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Novel role for receptor dimerization in post-translational processing and turnover of the GR?.

ABSTRACT: Glucocorticoids (GCs), acting via the glucocorticoid receptor (GR?), remain the mainstay therapeutic choice for the treatment of inflammation. However, chronic GC use, aside from generating undesirable side-effects, results in GR? down-regulation, often coupled to a decrease in GC-responsiveness, which may culminate in acquired GC resistance. The current study presents evidence for a novel role of the dimerization state of the GR? in mediating GC-mediated GR? turnover. Through comparing the effects of dimerization promoting GCs on down-regulation of a transfected human wild type GR? (hGRwt) or a dimerization deficient GR? mutant (hGRdim), we established that a loss of receptor dimerization restricts GR? turnover, which was supported by the use of the dimerization abrogating Compound A (CpdA), in cells containing endogenous GR?. Moreover, we showed that the dimerization state of the GR? influenced the post-translational processing of the receptor, specifically hyper-phosphorylation at Ser404, which influenced the interaction of GR? with the E3 ligase, FBXW7?, thus hampering receptor turnover via the proteasome. Lastly, the restorative effects of CpdA on the GR? pool, in the presence of Dex, were demonstrated in a combinatorial treatment protocol. These results expand our understanding of factors that contribute to GC-resistance and may be exploited clinically.

SUBMITTER: Wilkinson L

PROVIDER: S-EPMC6155283 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Novel role for receptor dimerization in post-translational processing and turnover of the GRα.

Wilkinson Legh L Verhoog Nicolette N Louw Ann A

Scientific reports 20180924 1

Glucocorticoids (GCs), acting via the glucocorticoid receptor (GRα), remain the mainstay therapeutic choice for the treatment of inflammation. However, chronic GC use, aside from generating undesirable side-effects, results in GRα down-regulation, often coupled to a decrease in GC-responsiveness, which may culminate in acquired GC resistance. The current study presents evidence for a novel role of the dimerization state of the GRα in mediating GC-mediated GRα turnover. Through comparing the effe ...[more]

PMID: 30250038

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Project description:Pharmacologically, glucocorticoids, which mediate their effects via the glucocorticoid receptor (GR), are a most effective therapy for inflammatory diseases despite the fact that chronic use causes side-effects and acquired GC resistance. The design of drugs with fewer side-effects and less potential for the development of resistance is therefore considered crucial for improved therapy. Dimerization of the GR is an integral step in glucocorticoid signaling and has been identified as a possible molecular site to target for drug development of anti-inflammatory drugs with an improved therapeutic index. Most of the current understanding regarding the role of GR dimerization in GC signaling derives for dimerization deficient mutants, although the role of ligands biased toward monomerization has also been described. Even though designing for loss of dimerization has mostly been applied for reduction of side-effect profile, designing for loss of dimerization may also be a fruitful strategy for the development of GC drugs with less potential to develop GC resistance. GC-induced resistance affects up to 30% of users and is due to a reduction in the GR functional pool. Several molecular mechanisms of GC-mediated reductions in GR pool have been described, one of which is the autologous down-regulation of GR density by the ubiquitin-proteasome-system (UPS). Loss of GR dimerization prevents autologous down-regulation of the receptor through modulation of interactions with components of the UPS and post-translational modifications (PTMs), such as phosphorylation, which prime the GR for degradation. Rational design of conformationally biased ligands that select for a monomeric GR conformation, which increases GC sensitivity through improving GR protein stability and increasing half-life, may be a productive avenue to explore. However, potential drawbacks to this approach should be considered as well as the advantages and disadvantages in chronic vs. acute treatment regimes.

Project description:Post-translational modifications (PTMs) on the N-terminal tails of histones and histone variants regulate distinct transcriptional states and nuclear events. Whereas the functional effects of specific PTMs are the current subject of intense investigation, most studies characterize histone PTMs/variants in a non-temporal fashion and very few studies have reported kinetic information about these histone forms. Previous studies have used radiolabeling, fluorescence microscopy and chromatin immunoprecipitation to determine rates of histone turnover, and have found interesting correlations between increased turnover and increased gene expression. Therefore, histone turnover is an understudied yet potentially important parameter that may contribute to epigenetic regulation. Understanding turnover in the context of histone modifications and sequence variants could provide valuable additional insight into the function of histone replacement.In this study, we measured the metabolic rate of labeled isotope incorporation into the histone proteins of HeLa cells by combining stable isotope labeling of amino acids in cell culture (SILAC) pulse experiments with quantitative mass spectrometry-based proteomics. In general, we found that most core histones have similar turnover rates, with the exception of the H2A variants, which exhibit a wider range of rates, potentially consistent with their epigenetic function. In addition, acetylated histones have a significantly faster turnover compared with general histone protein and methylated histones, although these rates vary considerably, depending on the site and overall degree of methylation. Histones containing transcriptionally active marks have been consistently found to have faster turnover rates than histones containing silent marks. Interestingly, the presence of both active and silent marks on the same peptide resulted in a slower turnover rate than either mark alone on that same peptide. Lastly, we observed little difference in the turnover between nearly all modified forms of the H3.1, H3.2 and H3.3 variants, with the notable exception that H3.2K36me2 has a faster turnover than this mark on the other H3 variants.Quantitative proteomics provides complementary insight to previous work aimed at quantitatively measuring histone turnover, and our results suggest that turnover rates are dependent upon site-specific post-translational modifications and sequence variants.

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Novel role for receptor dimerization in post-translational processing and turnover of the GR?.

Publications

Novel role for receptor dimerization in post-translational processing and turnover of the GRα.

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