Multiple glucocorticoid receptor isoforms and mechanisms of post-translational modification (original) (raw)
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Biochimica et biophysica acta, 2014
GR transcripts display a remarkable heterogeneity in their 5' untranslated regions (5'UTRs). These variable 5'UTRs are encoded by a series of alternative 1st exons, and together with their associated promoters they maintain tissue-specific GR expression levels. In this study we over-expressed GR transcripts containing individual 1st exons, and assessed their effect on RNA stability, 3'-splicing, translation initiation and protein isoform production. We showed that these alternative 5'UTRs influence the predicted mRNA structure and free energy, and were associated with differential levels of functional spliced mRNA. However, the 5'UTR had little influence on the relative levels of the two principal 3' splice transcripts, GR-α and -β. The overall mRNA length, the free energy of the transcript and the translational efficiency directly influenced total GR levels. However, individual N-terminal protein isoform levels appeared to depend upon elements within the...
Molecular Cell, 2005
and rheumatoid arthritis often have altered glucocorticoid sensitivity (Leung et al., 1998). Moreover, how one Molecular Endocrinology Group Laboratory of Signal Transduction hormone acts via one receptor to induce apoptosis in lymphocytes while inhibiting the same process in hepa-Department of Health and Human Services National Institute of Environmental Health Sciences tocytes remains a mystery. Clearly, the molecular basis for these cell-type specific effects of glucocorticoids is National Institutes of Health Research Triangle Park, North Carolina 27709 not well understood. Although differential cofactor recruitment has been proposed to contribute to variations in glucocorticoid sensitivities among different cells, strong evidence supports that a cell's sensitivity Summary is directly determined by the level and molecular nature of the GR therein (Lu and Cidlowski, 2004). Glucocorticoids regulate diverse physiological func-We report here the discovery of translational regulations ranging from mitosis to apoptosis, although tory mechanisms that generate eight N-terminal GR only one glucocorticoid receptor (GR) gene has been isoforms from a single GR mRNA species. Furthermore, discovered. We report here that one single GR mRNA we show that these GR isoforms all have unique tissue species unexpectedly produces at least eight functional distribution patterns and transcriptional regulatory pro-GR N-terminal isoforms via translational mechanisms. files. These data provide a basis for a mechanism in These GR isoforms display diverse cytoplasm-togenerating specificity in GR, and possibly other nuclear nucleus trafficking patterns and distinct transcripreceptor, signaling. tional activities. In human osteosarcoma cells, the transcriptional responses to glucocorticoids closely reflect the identity and abundance of the GR iso-Results forms. In addition, each GR isoform regulates both a common and a unique set of genes in the same cell. Detection of Multiple GR Isoforms Interestingly, the levels of these GR isoforms differ When the human (h)GRα was expressed in COS-1 cells, significantly among tissues. Based on these observawhich lack endogenous GR, multiple proteins of 94, 91, tions, we propose that cell-type specific GR isoforms 82-84, and 53-56 kDa were detected by using a GRgenerate specificity in glucocorticoid control of transelective anti-peptide antibody 57 (Cidlowski et al., scription in different tissues. 1990) in the absence and presence of the cognate hormone (Figure 1A). Therefore, ligand binding and its con-Introduction sequences on receptor turnover are not contributing factors to the expression of multiple GR proteins. The Glucocorticoids are essential for embryo maturation, 94 and 91 kDa bands correspond to the previously dedevelopment, metabolism, and survival (Barnes, 1998; scribed hGRα-A and hGRα-B isoforms, the latter of Tronche et al., 2004). Drugs that mimic the actions of which lacks the N-terminal 27 amino acids (aa) (Yudt these hormones are widely used to treat diseases such and Cidlowski, 2001). The 82-84 and 53-56 kDa proas cancer, inflammation, and autoimmune disorders. teins were previously thought to reflect degradation Glucocorticoids bind and activate the GR, which mediproducts of GR (Hollenberg et al., 1985; Yudt and Cidates the physiological and therapeutic actions of the lowski, 2001). However, when the wild-type (wt) hGRα ligand by directly or indirectly regulating transcription cDNA was expressed in vitro by using the TNT T7-couof target genes. Like other members of the nuclear repled rabbit reticulocyte lysate system (Promega, Madiceptor family, GR is a modular protein comprised of a son, WI), all protein bands observed in vivo were demajor transactivation activity (τ1) domain, a DNA bindtected as well, suggesting that the 82-84 and 53-56 ing domain, and a ligand binding domain that also has kDa proteins are unlikely to be degradation products a minor transactivation domain (τ2). Depending on the (Figure 1B). Similar results were also obtained with cell type and promoter context, GR can either up-or cDNAs for the rat GR and mouse GR (data not shown). downregulate gene expression via coordinated recruit-To elucidate the identities of these GR immunoreactive ing of cofactors and interaction with transcriptional maproteins, three specific anti-GR antibodies were used: chinery. 59, 57 (Cidlowski et al., 1990), and the C terminus-spe-Sensitivity to glucocorticoids varies considerably cific AshGR (Oakley et al., 1999), whose epitopes correamong individuals and, even within the same individual, spond to distinct peptide segments aa 245-259, 346responsiveness to glucocorticoids differs among tis-367, and 755-771 on the hGRα, respectively (Figure sues (Bronnegard et al., 1996). Within a cell, glucocorti-1C). Both antibodies 57 and AShGR detected all procoid responsiveness shifts dynamically during the cell teins, whereas anti-GR 59 detected all, but not the 53cycle (King and Cidlowski, 1998). In addition, patients 56 kDa proteins, suggesting that like hGRα-B, the 82with certain inflammatory conditions such as asthma 84, and 53-56 kDa proteins are likely N-terminal shortened forms of hGR. In addition, we performed experiments to exclude
The human glucocorticoid receptor: One gene, multiple proteins and diverse responses
Steroids, 2005
Glucocorticoids are a vital class of endogenous steroid hormones that regulate essential biological processes including growth, development, metabolism, behavior and apoptosis. Most, if not all, of these actions are thought to be mediated through the glucocorticoid receptor. The exact mechanisms of how one hormone, via one receptor, modulates such diverse biological functions are largely unknown. However, recent studies from our lab and others have suggested that a contribution for the diversity results from multiple isoforms of the glucocorticoid receptor that result from alternative RNA splicing and translation initiation of the glucocorticoid receptor mRNA. Additionally, each isoform is subject to several post-translational modifications, including phosphorylation, ubiquitination and sumoylation, which have been shown to modulate the receptor protein stability and/or function. Together these data provide potentially diverse mechanisms to establish cell type specific regulation of gene expression by a single transcription factor. Here, we summarize the recent advances and processes that generate these receptor isoforms and these post-translational modifications. We speculate that the composition and proportion of individual isoforms expressed in particular cellular contexts account for the diverse effects of glucocorticoid hormones.
Glucocorticoid receptor isoforms generate transcription specificity
Trends in Cell Biology, 2006
Glucocorticoids are necessary for life and are essential in all aspects of health and disease as they regulate processes from mitosis to apoptosis, from metabolism to growth and development. However, responses to glucocorticoids vary among individuals, cells and tissues. Recent evidence indicates that multiple glucocorticoid receptor (GR) isoforms are generated from one single GR gene by alternative splicing and alternative translation initiation. These isoforms all have unique tissue distribution patterns and transcriptional regulatory profiles. Furthermore, each is subject to various post-translational modifications that affect receptor function. Thus, increasing evidence suggests that unique GR isoform compositions within cells could determine the cell-specific response to glucocorticoids. Here, we discuss a new molecular model potentially underlying tissue-specific glucocorticoid resistance and selectivity.
The Origin and Functions of Multiple Human Glucocorticoid Receptor Isoforms
Annals of the New York Academy of Sciences, 2004
Glucocorticoid hormones are necessary for life and are essential in all aspects of human health and disease. The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), which binds glucocorticoid hormones and regulates gene expression, cell signaling, and homeostasis. Decades of research have focused on the mechanisms of action of one isoform of GR, GR␣. However, in recent years, increasing numbers of human GR (hGR) isoforms have been reported. Evidence obtained from this and other laboratories indicates that multiple hGR isoforms are generated from one single hGR gene via mutations and/or polymorphisms, transcript alternative splicing, and alternative translation initiation. Each hGR protein, in turn, is subject to a variety of posttranslational modifications, and the nature and degree of posttranslational modification affect receptor function. We summarize here the processes that generate and modify various hGR isoforms with a focus on those that impact the ability of hGR to regulate target genes. We speculate that unique receptor compositions and relative receptor proportions within a cell determine the specific response to glucocorticoids. Unchecked expression of some isoforms, for example hGR, has been implicated in various diseases.
Hormone Molecular Biology and Clinical Investigation, 2010
Glucocorticoids are necessary for life after birth and regulate numerous homeostatic functions in man, including glucose homeostasis, protein catabolism, skeletal growth, respiratory function, inflammation, development, behavior, and apoptosis. In a clinical setting, they are widely used as anti-inflammatory agents to control both acute and chronic inflammation. Unfortunately, owing to their broad range of physiological actions, patients treated with glucocorticoids for long periods of time experience a variety of serious side effects, including metabolic syndrome, bone loss, and psychiatric disorders including depression, mania, and psychosis. Our understanding of how one hormone or drug regulates all of these diverse processes is limited. Recent studies have shown that multiple glucocorticoid receptor isoforms are produced from one gene via combinations of alternative mRNA splicing and alternative translation initiation. These isoforms possess unique tissue distribution patterns a...
The biology of the glucocorticoid receptor: New signaling mechanisms in health and disease
Journal of Allergy and Clinical Immunology, 2013
Glucocorticoids are primary stress hormones necessary for life that regulate numerous physiological processes in an effort to maintain homeostasis. Synthetic derivatives of these hormones have been mainstays in the clinic for treating inflammatory diseases, autoimmune disorders, and hematological cancers. The physiological and pharmacological actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily of ligand-dependent transcription factors. Ligand-occupied GR induces or represses the transcription of thousands of genes by direct binding to DNA response elements and/or by physically associating with other transcription factors. The traditional view that glucocorticoids act through a single GR protein has changed dramatically with the discovery of a large cohort of receptor isoforms with unique expression, gene-regulatory, and functional profiles. These GR subtypes are derived from a single gene by alternative splicing and alternative translation initiation mechanisms. Post-translational modification of these GR isoforms further expands the diversity of glucocorticoid responses. Here, we discuss the origin and molecular properties of the GR isoforms and their contribution to the specificity and sensitivity of glucocorticoid signaling in healthy and diseased tissues.
The Human Glucocorticoid Receptor Isoform: EXPRESSION, BIOCHEMICAL PROPERTIES, AND PUTATIVE FUNCTION
Carolina Digital Repository (University of North Carolina at Chapel Hill), 1996
Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two receptor isoforms, hGR␣ and hGR, which differ at their carboxyl termini. The hGR␣ isoform conveys endocrine information to target tissues by altering patterns of gene expression in a hormone-dependent fashion. In contrast to hGR␣, very little is known about the hGR splice variant. Using hGR␣and hGR-specific riboprobes on human multiple tissue Northern blots, we show that the hGR message has a widespread tissue distribution. We also prove by reverse transcriptase-polymerase chain reaction that the alternative splicing event underlying the formation of the hGR message occurs in these tissues. Because the hGR protein differs from hGR␣ at the extreme COOH terminus, we investigated several of the biochemical properties of hGR expressed in transfected cells. hGR does not bind the glucocorticoid agonist dexamethasone nor the glucocorticoid antagonist RU38486 in vivo. Moreover, in contrast to hGR␣, hGR is located primarily in the nucleus of transfected cells independent of hormone administration. Finally, in the absence of hGR␣, hGR is transcriptionally inactive on a glucocorticoid-responsive enhancer. However, when both isoforms are expressed in the same cell, hGR inhibits the hormone-induced, hGR␣-mediated stimulation of gene expression. Thus, hGR potentially functions as a dominant negative inhibitor of hGR␣ activity.
Glucocorticoid receptor (GR) β has intrinsic, GRα-independent transcriptional activity
Biochemical and Biophysical Research Communications, 2009
The human glucocorticoid receptor (GR) gene produces C-terminal GRβ and GRα isoforms through alternative use of specific exons 9β and α, respectively. We explored the transcriptional activity of GRβ on endogenous genes by developing HeLa cells stably expressing EGFP-GRβ or EGFP. Microarray analyses revealed that GRβ had intrinsic gene-specific transcriptional activity, regulating mRNA expression of a large number of genes negatively or positively. Majority of GRβ-responsive genes was distinct from those modulated by GRα, while GRβ and GRα mutually modulated each other's transcriptional activity in a subpopulation of genes. We did not observe in HCT116 cells nuclear translocation of GRβ and activation of this receptor by RU 486, a synthetic steroid previously reported to bind GRβ and to induce nuclear translocation. Our results indicate that GRβ has intrinsic, GRα-independent, gene-specific transcriptional activity, in addition to its previously reported dominant negative effect on GRα-induced transactivation of GRE-driven promoters.