a potential mechanism to increase the diversity of GR signaling.
THE GRONE GENE
To date there is only one known GR gene. Nevertheless, cross-specificity, where one ligand can bind more than one type of receptor, is both physiologically important and experimentally confounding. For example, the mineralocorticoid receptor displays high affinity binding for endogenous glucocorticoids yet is a distinct gene product with an entirely different physiological role. Another example is the high affinity glucocorticoid antagonist, RU486, which is similarly efficacious on the progesterone receptor. Additionally, the xenobiotic binding orphan receptor, pregnane X receptor (PXR), binds the synthetic glucocorticoid dexamethasone and RU486 as well as other steroid hormone metabolites. Nevertheless, only a single GR gene has been identified in every species examined to date. We define the human GR (hGR) as the ubiquitous product(s) of the gene located on chromosome 5 q11q13 (16).
THE GRMULTIPLE PROMOTERS
The genomic organization of the human GR is shown in Fig. 1. The gene is comprised of nine exons, with protein coding regions beginning in exon 2. The hGR promoter region contains multiple GC boxes and lacks both a TATA and CCAAT boxes. Footprint and functional analysis of the promoter region (from -2300 bp) reveals up to 15 unique binding sites for transcription factors including Sp1, AP-1, YY 1, NF-
B, and even the GR itself (17, 18, 19). Thus, like most important regulatory proteins, GR expression, although considered ubiquitous, is regulated by a variety of transcription factors binding to their response elements within its promoter. This variety clearly provides the potential to translate into both cell type- and tissue-specific regulation of this otherwise widely expressed gene. How GR gene expression ultimately relates to the complex physiological outputs such as immune regulation and the stress response remains largely unknown.
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THE GRMULTIPLE mRNAs
Alternative splicing also gives rise to numerous GR isoforms. When cloned in 1985, two GR gene splicing products were identified and termed hGR and hGRß. Several other splicing variants have since been described. Immediately, hGR
was recognized as the classical GR composed of a single polypeptide chain of 777 amino acids, located in the cells cytoplasm without hormone and is the primary mediator of glucocorticoid action (16). A schematic representation of the primary GR gene and the alternative splicing event that produces hGR
and other variants is shown in Fig. 1B
. As described earlier, the sequence, structural organization, and mechanism of action of hGR
is highly homologous with other steroid receptor superfamily members and most of our understanding of GR physiology comes from the study of this receptor isoform.
Also shown in Fig. 1B is hGRß, which is generated through alternative splicing of the ninth and final exon, resulting in a protein sequence divergent only at the carboxy terminal. Because of its failure to bind hormone and activate gene transcription, hGRß was originally designated as a minor gene product and perhaps even a cloning artifact. It took another 10 yr for hGRß to be recognized as an endogenous hGR gene product and its function and expression further characterized (26, 27). hGRß is a single chain polypeptide with 742 amino acids. Although identical through amino acid 727, the 15 carboxy-terminal amino acids in hGRß are unique and replace the carboxy-terminal 50 amino acids in hGR
. The hGRß isoform is transcriptionally inactive and is unable to bind agonists or antagonists in all systems tested to date. Interestingly, despite binding to hsp90, hGRß is located in the nucleus in the absence of ligand. However, it is the dominant negative effect of hGRß on hGR
-mediated transactivation that is most intriguing, and potentially physiologically important. Recent studies investigating the mechanism of hGRß dominant negative activity suggest it is the formation of hGR
/ß heterodimers, incapable of binding coactivators as a ligand activated hGR
homodimer would, that causes the attenuated hGR
response (Ref. 28 ; and Yudt, M. R., C. M. Jewell, and J. A. Cidlowski, unpublished results).
Despite the biochemical and genetic dissection of hGRß function and expression, the physiological relevance and importance of hGRß remains controversial. First of all, there are often large differences in expression of endogenous hGR relative to hGRß. Cell-based transient transfection studies have suggested the need for 5-fold or more excess of hGRß for significant dominant negative effect on hGR
. Whereas data from whole organs suggests these proportions are not met, individual cells show significantly higher hGRß compared with neighboring cells within the same tissues or organs (29). This cell type-specific expression of hGRß combined with its constitutive nuclear localization indicate that nuclear levels of hGRß may be in high enough excess to mediate a dominant negative effect in certain cells. In addition, the lack of ligand-induced down-regulation of hGRß may further enhance the ratio of hGRß to hGR
in cells exposed to glucocorticoids (28). Nevertheless, several groups have been unable to replicate the dominant negative activity of hGRß, although the experimental conditions and cell types differ in many of the studies (30, 31). A second criticism regarding the significance of hGRß is the lack of a mouse homolog (32). Although the structural organization of the GR gene appears to be conserved between human and mouse, the alternative splicing event does not occur in the mouse. However, the presence of GRß mRNA has recently been reported in the rat (33). We speculate that the absence of GRß in mouse may be partially responsible for differences in glucocorticoid physiology between mice and men (34). For example, mouse lymphocytes are much more sensitive to glucocorticoid induced apoptosis than are human lymphocytes.
Additional data suggesting a pathophysiological role of hGRß has been reported recently. For example, several inflammatory conditions are associated with an elevated expression of hGRß in particular cells (35, 36, 37, 38). Additionally, the discovery of a polymorphism in the hGRß 3' untranslated region of rheumatoid arthritis patients, which stabilizes the mRNA and increases hGRß protein levels, suggests there is an important pathological role for this GR isoform (39). Recently, proinflammatory cytokines such as IL-8, TNF, and IL-1 were shown to increase hGRß expression (18, 38). Changing the ratio of hGRß to hGR
, where hGRß predominates, may promote glucocorticoid resistance (18). These pathological conditions, as well as the observation of highly variable cell and tissue expression profiles, suggest an important role for hGRß in human glucocorticoid physiology.
A third GR mRNA splice variant, termed GR-P, was found in tumor cells from a glucocorticoid resistant myeloma patient (40). The GR-P isoform is 676 amino acids and is encoded by exons 27 and part of intron 7. This truncated GR is missing a large portion of the LBD and, like hGRß, encodes a unique carboxy- terminal tail because of the inclusion of the intron fragment in the mRNA. In contrast to hGRß, GR-P has been found to up-regulate GR-mediated gene expression in some but not all cell lines. Interestingly, the levels of this isoform can represent from 1050% of the total GR in certain myeloma and other GR-resistant hematological malignancies. Its role in these cells, however, has not been clearly established (41). A similar GR splicing variant was also found in corticotroph adenomas (42) where alternative splicing retained three bases from the intron separating exons 3 and 4. The resulting protein, termed GR
has a single amino acid (arginine) insertion in the DNA binding domain. The functional consequences of this insertion are unknown.
THE GRTRANSLATIONAL ISOFORMS
Various GR isoforms are also produced by alternative translation initiation. Receptors derived from in vivo sources such as rat liver and various cell lines, as well as GR produced in vitro, fractionate into several bands upon electrophoresis. The major protein product, with an apparent molecular mass of 94 kDa, represents translation from the first initiator AUG codon, termed GR-A. However, this start codon lies within a weak Kozak translation initiation consensus sequence, which appears to result in leaky ribosomal scanning and translation initiation from downstream AUG codon(s) (43). The next downstream start codon (met 27 in human, or met 28 in rodents) results in production of a 91-kDa GR species, termed GR-B (Fig. 1C). Recent characterization of these two GR isoforms shows significant biochemical differences in gene trans-activation potential. The shorter GR-B species is nearly twice as efficient in glucocorticoid response element-mediated transactivation as the longer GR-A. This alternative AUG usage was first postulated after cloning of the GR gene (44), and is consistent with observed GR proteins from multiple sources (45). The biological significance and potential regulation of alternative initiation remains unclear, although similar observations have been made for other nuclear receptors (46). However, the distinct biochemical activities for these isoforms suggests they may play a role in the diversity of glucocorticoid response.
THE GRPOSTTRANSLATIONALLY MODIFIED ISOFORMS
Many nuclear receptors, including the GR, are phosphoproteins (47) and phosphorylation of steroid receptors has been implicated as a potential mechanism for ligand-independent activity via cross-talk with other signal transduction pathways. However, the GR appears to be refractory to any such activation in the absence of ligand (48). Nevertheless, the GR is thought to be a substrate for several kinases and phosphatases and has been shown to be poly-phosphorylated on serine and threonine residues, in the amino-terminal portion of the protein (Fig. 1D) (49). Although the precise role of each specific phosphorylation event remains unclear, mutation of multiple phosphorylation sites has a profound impact on the receptor stability, protein half-life and signaling in a promoter-specific context (50). Both constitutive, or basal phosphorylation, as well as ligand-induced phosphorylation of the GR occur. In addition, there appears to be a strong cell-cycle dependency of GR phosphorylation (51), as well as a potential role of phosphorylation in nuclear-cytoplasmic shuttling (52).
A recent study has shown that the mouse GR is a substrate for posttranslational ubiquitination, targeting the receptor for proteosomal degradation. In this case, a lysine residue found in a PEST degradation motif in the amino-terminal domain was mutated to alanine. This mutation blocks ligand-dependent GR down- regulation and markedly enhances GR signaling (53). Another related posttranslational modification of the GR is sumoylation. The GR has recently been shown to be sumoylated in vitro and this modification may modulate transcriptional regulation (54).
THE GRPOLYMORPHISMS and MUTANTS
The study of polymorphisms of the GR gene presents a clear example of how small changes in receptor expression levels, functional, and/or protein interactions can result in diverse clinical manifestations. Mutant forms of the GR were characterized long before the receptor itself was cloned (55). Table 1 summarizes some well studied GR polymorphisms. Most of the mutations were found associated with glucocorticoid resistance syndromes (56, 57). However, not all polymorphisms are associated with glucocorticoid resistance. For example, the N363S mutant results in glucocorticoid hypersensitivity and may contribute to male obesity (58). In addition, long-term treatment with glucocorticoids results in several types of resistance that appear to reflect selection of cells with mutant or absent receptors (59, 60).
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The full-length GR has been cloned and sequenced from over a dozen unique species including human, several nonhuman primates, rat, mouse, guinea pig, African frog, rainbow trout, flounder, and others (see the GR Resource: http://nrr.georgetown.edu/GRR/GRR.html and references therein). In addition, partial sequences exist for several species including chicken, sheep, and pig. The homology among mammalian species is relatively high, with nonmammalian species exhibiting significant heterogeneity in both sequence and length (61, 62). Just as among classes of nuclear receptor superfamily members, greater sequence homology between species lies within the GR DNA and ligand binding domains. However, rather profound differences in glucocorticoid sensitivity are observed among species. For example, the guinea pig is a well known example of a cortico-resistant species (63) as are several New World primates, such as the squirrel monkey and the marmoset (64). However, molecular analysis of these receptors suggests the underlying mechanism of glucocorticoid resistance in these species are distinct. For example, within the amino-terminal portion of the guinea pig GR LBD are a cluster of nonconserved changes from human and other species that appear to cause glucocorticoid resistance in that species (65). In contrast, New World primates GR LBD is highly homologous to that of both human and Old World primates. Glucocorticoid resistance in New World primates has been linked to a cytosolic factor reducing the sensitivity to circulating glucocorticoids in these species (66).
THE GRNONGENOME-ACTIVE ISOFORMS
A notable paradox of glucocorticoid biology is the rapid release of the hormone during a stress response (seconds) and the perceived delay (hours) in eliciting a genomic response consistent with the classical action of steroid and other nuclear hormone receptors. In the past several years, there has been an increased interest in nongenomic and/or membrane-associated actions of steroids and their receptors. This is the subject of several reviews (67, 68, 69). Distinct GR forms could presumably mediate the rapid actions of glucocorticoids. One possibility is the presence of a membrane bound or associated form of the GR (23, 70). This receptor may either be a unique gene product, as proposed for the progesterone receptor (71), or a modified version of the classical GR capable of integrating into the plasma membrane. Another possibility is that a cytosolic subset of GR mediates the rapid actions of glucocorticoids by participating in signal transduction pathways usually associated with membrane receptor signaling events (72). Whether this receptor turns out to be a unique gene product, specific isoform, or merely a subset of the classical receptor that alters cellular phenotype through protein-protein interactions with factors such as NF-B etc., remains to be determined.
CONCLUSIONS
Today in the pharmaceutical industry, small molecule ligands are being developed to interact with nuclear receptors with greater specificity and to promote particular biological functions of the GR without eliciting side effects. We propose that nature has chosen an alternative pathway toward specificity and diversity in GR signaling. The complexity of glucocorticoid biology lies more in the variety of receptors themselves rather than in the ligands to which they bind. Assuming that there are four or perhaps more GR isoforms in a cell, combined with up to eight phosphorylation sites, at least one ubiquitination site, and perhaps several sumoylation sites, the capacity to generate dozens of unique GRs in a single cell presents an enormous potential for signaling diversity. In that way, cells could produce unique biological response to the same hormone. The value of this hypothesis will come if specific isoforms can be linked to the control of a specific gene or sets of genes.
FOOTNOTES
1 Current address: Wyeth Research, P.O. Box 42528, Philadelphia, Pennsylvania 19101.
Abbreviations: GR, Glucocorticoid receptor; hGR, human GR; HPA, hypothalmic-pituitary-adrenal axis; LBD, ligand binding domain; NF-B; nuclear factor-
B.
Received for publication March 14, 2002. Accepted for publication April 25, 2002.
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