Characterization of two functional glucocorticoid receptors in the marine medaka Oryzias dancena

The cDNAs that encode the glucocorticoid receptors odGR1 and odGR2 were cloned from a euryhaline teleost, the marine medaka (Oryzias dancena). The open reading frames of odGR1 and odGR2 encode 790 and 783 amino acids, respectively, and show a sequence identity of 46% with each other. When inter- and intra-species comparisons of the GR domains were made, the N-terminal AF-1 (A/B) and hinge (D) domains showed relatively low identities, whereas the DNA-binding (C) domain (DBD) and ligand-binding (E) domain showed relatively high identities. Through phylogenetic analysis, we revealed that odGR1 and odGR2 belong to the teleost GR1 and GR2 groups, respectively. Transfection of odGR1 or odGR2 expression vectors into COS-7 cells along with a reporter vector demonstrated that cortisol and dexamethasone dose-dependently induce transcriptional activity in both GRs. As described in other teleostean fish, the transactivity of odGR2 was more sensitive at far lower concentrations of ligands than the transactivity of odGR1. When treated with aldosterone, the reporter gene was activated in COS-7 cells transfected with odGR2 but not in cells transfected with odGR1. RU486 inhibited transactivation by both GRs, but odGR2 was less sensitive to the inhibitor. Interestingly, alterations in coregulators, GRIP-1 and SMILE, mediated transactivation that was more drastic for odGR2 than odGR1. A nine-amino acid insertion (WRARQNTDG) in the DBD of odGR1 had a weak but significant influence on the transactivity of odGR2 with respect to responsiveness to agonists or coregulators. Taken together, these results indicate that the two odGRs possess distinct features not only for ligand sensitivity but also for preferential coregulator recruitment.

► Medaka GR2 is more sensitive in responding to cortisol and dexamethasone (DEX) than GR1.
► Aldosterone is a weaker agonist for medaka GR2 compared to cortisol and DEX.
► Although RU486 inhibited transactivation in both GRs, GR2 was more resistant.
► Mammalian coregulators, GRIP-1 and SMILE, modulate agonist-activated medaka GR1 and GR2.
► Medaka GR2 is a functionally closer ortholog to mammalian GRα.