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

Abstract

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 (Kd) 4.3 nM compared with Kd 4.6 nM for wild-type] and did not alter transrepression of an nuclear factor-kappaB (NF-kappaB 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 (Kd 6 nM compared with 4.6 nM) and did not alter transrepression of NF-kappaB. 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-kappaB 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.

Genetic engineering of the glucocorticoid receptor by fusion with the herpes viral protein VP22 causes selective loss of transactivation

“…The MMTV-luc plasmid consisted of 1·5 kb of the mouse mammary tumour virus long terminal repeat fused to the firefly luciferase gene in the pXP-2 backbone (Ray et al 1996a). The full-length wild-type human GR expression vector (wtGR) had the human GR cDNA inserted into the pcDNA3 plasmid (Ray et al 1999). The full-length rat GR expression vector, p6RGR, consisted of the rat GR cDNA driven by the Rous sarcoma virus (RSV) promoter.…”

“…The estrogen reporter gene (ERE-luc) consisted of three copies of a consensus estrogen response element (ERE) upstream of a minimal tk promoter and the firefly luciferase gene (luc), and was the kind gift of Dr Maurice Needham. A CMV-Gal plasmid was used to control for differences in transfection efficiency, as previously described (Ray et al 1999).…”

“…COS 7, HeLa and A549 cells were obtained from ECACC (Salisbury, UK), and were cultured as previously described (Ray et al 1999) in DMEM with glutamax (Gibco, Paisley, Strathclyde, UK) supplemented with 10% fetal calf serum. No antibiotics were used.…”

“…All experiments were performed on at least three separate occasions. Cells were routinely co-transfected with a CMVGal plasmid, and the results were expressed as corrected light units by dividing the luciferase assay light units by the optical density 570 from the Gal assay using CPRG as substrate as previously described (Ray et al 1999).…”

A Point Mutation of the AF2 Transactivation Domain of the Glucocorticoid Receptor Disrupts Its Interaction with Steroid Receptor Coactivator 1

“…6). There is specificity to this reaction because another AF2 mutation, Y753F, which displays reduced activation of the MMTV promoter (42), has no influence on the GR⅐SRC-1 interaction in the yeast two-hybrid system (Fig. 5).…”