br Glucocorticosteroid receptor structure and function The g
Glucocorticosteroid receptor structure and function The gene encoding for the human GR is composed of 9 exons (Oakley and Cidlowski, 2011) and the translated protein is described as having 4 functional regions. The protein contains a highly conserved central DNA-binding domain (DBD) or C-domain, encoded on exon 3 and 4, which contains two zinc-fingers and recognises specific palindromic DNA sequences of the glucocorticoid response element (GRE) upstream of the target genes, and is also a region important for homodimer formation. The hormone binding domain (HBD), or E-domain, and hinge region, or D-domain, are encoded on exons 5–9. The C-terminus HBD is also highly conserved between GRs, specifically the 22 ochratoxin a that interact with the glucocorticoid hormones, to form the hydrophobic ligand binding pocket that characterises all GRs (Bledsoe et al., 2002). The HBD is also the sight of two transactivation functional sites, named activation function 2 (AF2) and τ2 (Hollenberg and Evans, 1988, Kucera et al., 2002). The D-domain, located between the DBD and HBD, is the least conserved region and is involved in protein folding. Additionally, one of two nuclear localisation signals, NL1, spans the DBD/hinge region transition; the other, NL2, is present in the HBD (Bamberger et al., 1996). The N-terminal transactivation domain (NTD) also known as the A/B domain, is encoded on exon 2; it is also not well conserved between the vertebrate GR, but is the site of the ligand-independent AF1 site (Giguere et al., 1986, Oka et al., 2015, Sturm et al., 2011). Classically, the inactive GRs reside in the cytoplasm as part of a large heteromeric complex which includes HSP90 and immunophilins (Heitzer et al., 2007). Following ligand/receptor binding, the GR dissociates and is transferred to the nucleus where it forms homodimers, interacts with GREs and stimulates gene expression. Alternately, the GR-ligand complex may interact with less well defined negative GREs to suppress gene expression. The GRs can also interact with other transcription factors to repress or enhance gene expression (Glass and Rosenfeld, 2000). However, alternate splice variants, different translation initiation sites, post-translation modifications (e.g. phosphorylation) and single nucleotide polymorphisms result in a diverse array of GR proteins (Oakley and Cidlowski, 2011). In recent years, it has become apparent that the plethora of GR isoforms have different functional properties when compared to the wild-type GR, termed GRα, and can influence GRα function or act independently (Oakley and Cidlowski, 2011). For example, in the human GR there is an acceptor splice site between exon 8 and 9 that results in a splice variant termed GRβ. GRα and β share the first 727 amino acids, thereafter the GRα possesses a further 50 amino acids and GRβ a non-homologous additional 15 amino acids. GRβ lacks the C-terminal helix 12 and the AF2 region, and so is unable to bind cortisol and induce transactivation. It does, however, form heterodimers with GRα to act as a dominant-negative inhibitor of GRα gene expression (Bamberger et al., 1995), and has also been shown to directly stimulate or repress a number of genes not regulated by GRα (Kino et al., 2009, Lewis-Tuffin et al., 2007). A similar variant is present in zebrafish (Danio rerio), but the acceptor splice site is absent in exon 8 of other fish species and thus the presence of GRβ may be restricted to the Ostariophysi superorder in the fishes (Schaaf et al., 2008). Zebrafish GRβ acts in a similar way as its vertebrate paralogue as a dominant-negative inhibitor of GRα, and similarly has recently been shown to regulate its own suite of genes (Chatzopoulou et al., 2015). Another splice variant of the human GRα sees three bases retained from the intron separating exon 3 and 4, which results in an additional amino acid, arginine, inserted between the two zinc fingers of the DBD this has been termed GRγ (Ray et al., 1996, Rivers et al., 1999). GRγ binds GCs with a similar affinity as GRα, but has an impaired ability to regulated GR transcription (Ray et al., 1996), despite this, in various tissues GRγ can regulate a different subset of genes to GRα (Oakley and Cidlowski, 2011) and has been associated with GC resistance in a number of cancers (Beger et al., 2003). The alternate translation start sites in exon 2 have results in 8 versions of GRα (Oakley and Cidlowski, 2011). One of these, GRα-D, lacks the A/B domain, but is still active, regulating around 1800 genes in response to GCs (Oakley and Cidlowski, 2011). Furthermore, single nucleotide polymorphisms in GRα can also significantly alter function. Such an example is in GRα ER22/23EK polymorphism within exon 2, where a G to A point mutation in codon 22 results in a change of arginine to lysine; in patients with this mutation there is a concurrent silent G to A point mutation in codon 23 (van Rossum et al., 2002). The result is a receptor with decreased hormone sensitivity, which is associated with patients with altered metabolism, risk of cardiovascular disease (DeRijk and de Kloet, 2005) and who are susceptible to depression (Panek et al., 2014). The list of GR isoforms demonstrates that a wide range of regulatory strategies, including difference protein-ligand, protein-protein and protein-DNA interactions that mediate cellular specific glucocorticoid actions.