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. 2013 Apr;98(4):E790-5.
doi: 10.1210/jc.2012-3549. Epub 2013 Feb 20.

A Novel Point Mutation in the DNA-binding Domain (DBD) of the Human Glucocorticoid Receptor Causes Primary Generalized Glucocorticoid Resistance by Disrupting the Hydrophobic Structure of Its DBD

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A Novel Point Mutation in the DNA-binding Domain (DBD) of the Human Glucocorticoid Receptor Causes Primary Generalized Glucocorticoid Resistance by Disrupting the Hydrophobic Structure of Its DBD

Michael L Roberts et al. J Clin Endocrinol Metab. .
Free PMC article

Abstract

Context: Primary generalized glucocorticoid resistance is a rare genetic condition characterized by partial end-organ insensitivity to glucocorticoids. Most affected subjects present with clinical manifestations of mineralocorticoid and androgen excess. The condition has been associated with inactivating mutations in the human glucocorticoid receptor (hGR) gene, which impair the molecular mechanisms of hGRα action, thereby reducing tissue sensitivity to glucocorticoids.

Objective: ΤHE aim of our study was to investigate the molecular mechanisms through which one previously described natural heterozygous V423A mutation, the second mutation detected in the DNA-binding domain (DBD) of the hGRα, affects glucocorticoid signal transduction.

Design and results: Compared with the wild-type receptor, hGRαV423A demonstrated a 72% reduction in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to dexamethasone. The hGRαV423A receptor showed a significant reduction in its ability to bind to glucocorticoid-response elements of glucocorticoid-responsive genes, owing to structural alterations of the DBD confirmed by computer-based structural analysis. In addition, hGRαV423A demonstrated a 2.6-fold delay in nuclear translocation following exposure to the ligand, although it did not exert a dominant negative effect on the wild-type hGRα, had a similar affinity to the ligand with the wild-type receptor, and displayed a normal interaction with the GRIP1 coactivator in vitro.

Conclusions: The natural mutant receptor hGRαV423A causes primary generalized glucocorticoid resistance by affecting multiple steps in the cascade of glucocorticoid receptor action, which primarily involve decreased ability to bind to target glucocorticoid response elements and delayed translocation into the nucleus.

Figures

Figure 1.
Figure 1.
Transcriptional defects of hGRαV423A. A, hGRαV423A demonstrated a 72% reduction in the ability to transactivate the mouse mammary tumor virus promoter in response to dexamethasone. CV-1 cells were transfected with the wild-type (WT) or the mutant (V423A) hGRα-expressing plasmid together with reporter plasmids and were treated with indicated concentrations of dexamethasone. Circles indicate mean and SEM values of the firefly luciferase activity corrected with the renilla luciferase activity obtained in at least 5 independent experiments. **P < .01, compared to the wild-type hGRα. B and C, Ability of hGRαV423A to bind to DNA. The mutant receptor hGRαV423A demonstrates reduced binding to GREs of endogenous hGR target genes. HCT116 human colon carcinoma cells defective in endogenous GR proteins were transfected with WT hGRα-expressing or the mutant receptor hGRαV423A-expressing plasmid and were treated with 10−6 M of dexamethasone. Cross-linked chromatin from the cells was used in the ChIP assays employing anti-GRα antibody or control IgG. Primers specific for the amplification of GREs of the GILZ (B) and G6Pase (C) genes were used. Bars demonstrate mean and SEM values of specific enrichments (fold differences) vs input (ChIP/Input = 2Input Ct−ChIP Ct) obtained in at least 3 independent experiments. *P = .010 and **P = .046. Ct, threshold cycle.
Figure 2.
Figure 2.
The V423A mutation alters the specific binding of hGR DBD to GREs. (A) Replacement of valine (V) by alanine (A) at position 423 in the hGR DBD reduces the hydrophobic nature of the site and permits water to diffuse into the ion-binding region of the protein, where it is captured by hydrogen bonds to C424 and C441 among others. Water is almost never observed in this area in the WT simulation. The structures in green indicate the WT receptor, whereas those in blue represent the mutant receptor. Water molecules are indicated by capsule-like molecules in gray and red. B, The most significant changes in hydrogen bonding in this area occur to C424, C441, and R477. C and D, The energy penalty in the V423A mutant for these nonnative interactions is greater than 15 kcal/mol. Given that R477 plays a key role for binding of the DBD of the receptor to GREs, the presence of the V423A mutation may explain the reduced binding of hGRαV423A to DNA and the subsequent reduction of its transcriptional activity.

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