A ligand-specific kinetic switch regulates glucocorticoid receptor trafficking and function

Abstract

The ubiquitously expressed glucocorticoid receptor (GR) is a major drug target for inflammatory disease, but issues of specificity and target tissue sensitivity remain. We now identify high potency, non-steroidal GR ligands, GSK47867A and GSK47869A, which induce a novel conformation of the GR ligand-binding domain (LBD) and augment the efficacy of cellular action. Despite their high potency, GSK47867A and GSK47869A both induce surprisingly slow GR nuclear translocation, followed by prolonged nuclear GR retention, and transcriptional activity following washout. We reveal that GSK47867A and GSK47869A specifically alter the GR LBD structure at the HSP90-binding site. The alteration in the HSP90-binding site was accompanied by resistance to HSP90 antagonism, with persisting transactivation seen after geldanamycin treatment. Taken together, our studies reveal a new mechanism governing GR intracellular trafficking regulated by ligand binding that relies on a specific surface charge patch within the LBD. This conformational change permits extended GR action, probably because of altered GR-HSP90 interaction. This chemical series may offer anti-inflammatory drugs with prolonged duration of action due to altered pharmacodynamics rather than altered pharmacokinetics.

Keywords: Crystal structure; GR; Glucocorticoid; Heat shock protein 90; Nuclear receptor; Subcellular trafficking.

Fig. 1.

GSK47867A and GSK47869A are highly potent GR agonists. Structure of steroidal and non-steroidal glucocorticoids (A). HeLa cells were transfected with a positive GR reporter gene (TAT3-luc) (B) or with a glucocorticoid-repressed NFκB reporter gene (NRE-luc) (C). At 24 hours post-transfection, NRE-Luc transfected cells were pre-treated with TNF-α (0.5 ng/ml) for 30 minutes. Subsequently all transfected cells were treated with 0.01–1000 nM Dex, hydrocortisone (HC), GSK47867A (67A) or GSK47869A (69A) for 18 hours, and were then lysed and subjected to analysis using a luciferase assay. The graphs (mean±s.d.) show the relative light units (RLU) (B) or percentage inhibition (C) from one of three representative experiments performed in triplicate.

Fig. 2.

Dex and GSK47867A binding induces different GR LBD structures. (A,B) Comparison of the crystal structures of the GR LBD bound to Dex (A, purple) and GSK47867A (67A) (B, blue). The residues in the binding pocket that show significant movement upon 67A binding are highlighted in yellow. When 67A binds to the GR LBD the head region causes movement of residues Gln570, Met604 and Arg611 (D) when compared with Dex binding (C).

Fig. 3.

GR LBD surface charge is altered by GSK47867A binding. (A,C) The region of the GR LBD surface where residues Gln570, Met604 and Arg611 are exposed is highlighted [A, with Dex in purple and C, GSK47867A (67A) in blue]. (B,D) A close-up of this region is shown with an electrostatic charge map that reveals the creation of a patch of positive surface charge due to the movement of Arg611 upon 67A binding.

Fig. 4.

GSK47867A and GSK47869A induce slow kinetics of GR activation. HeLa cells were treated with DMSO vehicle, 100 nM Dex, 3 nM GSK47867A (67A) or 3 nM GSK47869A (69A) for 1, 4 or 24 hours. Cells were then lysed and RNA was extracted using an RNeasy kit. RNA was reverse transcribed and subjected to qPCR for FKBP5 (A) and GILZ (B) using Sybr Green detection in an ABI q-PCR machine and with data analysed by the ΔΔCT method. Graphs (mean±s.e.m.) combine data from three separate experiments and display fold change over that in vehicle-treated control. (C) Following transfection with HaloTag-GR, HeLa cells were incubated with 100 nM Dex, 3 nM FP, 3 nM 67A or 69A. Cells were imaged in real-time at 37°C to determine the subcellular localisation of the GR (white) at the times indicated. Scale bars: 25 µm. Images are representative of three independent experiments. (D) HeLa cells transfected with a TAT3-Luc reporter plasmid were treated with 100 nM Dex, 3 nM FP, 3 nM 67A or 69A for up to 24 hours. The production of luciferase was tracked by measuring the relative light units (RLU) emitted from each sample; D tracks RLU production over the first 5 hours following addition of treatment and is representative of three separate experiments. The time taken to reach half the maximal light output was measured for all treatments (E). Statistical significance was evaluated by one-way ANOVA followed by Tukey post-test. *P<0.005 compared with control; **P<0.001 compared with Dex.

Fig. 5.

GSK47867A and GSK47869A rapidly accumulate in cells. A549 cells were treated with 10 µM Dex, FP, GSK47867A (67A) or GSK47869A (69A) for 10 minutes and subsequently washed and lysed. The cell samples were analysed for ligand uptake by mass spectrometry (A). HeLa cells were treated with DMSO vehicle (not shown), 100 nM Dex, 3 nM FP, 3 nM 67A or 3 nM 69A either for 4 hours or for 10 minutes followed by washout (WO) and culture in ligand-free medium for 4 hours. Subsequently cells were lysed and RNA extracted using an RNeasy kit. RNA was reverse transcribed and subjected to qPCR for GILZ (B), FKBP5 (C) and IGFBP1 (D) using Sybr Green detection in an ABI q-PCR machine and data were analysed by the ΔΔCT method. Graphs (mean±s.e.m.) combine data from three separate experiments and display percentage induction compared with the equivalent 4 hours of constant treatment. (E,F) Following transfection with HaloTag-GR HeLa cells were placed on ice for 10 minutes and subsequently incubated with 100 nM Dex, 3 nM FP, 3 nM 67A or 69A for 1 hour on ice. Following treatment, cell images were captured in real-time at 37°C to determine the subcellular localisation of the GR (white, E). Scale bars: 25 µm. F displays the average time (mean±s.e.m) to exclusively nuclear GR following 1 hour with ligand on ice, calculated from three separate experiments. Statistical significance was evaluated by one-way ANOVA followed by Tukey post-test. *P<0.001 compared with Dex.

Fig. 6.

Disruption of the microtubule network increases the rate of GR translocation in a ligand-specific manner. (A) The ribbon structure of the GR LBD bound to Dex. The residues highlighted in yellow were identified by Ricketson et al. (Ricketson et al., 2007) as important for interaction between GR and HSP90. (B) The region of the GR LBD surface where the NSGs cause an alteration in surface charge. (C) The region of the GR LBD surface where Met604 is exposed is highlighted in yellow. This area overlaps the region identified as having altered surface charge upon binding NSG, supporting the lack of HSP90 engagement with NSG treatment. (D) Untreated HeLa cells with GFP-labelled microtubules. Incubation for 1 hour with 2 µM colcemid disrupts the microtubule network (E). Following transfection with a halo-tagged GR, HeLa cells were incubated with 2 µM Colcemid for 1 hour then subsequently co-treated with 100 nM Dex, 3 nM FP, 3 nM GSK47867A (67A) or 3 nM GSK47869A (69A) (F). Cells were imaged in real-time and analysed for subcellular localisation of the GR (white). Scale bars: 25 µm. (G) The average time taken (mean±s.e.m) for the GR to be exclusively nuclear. Statistical significance was evaluated by one-way ANOVA followed by Tukey post-test. *P<0.005 compared with treatment without colcemid.

Fig. 7.

Antagonism of HSP90 has less impact on the activity of NSG ligands. (A,D) HeLa cells transfected with a TAT3-Luc reporter plasmid were treated with 100 nM Dex, 3 nM GSK47867A (67A) or 3 nM GSK47869A (69A) for 24 hours. Subsequently cells were either co-treated with 10 mM geldanamycin (GA) (D) or washed (WO) and placed in serum-free recording medium (A) for a further 24 hours. The production of luciferase was tracked by measuring the relative light units (RLU) emitted from each sample. Graphs tracks RLU production for 24 hours following GA addition or ligand removal. Graphs are representative of three separate experiments. (B,C) HeLa cells were treated with DMSO vehicle (not shown), 100 nM Dex, 3 nM FP, 3 nM 67A or 3 nM 69A for 24 hours or 1 hour followed by washes (WO) and then cultured in ligand-free medium for 24 hours. Subsequently cells were lysed and RNA was extracted using an RNeasy kit. RNA was reverse transcribed and subjected to qPCR of GILZ (B) and FKBP5 (C) using Sybr Green detection in an ABI q-PCR machine and data analysed by the ΔΔCT method. Graphs (mean±s.e.m.) combine data from three separate experiments and display percentage induction compared with the equivalent constant treatment for 24 hours. HeLa cells were treated with 100 nM Dex, 3 nM 67A or 69A for 2 hours and then co-treated with 10 mM GA for a further 2 hours (E) or 22 hours (F), and a constant 4-hour or 24-hour treatment was used as a comparison. Following treatment, cells were lysed in RIPA buffer containing phosphatase and protease inhibitors and analysed by immunoblotting for GR abundance and GR Ser211 phosphorylation. α-Tubulin was used as a loading control. Statistical significance was evaluated by one-way ANOVA followed by Tukey post-test. *P<0.01 compared with both Dex and FP. (G) Mechanism of GR action. Upon binding glucocorticoids (Gc) (1) the GR interacts with the translocation machinery enabling nuclear import (2). In the nucleus, GR binds to cis-elements to activate or repress target gene expression (3). The GR undergoes dynamic cycles of dissociation, and re-binding of ligand, which occurs in an HSP90-dependent manner (4). Interaction with PP5 facilitates nuclear export of the GR (5) enabling it to be recycled or targeted for degradation by the proteasome (6).