Mutations of glucocorticoid receptor differentially affect AF2 domain activity in a steroid-selective manner to alter the potency and efficacy of gene induction and repression

Abstract

The transcriptional activity of steroid hormones is intimately associated with their structure. Deacylcortivazol (DAC) contains several features that were predicted to make it an inactive glucocorticoid. Nevertheless, gene induction and repression by complexes of glucocorticoid receptor (GR) with DAC occur with potency (lower EC 50) greater than and efficacy (maximal activity, or A max) equal to those of the very active and smaller synthetic glucocorticoid dexamethasone (Dex). Guided by a recent X-ray structure of DAC bound to the GR ligand binding domain (LBD), we now report that several point mutants in the LBD have little effect on the binding of either agonist steroid. However, these same mutations dramatically alter the A max and/or EC 50 of exogenous and endogenous genes in a manner that depends on steroid structure. In some cases, Dex is no longer a full agonist. These properties appear to result from a preferential inactivation of the AF2 activation domain in the GR LBD of Dex-bound, but not DAC-bound, receptors. The Dex-bound receptors display normal binding to, but a greatly reduced response to, the coactivator TIF2, thus indicating a defect in the transmission efficiency of GR-steroid complex information to the coactivator TIF2. In addition, all GR mutants that are active in gene induction with either Dex or DAC have greatly reduced activity in gene repression. This contrasts with the reports of GR mutations preferentially suppressing GR-mediated induction. The properties of these GR mutants in gene induction support the hypothesis that the A max and EC 50 of GR-controlled gene expression can be independently modified, indicate that the receptor can be modified to favor activity with a specific agonist steroid, and suggest that new ligands with suitable substituents may be able to affect the same LBD conformational changes and thereby broaden the therapeutic applications of glucocorticoid steroids.

Fig. 1

Structures of agonist glucocorticoids Dex and DAC.

Fig. 2

Effects of GR mutations on level of gene induction. A. Schematic of position of point mutations in LBD with numbers above the drawing indicating the amino acids of the LBD with AF2 (cross hatched), AF1 (core is striped), and DBD (dashes) domains. The affinity of Dex binding (relative to wt GR) was determined by Scatchard assays (see Materials and Methods). B-D. The A_max, or total amounts of reporter gene induction (GREtkLUC in B, MMTVLUC in C), by EtOH ± saturating concentrations of Dex or DAC was determined in CV-1 cells as described in Materials and Methods. For the endogenous gene LAD1, the fold induction by 1 μM Dex or DAC in U2OS cells was determined by qRT-PCR as presented in Materials and Methods. The average values ± S.E.M. from 7 (GREtkLUC; 2 for Class I mutants), 2 (MMTVLUC) and 4 (LAD1) independent experiments are plotted. ND = not determined. Relative to wild type GR, * P < 0.050, ** P = 0.0070, *** P = 0.0006.

Fig. 3

Dose-response curves for mutant GR induction of reporter genes. A. Dose-response curves for DAC induction of exogenous GREtkLUC in CV-1 cells. For each receptor, the data of one experiment are plotted as percent of the maximal level of induction by DAC, as described in Materials and Methods. B-C. Change in EC₅₀ with receptor mutation for induction of exogenous reporter genes by Dex or DAC. D. Dose-response curves for DAC induction of endogenous LAD1 gene in U2OS cells. A representative experiment is shown. E. Change in EC₅₀ with receptor mutation for induction of LAD1 gene by Dex or DAC. Note that the average EC₅₀ values (bar graphs) ± S.E.M. are plotted on a logarithmic y-axis for n = 5–8 (GREtkLUC; relative to wt GR, P = 0.0016 for all Dex-bound receptors and P = 0.0025 for all DAC-bound receptors), 2 (MMTVLUC) and 4 (LAD1; relative to wt GR, P = 0.029 for all Dex- and DAC-bound receptors) independent experiments. ND = not determined.

Fig. 4

Repression of the endogenous Coll3 gene by mutant GRs in U2OS cells. A. Effect of mutations on fold repression of Coll3 mRNA by Dex and DAC. Data represent average of 4 independent experiments ± S.E.M. (relative to wt GR, P <0.05 for all except DAC with E558A and L626I). B. Dose-response curves for Dex or DAC repression of endogenous Coll3 by indicated mutant GRs. The Coll3 mRNA obtained with increasing steroid, expressed as percent of Coll3 mRNA remaining with a saturating concentration of steroid, is plotted for each mutant receptor in one representative experiment. C. Change in EC₅₀ for repression of Coll3 mRNA expression with receptor mutation in presence of Dex or DAC. See Materials and Methods for experimental details. The average values ± S.E.M. of 4 independent experiments are plotted on a logarithmic y-axis (relative to wt GR, P <0.05 for all). ND = not determined.

Fig. 5

Two-hybrid assays for VP16/GR chimeras with GAL/coactivator or GAL/corepressor in CV-1 cells. A&C. Total Luciferase activity induced from FRLuc reporter by GAL ± GRIP1 (A) or NCoR-RID (C) with indicated mutant VP16/GRs plus EtOH, 1 μM Dex, or 0.1 μM DAC was determined, normalized to the value for GAL/EtOH with the Q588K mutant, and plotted as described in Materials and Methods. *P < 0.05 for mutant vs. wt GR with GAL/GRIP1 or GAL/NCoR-RID and steroid. B&D. EC₅₀ values for Dex or DAC induction of FRLuc reporter by GAL/GRIP1 (B) or NCoR-RID (D) with indicated mutant VP16/GRs was determined and plotted as described in Materials and Methods. Error bars for all graphs are S.E.M of 5 independent experiments (4 experiments for pGAL controls for NCoR-RID; 3 experiments for VP16/GRR629Y). NMF = no meaningful figure because the fold induction approached the usual error bars in the data points, thereby precluding a meaningful dose-response curve. In all cases, P = 0.029 for mutant vs. wt GR with GAL/GRIP1 or GAL/NCoR-RID and steroid.

Fig. 6

Transcriptional properties of GAL/GR525C mutants ± coactivator TIF2 in CV-1 cells. Total Luciferase activity (A_max) (A) and EC₅₀ (B) for induction by GAL/GR525C alone from the FRLuc reporter with Dex or DAC ± TIF2 was determined and plotted as described in Materials and Methods. Error bars are S.E.M of 4 (A) or 5 (B) independent experiments. For all cases, † = not significant (P > 0.05) for mutant vs. wt GR ± GRIP1 and steroid. No symbol above bar indicates P ≤ 0.029.

Fig. 7

Transcriptional properties of full-length GR mutants ± coactivator TIF2 in CV-1 cells. Total Luciferase activity (A_max) (A) and EC₅₀ (B) for induction of GREtkLUC reporter by Dex or DAC ± TIF2 was determined and plotted as described in Materials and Methods. Error bars are S.E.M of 5 independent experiments. In all cases either with or without TIF2, *P < 0.05, **P < 0.0005 for mutant vs. wt GR in (A). In (B), P = 0.0079 for all comparisons of mutant vs. wt GR.

Fig. 8

Predicted structures of mutant GR LBDs. Computer modeling of Dex and DAC (both in yellow) binding to GR LBD of wt vs. A625I (top) and wt vs. R629Y (bottom) mutants. See Discussion for further details.