Sequence and expression of a cDNA encoding Japanese flounder glucocorticoid receptor

The cDNA of the glucocorticoid receptor (GR) has been isolated from Japanese flounder liver and sequenced. The cDNA contained a complete open reading frame encoding 807 amino acid residues. The structural homology indicated that the Japanese flounder GR (jfGR) was divided into six different regions, A to F. Comparison of these regions of jfGR to those of rainbow trout GR showed a homology of 37% for A/B region, 98% for C region, 53% for D region, 93% for E region, and 82% for F region. Especially, eight cysteines in two zinc-fingers and the P-box in C region (DNA binding domain) were completely conserved. However, it contained an additional 9 amino acids insertion (WRARQNTDG) between the two zinc fingers which was previously detected in rainbow trout and eel GRs. Such insertion reported here could be for only fish GRs among any of steroid hormone receptors, suggesting that this amino acid insertion was a specific feature of fish GRs. The trans-activation function of this clone was verified by transfection into COS-7 cell, which lacks endogenous GR. A CMV-based expression vector consisting of jfGR cDNA was cotransfected with a reporter plasmid consisting of an glucocorticoid-responsive element fused to the chloramphenicol acetyl transferase (CAT) gene. As a result, the expression of CAT protein was stimulated by the addition of dexamethasone or cortisol. This evidence showed that this clone encoded the functional receptor.