scholarly journals The Helix 1-3 Loop in the Glucocorticoid Receptor LBD Is a Regulatory Element for FKBP Cochaperones

2013 ◽  
Vol 27 (7) ◽  
pp. 1020-1035 ◽  
Author(s):  
Carmel Cluning ◽  
Bryan K. Ward ◽  
Sarah L. Rea ◽  
Ajanthy Arulpragasam ◽  
Peter J. Fuller ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Receptor Activity ◽  
Hek293 Cells ◽  
Global Changes ◽  
Nuclear Targeting ◽  
Receptor Complexes

Abstract The heat-shock protein 90 (Hsp90) cochaperone FK506-binding protein 52 (FKBP52) upregulates, whereas FKBP51 inhibits, hormone binding and nuclear targeting of the glucocorticoid receptor (GR). Decreased cortisol sensitivity in the guinea pig is attributed to changes within the helix 1 to helix 3 (H1-H3) loop of the guinea pig GR (gpGR) ligand-binding domain. It has been proposed that this loop serves as a contact point for FKBP52 and/or FKBP51 with receptor. We examined the role of the H1-H3 loop in GR activation by FKBP52 using a Saccharomyces cerevisiae model. The activity of rat GR (rGR) containing the gpGR H1-H3 loop substitutions was still potentiated by FKBP52, confirming the loop is not involved in primary FKBP52 interactions. Additional assays also excluded a role for other intervening loops between ligand-binding domain helices in direct interactions with FKBP52 associated with enhanced receptor activity. Complementary studies in FKBP51-deficient mouse embryo fibroblasts and HEK293 cells demonstrated that substitution of the gpGR H1-H3 loop residues into rGR dramatically increased receptor repression by FKBP51 without enhancing receptor-FKBP51 interaction and did not alter recruitment of endogenous Hsp90 and the p23 cochaperone to receptor complexes. FKBP51 suppression of the mutated rGR did not require FKBP51 peptidylprolyl cis-trans isomerase activity and was not disrupted by mutation of the FK1 proline-rich loop thought to mediate reciprocal FKBP influences on receptor activity. We conclude that the gpGR-specific mutations within the H1-H3 loop confer global changes within the GR-Hsp90 complex that favor FKBP51 repression over FKBP52 potentiation, thus identifying the loop as an important target for GR regulation by the FKBP cochaperones.

scholarly journals SAT-718 Inhibition of Androgen Receptor Activity by DDE Is Affected by Mutations in the BF3 Site

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Andrea Lozano ◽  
Evangelia Kotsikorou ◽  
Frank B Dean
Keyword(s):  
Androgen Receptor ◽  
Ligand Binding ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Hek293 Cells ◽  
Luciferase Assay ◽  
Breakdown Product ◽  
Reporter System ◽  
Surface Binding ◽  
Endocrine Disrupting

Abstract The androgen receptor (AR) plays an important role in the development of the male phenotype and traits. Some diphenyl compounds inhibit AR activity by binding to a hydrophobic surface binding site, BF3. A similar diphenyl structure is found in 4,4’ DDT and its breakdown product 4,4’ DDE. Previous results showed that DDT and DDE induced the release of bound dihydrotestosterone from the AR ligand binding domain, with IC50 values ranging from 54 to 82uM. This suggested that DDT and related compounds may act as endocrine disrupting chemicals by binding to the BF3 site and inducing allosteric changes in the AR structure, disrupting binding of the steroid to the ligand binding domain. Here, an AR reporter system was transiently transfected into HEK293 cells and AR activity was measured using a dual luciferase assay. The system was used to measure the response of the AR protein to varying concentrations of dihydrotestosterone in the presence and absence of DDE. DDE inhibited the activation of AR by dihydrotestosterone under these conditions. Five mutant AR genes with amino acid changes in the BF3 site were tested for alterations in the ability of DDE to disrupt AR activity. The five mutations tested were F673K, F673W, G724R, G724M, and L830D. The ability of DDE to inhibit AR activity was reduced by the mutations in the BF3 site. These results suggest that DDE acts as an endocrine disrupting chemical (EDC) by binding to the BF3 site and allosterically regulating AR activity.

scholarly journals Folding and stability of the ligand-binding domain of the glucocorticoid receptor

2002 ◽  
Vol 11 (8) ◽  
pp. 1926-1936 ◽  
Author(s):  
Stephen H. McLaughlin ◽  
Sophie E. Jackson
Keyword(s):  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Binding Domain ◽  
Ligand Binding Domain

scholarly journals Molecular Determinants of Glucocorticoid Receptor Mobility in Living Cells: the Importance of Ligand Affinity

2003 ◽  
Vol 23 (6) ◽  
pp. 1922-1934 ◽  
Author(s):  
Marcel J. M. Schaaf ◽  
John A. Cidlowski
Keyword(s):  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Conformational Change ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Dependent Manner ◽  
Dna Binding Domain ◽  
Ligand Affinity ◽  
High Affinity

ABSTRACT The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), which is activated upon ligand binding, and can alter the expression of target genes either by transrepression or transactivation. We have applied FRAP (fluorescence recovery after photobleaching) to quantitatively assess the mobility of the yellow fluorescent protein (YFP)-tagged human GR α-isoform (hGRα) in the nucleus of transiently transfected COS-1 cells and to elucidate determinants of its mobility. Addition of the high-affinity agonist dexamethasone markedly decreases the mobility of the receptor in a concentration-dependent manner, whereas low-affinity ligands like corticosterone decrease the mobility to a much lesser extent. Analysis of other hGRα ligands differing in affinity suggests that it is the affinity of the ligand that is a major determinant of the decrease in mobility. Similar results were observed for two hGRα antagonists, the low-affinity antagonist ZK98299 and the high-affinity antagonist RU486. The effect of ligand affinity on mobility was confirmed with the hGRα mutant Q642V, which has an altered affinity for triamcinolone acetonide, dexamethasone, and corticosterone. Analysis of hGRα deletion mutants indicates that both the DNA-binding domain and the ligand-binding domain of the receptor are required for a maximal ligand-induced decrease in receptor mobility. Interestingly, the mobility of transfected hGRα differs among cell types. Finally, the proteasome inhibitor MG132 immobilizes a subpopulation of unliganded receptors, via a mechanism requiring the DNA-binding domain and the N-terminal part of the ligand-binding domain. Ligand binding makes the GR resistant to the immobilizing effect of MG132, and this effect depends on the affinity of the ligand. Our data suggest that ligand binding induces a conformational change of the receptor which is dependent on the affinity of the ligand. This altered conformation decreases the mobility of the receptor, probably by targeting the receptor to relatively immobile nuclear domains with which it transiently associates. In addition, this conformational change blocks immobilization of the receptor by MG132.

Enhancing the Stability and Solubility of the Glucocorticoid Receptor Ligand-Binding Domain by High-Throughput Library Screening

2010 ◽  
Vol 403 (4) ◽  
pp. 562-577 ◽  
Author(s):  
Tobias Seitz ◽  
Ralf Thoma ◽  
Guillaume A. Schoch ◽  
Martine Stihle ◽  
Jörg Benz ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
High Throughput ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Library Screening ◽  
Receptor Ligand ◽  
The Stability
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