Fish glucocorticoid receptor with splicing variants in the DNA binding domain

Abstract

Here we describe the isolation of a rainbow trout cDNA containing an entire GR coding region. Although the encoded protein is highly homologous to other GRs, especially in its DNA binding domain, it contains a nine amino acid insertion between the two zinc fingers. This novel form is found in all rainbow trout tissues examined; however, the testis also contains a splice variant lacking this insert, making it completely continuous to other GRs. In transient transfection assays of cultured cells, the two rainbow trout GR variants activated transcription from the glucocorticoid-responsive mouse mammary tumor virus promoter to comparable levels.

Evolution of glucocorticoid receptors with different glucocorticoid sensitivity

“…In in vitro experiments in rainbow trout, the GR agonist dexamethasone activated the tested downstream gene at lower concentrations than cortisol in the same experiment: EC 50 for GR1 stimulated with cortisol was 46 nM while it was 4$3 nM with dexamethasone treatment; for GR2, this was 0$7 nM for cortisol and 0$35 nM for dexamethasone (Bury et al 2003). In other experiments, it was shown that, with the same concentration of ligand, dexamethasone gave a stronger activation of the reporter gene than cortisol (Takeo et al 1996, Tokuda et al 1999, Greenwood et al 2003.…”

“…the nine-amino-acid insert exceeds that of the non-spliced form (Takeo et al 1996, Bury et al 2003, Greenwood et al 2003. In Burtons' mouthbrooder, the variant without the insert was found in all organs tested in rainbow trout; however, it could only be detected in testis (Takeo et al 1996, Greenwood et al 2003.…”

“…These nine amino acid inserts between the two zinc fingers are also remarkably conserved among teleostean fish species. In rainbow trout (Ducouret et al 1995, Takeo et al 1996, Japanese eel (Anguilla japonica) (Todo & Nagahama 1998), Japanese flounder (Paralichthys olivaceus) (Tokuda et al 1999), Burtons' mouthbrooder (Greenwood et al 2003), brown trout (Salmo trutta; AY863149), European sea bass (Dicentrarchus labrax; AY549305) and fugu (T. rubripes) a WRARQNTDG insert is present. A slightly different insert is found in tetraodon (T. nigroviridis): WRARQNTVC.…”

“…In all these species, GR1 (the gene showing the nine-amino-acid insert) is more strongly expressed than the GR2 gene (Bury et al 2003, Greenwood et al 2003. Moreover, most first-reported teleostean fish GR sequences have the nine-amino-acid insert and would therefore be classified as GR1, such as in European sea bass and brown trout (Ducouret et al 1995, Takeo et al 1996, Todo & Nagahama 1998, Tokuda et al 1999. Whether this reflects relative abundance of the GR1 messenger or preferential primer design is unclear.…”

“…Moreover, alternative splicing occurs in these fish GR genes (Takeo et al 1996, Greenwood et al 2003, which leads to functional transcripts with different transactivation properties compared to wild-type. Both the duplicate GR genes as well as the splice variants of these GR genes in fish are differentially expressed in tissues and differ in their affinity for cortisol, the single dominant GC in fish (Bury et al 2003, Greenwood et al 2003.…”