Impact of Glucocorticoid Receptor Density on Ligand-Independent Dimerization, Cooperative Ligand-Binding and Basal Priming of Transactivation: A Cell Culture Model
Open Access
- 22 May 2013
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 8 (5), e64831
- https://doi.org/10.1371/journal.pone.0064831
Abstract
Glucocorticoid receptor (GR) levels vary between tissues and individuals and are altered by physiological and pharmacological effectors. However, the effects and implications of differences in GR concentration have not been fully elucidated. Using three statistically different GR concentrations in transiently transfected COS-1 cells, we demonstrate, using co-immunoprecipitation (CoIP) and fluorescent resonance energy transfer (FRET), that high levels of wild type GR (wtGR), but not of dimerization deficient GR (GRdim), display ligand-independent dimerization. Whole-cell saturation ligand-binding experiments furthermore establish that positive cooperative ligand-binding, with a concomitant increased ligand-binding affinity, is facilitated by ligand-independent dimerization at high concentrations of wtGR, but not GRdim. The down-stream consequences of ligand-independent dimerization at high concentrations of wtGR, but not GRdim, are shown to include basal priming of the system as witnessed by ligand-independent transactivation of both a GRE-containing promoter-reporter and the endogenous glucocorticoid (GC)-responsive gene, GILZ, as well as ligand-independent loading of GR onto the GILZ promoter. Pursuant to the basal priming of the system, addition of ligand results in a significantly greater modulation of transactivation potency than would be expected solely from the increase in ligand-binding affinity. Thus ligand-independent dimerization of the GR at high concentrations primes the system, through ligand-independent DNA loading and transactivation, which together with positive cooperative ligand-binding increases the potency of GR agonists and shifts the bio-character of partial GR agonists. Clearly GR-levels are a major factor in determining the sensitivity to GCs and a critical factor regulating transcriptional programs.Keywords
This publication has 108 references indexed in Scilit:
- Glucocorticoid Receptor–Promoter Interactions: Energetic Dissection Suggests a Framework for the Specificity of Steroid Receptor-Mediated Gene RegulationBiochemistry, 2012
- Transcription Factor AP1 Potentiates Chromatin Accessibility and Glucocorticoid Receptor BindingMolecular Cell, 2011
- Chromatin accessibility pre-determines glucocorticoid receptor binding patternsNature Genetics, 2011
- A theoretical framework for gene induction and experimental comparisonsProceedings of the National Academy of Sciences of the United States of America, 2010
- Repression of DNA-binding dependent glucocorticoid receptor-mediated gene expressionProceedings of the National Academy of Sciences of the United States of America, 2009
- Ultradian hormone stimulation induces glucocorticoid receptor-mediated pulses of gene transcriptionNature, 2009
- Modulation of transcription parameters in glucocorticoid receptor-mediated repressionMolecular and Cellular Endocrinology, 2008
- What goes on behind closed doors: physiological versus pharmacological steroid hormone actionsBioEssays, 2008
- The RXR heterodimers and orphan receptorsCell, 1995
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976