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Structure/Function of the Human Glucocorticoid Receptor: Tyrosine 735 Is Important for Transactivation

Department of Medicine (D.W.R., J.S., A.W.) and School of Biological Sciences (C.-S.S., A.B., A.W.) University of Manchester Manchester, M13 9PT, United Kingdom

Ligand-induced activation of the glucocorticoid receptor (GR) is not well understood. The GR ligand-binding domain was modeled, based on homology with the progesterone receptor. Tyrosine 735 interacts with the D ring of dexamethasone, and substitution of D ring functional groups results in partial agonist 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 compared with K_d 4.6 nM for wild-type] and did not alter transrepression of an nuclear factor-B (NF-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), retained high-affinity ligand binding for dexamethasone (K_d 6 nM compared with 4.6 nM) and did not alter transrepression of NF-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 significantly lower affinity for dexamethasone (10.4 nM). Maximal transrepression of NF-B was unaltered, but the IC₅₀ for this effect was increased. Tyr735Ser had a major shift in EC₅₀ (118 nM) for transactivation of an MMTV reporter.

Maximal transactivation of MMTV induced by the natural ligand cortisol was reduced to 60% by Tyr735Phe and Tyr735Val and was completely absent by Tyr735Ser. These data suggest that tyrosine 735 is important for ligand interpretation and transactivation.

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