A critical role of helix 3-helix 5 interaction in steroid hormone receptor function

Abstract

The ligand-binding domains of steroid hormone receptors possess a conserved structure with 12 alpha-helices surrounding a central hydrophobic core. On agonist binding, a repositioned helix 12 forms a pocket with helix 3 (H3) and helix 5 (H5), where transcriptional coactivators bind. The precise molecular interactions responsible for activation of these receptors remain to be elucidated. We previously identified a H3-H5 interaction that permits progesterone-mediated activation of a mutant mineralocorticoid receptor. We were intrigued to note that the potential for such interaction is widely conserved in the nuclear receptor family, indicating a possible functional significance. Here, we demonstrate via transcriptional activation studies in cell culture that alteration of residues involved in H3-H5 interaction consistently produces a gain of function in steroid hormone receptors. These data suggest that H3-H5 interaction may function as a molecular switch regulating the activity of nuclear receptors and suggest this site as a general target for pharmacologic intervention. Furthermore, they reveal a general mechanism for the creation of nuclear receptors bearing increased activity, providing a potentially powerful tool for the study of physiologic pathways in vivo.

Fig. 1.

Structural model for H3–H5 interaction in GR_L604. (A) Coconservation of H3–H5 residues in NRs. The H3 and H5 amino acid sequences of selected NRs were aligned by using clustal w (26), and the residues corresponding to serine 810 on H5 and alanine 773 on H3 in MR are highlighted. Receptors bearing leucine at the H5 position frequently have alanine at the H3 position, whereas receptors bearing methionine at the H5 position have glycine at the H3 position. ER, estrogen receptor; ERR2, estrogen-related receptor type 2; RXRG, rexinoid receptor γ, α, and β; AD4BP, adrenal 4 binding protein (also known as SF1, steroidogenic factor 1); PPARG, peroxisome proliferator-activated receptor γ; RARA, mouse retinoic acid receptor α; AR, androgen receptor. (B) Ribbon drawing of the wild-type GR-LBD (19). H3 is colored in purple, H5 in cyan, and dexamethasone in gray. The steroid, the side chain of Met-604, and the carbonyl oxygen of the Gly-567 main chain are shown as ball-and-stick models and are labeled accordingly. The figure shows that the Met-604 side chain points away from the ligand and is too far from Gly-567 to be in vdW contact. (C) Ribbon drawing of the GR_L604 mutant. Unlike Met-604, the side chain of Leu-604 is in vdW contact with the carbonyl oxygen of the Gly-567 main chain. The structure of the GR_L604 mutant was generated in silico by replacing Met-604 against leucine. Figures were prepared with the programs molscript, bobscript, and raster3d.

Fig. 2.

GR_L604 is active at 10-fold lower steroid concentrations than GR_WT. The ability of wild-type and mutant GRs to induce luciferase expressed under control of the mouse mammary tumor virus promoter was assessed in Cos7 cells in the absence or presence of the indicated steroids. Luciferase activity is expressed as percent of maximal induction of GR_WT by dexamethasone (10 nM). All data points represent the mean of at least nine independent transfections. (A) Schematic of the GR mutants studied. (B–D) Dose–response curves for induction of luciferase by GR_WT, GR_L604, GR_L604A567, and GR_A567 in response to cortisol (B), dexamethasone (C), and corticosterone (D). (E) Dose–response curves for induction of luciferase by GR_WT, GR_A604, and GR_A604A567 by dexamethasone. *, P < 0.001 vs. GR_WT...., P < 0.01 vs. GR_WT.

Fig. 3.

GR_L604 has increased affinity for glucocorticoids. (A) Scatchard analysis of the binding of [³H]dexamethasone in extracts expressing GR_WT, GR_L604, GR_L604A567, or GR_A604. Each data point was assayed at least four times; one representative set of data for binding to GR_WT, GR_L604, GR_L604A567, and GR_A604 is shown, and the mean K_d values are indicated. (B) Competition of varying concentrations of corticosterone and 5 nM [³H]dexamethasone for binding to GR_WT, GR_L604, and GR_L604A567 in Cos-7 cell extracts. Each data point represents the mean ± SEM of at least four independent experiments. (C) Competition of varying concentrations of 11-deoxycorticosterone and 5 nM [³H]dexamethasone for binding to GR_WT, GR_L604, and GR_L604A567 in Cos-7 cell extracts. Each data point represents the mean ± SEM of at least four independent experiments.

Fig. 4.

PR_L759 and PR_A759 are constitutively active. The ability of PR mutants to induce luciferase expressed under control of the PRE promoter was assessed in Cos7 cells in the presence of progesterone and 19-norprogesterone. Luciferase activity is expressed as percent of maximal induction of PR_WT by progesterone. (A) Transcriptional activity of PR_WT, PR_L759, PR_L759A722, and PR_A722 in the presence of progesterone and 19-norprogesterone. (B) Transcriptional activity of PR_WT, PR_I759, and PR_I759A722 in the presence of progesterone and 19-norprogesterone. (C) Transcriptional activity of PR_WT, PR_V759, and PR_V759A722 in the presence of progesterone and 19-norprogesterone. (D) Transcriptional activity of PR_WT, PR_A759, and PR_A759A722 in the presence of progesterone and 19-norprogesterone. (E) RU486 inhibits constitutive activity of PR_L759 and PR_A759. The ability of RU486 to inhibit PR_WT, PR_L759, or PR_A759 induced luciferase expression from the PRE promoter was assessed in Cos-7 cells in the presence (PR_WT) or absence (PR_L759 or PR_A759) of 10 nM progesterone. Luciferase activity is expressed as percent of maximal induction of PR_WT by 10 nM progesterone. All data points represent the mean ± SEM of at least nine independent transfections. *, P < 0.001 vs. GR_WT...., P < 0.01 vs. GR_WT; ‡, P < 0.05 vs. GR_WT.