Table 1.
Primer information.
Fig 1.
Transcriptional activity of alternative GR variants harboring the Ala610Val substitution after treatment with different concentrations of steroid.
Presented are dose-response curves and EC50 estimations for the endogenous ligand cortisol in a wide (A) and narrow range (B). Additionally, the transactivation activity of aldosterone (C) and progesterone (D) is illustrated. The wild-type variant is labeled as Ala610 (blue) and the mutant GR variant as Val610 (red). Plotted are normalized means ± SEM of two separate experiments performed in triplicate.
Fig 2.
Cytosol-to-nucleus shuttle and subcellular localization of GFP-tagged GR variants.
Cos-7 cells were transiently transfected with either the wild-type Ala610 (A) or the mutant Val610 (B) GR variant and stimulated with 10 nM dexamethasone to induce translocation. Representative images of time-series showing the same cells are presented (A and B). The increase in fluorescence intensity in the nucleus after dexamethasone stimulation (C) and the comparison of the estimated half-maximal translocation time between both GR variants (D) based on observations of three independent transfection experiments are depicted.
Fig 3.
Stereogram of the interaction between glucocorticoid receptor ligand binding domain and dexamethasone.
Structural models of helix 3 to helix 7 of the ligand binding domain of the wild-type Ala610 (A) and the mutated Val610 (B) receptor variants are shown. The distances of the alternative alanine (blue) and valine (red) residue at amino acid position 610 with the two methyl carbon atoms of Val654, and the carbon C-6 and C-7 positions of the ligand dexamethasone (green), are indicated in angstroms (Å).
Fig 4.
In vitro and in vivo glucocorticoid receptor binding assays.
Dexamethasone binding assays were performed for transfected Cos-7 cells (in vitro; A and B) and for cytosolic fractions of pituitary glands (in vivo; C and D). For Cos-7 cells, specific binding curves (A) were plotted based on three independent experiments for Ala610 GR (blue) and Val610 GR (red). For pituitary glands, homozygous carriers of wild-type GR (Ala610 GR; blue) and heterozygous carriers of mutant GR (AlaVal610 GR; red) were analyzed (C). Dashed lines indicate the estimated dissociation constant (KD) and the maximum specific binding (Bmax), respectively. Comparisons of KD and Bmax between groups are illustrated for the in vitro (B) and in in vivo (D) binding assay as means ± SEM.