Insights into GR binding to coregulators. An experimental and theoretical approach.

MONCZOR FEDERICO; FITZSIMONS CARLOS; HOUTMAN RENE; VREUGDENHIL ERNO; MEIJER ONNO

Lunteren

Congreso; 9th Dutch Endo-Neuro-Psycho meeting; 2011

ENP Society

Introduction: A549 cells represent a well-characterized model to study regulation of gene expression by the glucocorticoid receptor (GR). In these cells, GR activation resulted in gene-specific induction of three primary target genes (GILZ, THBD, and SLC19A2) by the GR agonist DEX. The three curves differed both in their potency and efficacy. These differences could be attributed to several factors, including gene-specific coregulator recruitment. In order to test this hypothesis, we developed a theoretical model of GR activity.Results: Our model involves random sequential binding of ligands and coregulators to the GR. Two affinity constants and a parameter alpha, reflecting the possible allosterism between both binding events (ligand and coregulator), were used. The model is able to explain the observed differences in dose-response curves and to predict: 1) A decrease in GR expression would result in a decrease in efficacy, without associated changes in agonist potency, thus affecting low-responsive genes more than high-responsive ones. Decreasing GR expression by using siRNAs in A549 cells resulted in altered gene induction profiles, as predicted by the model. 2) A spontaneous binding of coregulators to GR, in the absence of any ligand. Using a cofactor peptide array and a GR ligand binding domain (GR-LBD) we show, in the absence of any ligand, a specific and spontaneous concentration-dependent coregulator binding that was differentially modulated by the presence of three GR ligands: DEX, RU486 and cyproterone. Surprisingly, the potency to recruit the GR-LBD was similar for all co-regulators.Discussion: Our simple model is sufficient to explain the experimental results in terms of differential coregulator recruitment and to predict spontaneous coregulator binding to the GR-LBD. Although the GR-LBD was unable to distinguish among co-regulators, we cannot exclude the possibility that other GR domains may contribute to co-factor discrimination. Our observations contribute to a deeper understanding of the molecular mechanisms that control GR-mediated gene transcription upstream of DNA binding.