title: Glucocorticoid ligands specify different interactions with NF-kappaB by allosteric effects on the glucocorticoid receptor DNA binding domain.
abstract: Glucocorticoids inhibit inflammation by acting through the glucocorticoid receptor (GR) and powerfully repressing NF-kappaB function. Ligand binding to the C-terminal of GR promotes the nuclear translocation of the receptor and binding to NF-kappaB through the GR DNA binding domain. We sought how ligand recognition influences the interaction between NF-kappaB and GR. Both dexamethasone (agonist) and RU486 (antagonist) promote efficient nuclear translocation, and we show occupancy of the same intranuclear compartment as NF-kappaB with both ligands. However, unlike dexamethasone, RU486 had negligible activity to inhibit NF-kappaB transactivation. This failure may stem from altered co-factor recruitment or altered interaction with NF-kappaB. Using both glutathione S-transferase pull-down and bioluminescence resonance energy transfer approaches, we identified a major glucocorticoid ligand effect on interaction between the GR and the p65 component of NF-kappaB, with RU486 inhibiting recruitment compared with dexamethasone. Using the bioluminescence resonance energy transfer assay, we found that RU486 efficiently recruited NCoR to the GR, unlike dexamethasone, which recruited SRC1. Therefore, RU486 promotes differential protein recruitment to both the C-terminal and DNA binding domain of the receptor. Importantly, using chromatin immunoprecipitation, we show that impaired interaction between GR and p65 with RU486 leads to reduced recruitment of the GR to the NF-kappaB-responsive region of the interleukin-8 promoter, again in contrast to dexamethasone that significantly increased GR binding. We demonstrate that ligand-induced conformation of the GR C-terminal has profound effects on the functional surface generated by the DNA binding domain of the GR. This has implications for understanding ligand-dependent interdomain communication.
authors.affiliation: Centre for Molecular Medicine and Endocrine Sciences Research Group, Stopford Building, Faculty of Medicine, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: Helen
authors.initials: H
authors.lastname: Garside
authors.forename: Adam
authors.initials: A
authors.lastname: Stevens
authors.forename: Stuart
authors.initials: S
authors.lastname: Farrow
authors.forename: Claire
authors.initials: C
authors.lastname: Normand
authors.forename: Benoit
authors.initials: B
authors.lastname: Houle
authors.forename: Andy
authors.initials: A
authors.lastname: Berry
authors.forename: Barbara
authors.initials: B
authors.lastname: Maschera
authors.forename: David
authors.initials: D
authors.lastname: Ray
mesh_terms: D000494:Allosteric Regulation; D004247:DNA / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D005938:Glucocorticoids / Q000378:metabolism* / Q000494:pharmacology; D006367:HeLa Cells; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D015735:Mifepristone / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000378:metabolism*; D051996:Transcription Factor RelA
chemical_list: D005938:Glucocorticoids; D006727:Hormone Antagonists; D016209:Interleukin-8; D008024:Ligands; D016328:NF-kappa B; D011965:Receptors, Glucocorticoid; D051996:Transcription Factor RelA; D015735:Mifepristone; D003907:Dexamethasone; D004247:DNA