Endocrine Abstracts

Glucocorticoids powerfully inhibit inflammation by repressing NFkB function. We sought how ligand recognition by the glucocorticoid receptor (GR) regulates this effect. In vivo some glucocorticoid ligands (eg RU486) have negligible anti-inflammatory activity, in contrast to the agonist dexamethasone, despite promoting nuclear translocation of the GR, and binding to DNA. In-vitro studies have not identified a role for ligand binding in allowing interaction between the GR and NFkB, which is mediated by the DNA binding domain of the GR, and so the role of ligand recognition is uncertain.

Using GST pull-down and BRET approaches we identify an unexpected effect of ligand binding on protein-protein interaction between the GR and the p65 component of NF-kB, with the antagonist RU486 inhibiting efficient recruitment. Dexamethasone and RU486 had similar EC50 (8.7nM and 6.88nM) values measured by BRET, but RU486 reduced maximal interaction by 50%.

In addition we identified significant differences in the spectrum of transcriptional co-modulators recruited to the C terminal of the GR when bound to either dexamethasone, or RU486. Taken together these data show that ligand recognition selects the profile of co-modulators recruited to the ligand binding domain, but also exerts an allosteric effect on the DNA binding domain to regulate binding of NFkB.

To investigate the additional constraints imposed by DNA binding we used chromatin immunoprecipitation to measure recruitment of the GR to a well-characterised NFkB response element from the IL-8 gene. This showed that dexamethasone promoted recruitment of the GR to DNA bound NFkB, but that RU486 had only a minimal effect. These data show that ligand recognition by the GR has profound effects not only on co-modulator recruitment to the GR C terminal, but also a major effect on the functional surface generated by the DNA binding domain. Understanding this allosteric switch has implications for anti-inflammatory therapeutic developments.