Impact of Glucocorticoid Receptor Density on Ligand-Independent Dimerization, Cooperative Ligand-Binding and Basal Priming of Transactivation: A Cell Culture Model

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.

Homodimerization of glucocorticoid receptor from single cells investigated using fluorescence correlation spectroscopy and microwells

“…The maximum amount of endogenous GR in COS-1 is 61 nM based on the previously reported 111 000 molecules per cell (1420 fmol/mg) [17] and our finding of a 3 pL cell volume. Moreover, 16 200 fmol/mg in cytotrophoblasts [18] was calculated to be 701 nM using the same method.…”

“…(13)(14)(15), the fractions of monomeric and homodimeric EGFP-GR (F m and F d ) are shown as Eqs. (16) and (17).…”

Medroxyprogesterone Acetate Differentially Regulates Interleukin (IL)-12 and IL-10 in a Human Ectocervical Epithelial Cell Line in a Glucocorticoid Receptor (GR)-dependent Manner

“…Considering that P 4 and MPA have previously been shown to have different binding affinities and transcriptional activities via the GR (11,92), it was surprising that these progestogens displayed similar GR-mediated effects in the Ect1/E6E7 cell line. However, the relative affinities of P 4 and MPA for the GR may be different in this cell line compared with other cell lines, as it has previously been shown that the concentration of GR determines the binding affinity of a ligand for the receptor (93). Moreover, as we have previously shown that P 4 and MPA differentially regulate cytokine gene expression in a cell-and promoter-specific manner (39), discrepancies between the results from this study and others using synthetic GRE-containing promoters in COS-1 cells, for example (11), may be due to either cell-or promoter-specific effects.…”

The Emerging Role of TPR-Domain Immunophilins in the Mechanism of Action of Steroid Receptors

“…The course of DNAprotein assembly has been discussed for a number of transcription factors, and the consensus is that there are three possible mechanisms. One mechanism implies that receptor dimerization could take place in the cytoplasm before its nuclear translocation [100,101]. The dimer pathway model sustains that transcription factors must dimerize on the nuclear environment to permit DNA binding, and the monomer pathway postulates that two monomers can bind sequentially to promoter sequences and protein assembly takes place during the in situ dimerization [102,103].…”

“…The dimer pathway model sustains that transcription factors must dimerize on the nuclear environment to permit DNA binding, and the monomer pathway postulates that two monomers can bind sequentially to promoter sequences and protein assembly takes place during the in situ dimerization [102,103]. Also, it has been suggested that the level of receptor expression affects the formation of homodimers [101]. Nevertheless, it is still unknown whether steroid receptor homodimers form before or as a result of binding to hormone-responsive elements because some studies have showed some evidence favoring the monomer pathway model [104,105], whereas others support the dimer pathway model [106][107][108].…”