Structure/function of the human glucocorticoid receptor: Tyrosine 735 is important for transactivation

Ligand-induced activation of the glucocorticoid receptor (GR) is not well u nderstood. The GR ligand-binding domain was modeled, based on homology with the progesterone receptor. Tyrosine 735 interacts with the D ring of dexam ethasone, and substitution of D ring functional groups results in partial a gonist steroids with reduced ability to direct transactivation. Loss of the Tyr735 hydroxyl group by substitution to phenylalanine (Tyr735Phe) did not reduce ligand binding affinity [dissociation constant (K-d) 4.3 nM compare d with K-d 4.6 nM for wild-type] and did not alter transrepression of an nu clear factor-kappa B (NF-kappa B reporter. But, there was a significant 30% reduction in maximal transactivation of a mouse mammary tumor virus (MMTV) reporter, although with an unchanged EC50 (8.6 nM compared with 6 nM). Substitution to a nonaromatic hydrophobic amino acid, valine (Tyr735Val), r etained high-affinity ligand binding for dexamethasone (K-d 6 nM compared w ith 4.6 nM) and did not alter transrepression of NF-kappa B, However, there was a 36% reduction in MMTV activity with a right shift in EC50 (14.8 nM). The change to serine, a small polar amino acid Tyr735Ser), caused signific antly lower affinity for dexamethasone (10.4 nM), Maximal transrepression o f NF-kappa B was unaltered, but the IC50 for this effect was increased. Tyr 735Ser had a major shift in EC50 (118 nM) for transactivation of an MMTV re porter. Maximal transactivation of MMTV induced by the natural ligand cortisol was reduced to 60% by Tyr735Phe and Tyr735Val and was completely absent by Tyr7 35Ser. These data suggest that tyrosine 735 is important for ligand interpr etation and transactivation.