Glucocorticoid Receptor (GR)
The gene NR3C1 encoding the glucocorticoid receptor (GR) is located on chromosome 5q31.3 in humans, chromosome 18 in rats and mice, and chromosome 13 in chickens (Flaherty et al. 2012). It counts 2201 bp and contains 15 exons. The five prime untranslated region consists of exon 1 while the protein-encoding region involves exons 2–9. Homologs are conserved in chimpanzees, dogs, rats, zebra fish, and frogs. The structural organization of the GR arises from exons 2 to 9 and includes a DNA-binding domain and hinge region between the N and C termini. Exon 2 codes the N-terminal domain. Exons 3 and 4 encode the DNA-binding domain consisting of two zinc fingers. The GR consists of 777 amino acids and is expressed throughout the body. Subcellular locations include the cytoplasm, mitochondrion, cell nucleus, and plasma membrane. It is presently unknown if different structural forms of the GR are associated with the cytoplasm and membrane.
The name glucocorticoids derive from original descriptions of GR function in the regulation of gluconeogenesis, however the diverse functions of the GR and now beginning to be understood. GRs bind the glucocorticoid hormones cortisol (humans) and corticosterone (rodents) and regulate genes facilitating processes such as energy metabolism, immune responses, growth and development, and brain and body responses to stress and challenge.
Glucocorticoids bind to the GR to regulate gene transcription, this regulation can result in either gene translation or transrepression (Prager and Johnson 2009). In addition, GR may also act as fast-acting membrane-associated receptors regulating cell structure and function (Prager and Johnson 2009; de Kloet 2014). Disorders potentially associated with mutations in the gene or dysregulation of the GR function include glucocorticoid resistance, Cushing’s syndrome lymphosarcoma, major depressive disorder, posttraumatic stress disorder, and other diverse disorders (Kadmiel and Cidlowski 2013).
Structure of GR
The GR protein has domains arising from exons 2 to 9. Exon 2 codes for the N-terminus, containing the main transcriptional domain. The central region of the protein which consists of two zinc fingers involved in DNA binding and homodimerization (see below under structural studies) are encoded by exons 3 and 4. The DNA binding domain is coded with the composition “zinc subdomain-helix-8-strand”. The two helixes are perpendicular to each other with the hydrophobic side chains forming a protein core. The zinc sites are located at an equidistance from the outside of the core. The first subdomain is folded onto the core and connects with the two helices and the C-terminal. The second protrudes out from the protein core, forms a loop, an α-strand and a short α-helix (Haerd and Gustafsson 1993). The C terminus includes the domains required for transcription and ligand binding.
The glucocorticoid receptor in humans has two splice variants labelled GRα and GRβ. The two isoforms are structured identically from amino acid 1 to 727 and then deviate. The GRα functions as a transcription factor, but GRβ does not bind glucocorticoid and lacks transcriptional functionality. It has been implicated in asthma-related glucocorticoid resistance due to its dominant-negative inhibition of GRα.
Both GR and the mineralocorticoid receptor (MR) have similar mechanisms of action and therefore similar functional architecture. The same receptor domains are responsible for ligand binding interactions with a variety of heat shock proteins, translocation to the nucleus, DNA binding, and other transcriptional regulatory protein interactions (Haerd and Gustafsson 1993). The DNA-binding domain is a highly conserved region of the GR, abundant in lysine, arginine, and cysteine. The 15 base pair glucocorticoid response element (GRE) core sequence (GGTACANNNTGTTCT) contains two partially palindromic hexamer sequences with three intervening nucleotides. This allows a recombinant DNA-binding domain molecule to associate with one half-site of the GRE while a second one binds cooperatively to the adjacent half-site. This action strongly depends on the three nucleotides of the intervening sequence. A section of five amino acid residues has been found to be essential for dimerization and the binding of GR to GRE (Haerd and Gustafsson 1993).
Function of GR
Influential early work from de Kloet and colleagues identified differences in receptor binding affinity between MR and GR (for review see Prager and Johnson (2009) and de Kloet (2014)). This finding leads to the important concept that at resting levels of adrenal corticosterone release, corticosterone bounds predominately to MR, while at periods to elevate corticosterone release including as a result of stress, corticosterone also bound to the lower affinity GR. Within the brain, GRs are found in dense concentrations in neurons of the hippocampus, amygdala, and the prefrontal cortex. Its abundant expression throughout the limbic system suggests an important role in stress and defense reactions (Wolf et al. 2016)
During times of stress, GR is recruited. Stress triggers the activation of the hypothalamic–pituitary–adrenal (HPA) axis. Corticotrophin-releasing hormone is secreted from the anterior hypothalamus stimulating the pituitary gland to release adrenocorticotropic hormone (ACTH) into the blood stream. ACTH stimulates the adrenal glands to release corticosteroids (cortisol in humans, corticosterone in rodents; CORT) into the blood stream (Reul and Kloet 1985). Once CORT enters the brain via the blood stream the elevated levels act on the HPA-axis to inhibit the release of more corticosteroids, reducing the initial stress reaction (transrepression). GR returns target cells back to baseline after an initial stress reaction and enhances recovery by increasing energy metabolism. Stress-related pathology may result from dysregulation of this CORT/HPA-axis interaction (for detailed review see Millan et al. (2012)). Depending on the amount of CORT exposure, the slow genomic action whereby CORT binding facilitates gene transcription may take up to 30 minutes from receptor activation and can last for days (see Joels et al. 2012).
Pharmacology of GR
The endogenous ligands of GR include aldosterone, corticosterone, cortisol, and deoxycortisone. Agonists include clobetasol, therapeutically used for skin disorders such as psoriasis; fluticasone propionate – asthma medication; desoximetasone – a metabolite; and methylprednisolone commonly used to treat immunodeficiency syndromes. Mifepristone is a GR antagonist marketed as RU-486; and a selective antagonist is onapristone, used to treat breast cancer and implicated as a possible treatment for hormone-dependent tumors (Vilasco et al. 2011). Since glucocorticoid therapy was instituted over 60 years ago it has become the pillar of anti-inflammatory modulators (Kadmiel and Cidlowski 2013). Synthetic glucocorticoids are now prescribed for conditions such as asthma and chronic obstructive pulmonary disease. Dexamethasone is used in psychiatry to test for functioning of the HPA axis and its feedback mechanisms (dexamethasone suppression test). Dexamethasone, a synthetic CORT, acts centrally to suppress the ongoing HPA activity and reduces endogenous CORT levels.
Glucocorticoids can affect neuronal activity within seconds of exposure to cells. When genomic regulation was found to be incompatible with rapid effects, studies suggested these actions were mediated by the activation of membrane-associated receptors (see Prager and Johnson (2009) and Wolf et al. (2016) for review). Reports to date of these actions involve the limbic system and brainstem, areas involving stress, learning, emotional memory, reproductive behavior, and movement. Studies involving the effects of glucocorticoids on learning, memory, and stress show both inhibitory and excitatory processes.
In a classic study, the reproductive behavior of male rough-skinned newts was shown to be suppressed by rapid corticosterone action that inhibited neural circuits of the brain stem (Orchinik et al. 1991). In 1996, Sandi et al. reported systemic glucocorticoid increased locomotor activity of rats in a novel environment, finding the effect was nitric oxide dependent (Sandi et al. 1996). Electrophysiological studies by Joels et al. (2012) and Tasker and Herman (2011) have identified fast-acting GR responses.
Direct anatomical localization of GR at neuron membranes including synapses was shown by Johnson et al. (2005) who identified GR receptors localized in the postsynaptic membranes of the lateral amygdala (Johnson et al. 2005; Prager et al. 2010). They established these receptors in the presynaptic terminals, the postsynaptic density, dendrites, dendrites spines, and soma of neurons. Emerging data suggest that these membrane GRs may rapidly regulate neuron dendrite spine structure (for review see Russo et al. (2016)). Synaptic GR may play a role in the modulation of synaptic plasticity related to memory (Prager and Johnson 2009; Wolf et al. 2016). Evidence for mGRs in other cells have also been demonstrated. For example, Gametchu and coworkers identified human mGR in leukemic cells membranes using peptide antibody labelling (Gametchu et al. 1993).
Cortisol (or corticosterone) activates the glucocorticoid receptor which functions both as a transcription factor itself, a regulator of other transcription factors, and also as a fast-acting membrane receptor. The genomic GR is located in the cytoplasm and it is transported to the cell nucleus when ligand bound, where it plays a role in transcription processes. Its functions include regulation of cell proliferation, tissue differentiation, inflammatory processes, and neuronal plasticity. GR gene mutations are linked with Cushing’s disease and glucocorticoid resistance and other disorders. As GRs are located in almost every tissue of the body and act in both genomic and nongenomic capacities, a comprehensive understanding of their mechanisms of action will ensure their role as therapeutic targets.
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