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

Abstract

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.

Recent advances in the molecular mechanisms causing primary generalized glucocorticoid resistance

“…Most of the reported NR3C1 gene mutations are located in the LBD of the receptor, three of them, however, the hGRαV423A, the hGRαR469X and the hGRαR477H, having been identified in the DBD (Figure 3). 19,21,22,[31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] Over the last three decades, advances in molecular and structural biology have enabled the study of the molecular mechanisms through which the mutant hGRs impair glucocorticoid signal transduction and cause the variable clinical phenotype of Primary Generalized Glucocorticoid Resistance (Table 2).…”

“…[45][46][47] Specifically, we investigated: i) the ability of the mutant receptors to induce glucocorticoid-responsive genes through reporter assays; ii) the expression of the mutant receptors at the protein level via Western blotting; iii) the ability of the mutant receptors to exert a dominant negative effect upon the hGRα-mediated transcriptional activity using reporter assays; iv) the transrepressive activity of the mutant receptors upon the NF-κB-mediated transcriptional activity through reporter assays; v) the affinity of the mutant receptors for the ligand via dexamethasonebinding assays; vi) the subcellular localization of the mutant receptors in the absence of ligand and the time required to complete nuclear translocation following exposure to dexamethasone using green fluorescent protein (GFP)-fused plasmids; vii) the binding of the mutant receptors to GREs through in vitro binding assays; viii) the ability of the mutant receptors to interact with coactivators, such as the glucocorticoid receptor-interacting protein 1 (GRIP1) coactivator, using Glutathione-S-Transferase (GST)-pull down assays; and ix) the conformational changes of the mutant receptors causing Primary Generalized Glucocorticoid Resistance through computer-based 3-dimensional simulation using crystallographic data available in public. [45][46][47] The first patient was a 9-year-old boy who presented with anxiety, fatigue, and hypertension. 45 He harbored a novel heterozygous mutation in the NR3C1 gene that resulted in substitution of valine (V) by alanine (A) at amino acid position 423 in the LBD of the receptor.…”

“…[45][46][47] The first patient was a 9-year-old boy who presented with anxiety, fatigue, and hypertension. 45 He harbored a novel heterozygous mutation in the NR3C1 gene that resulted in substitution of valine (V) by alanine (A) at amino acid position 423 in the LBD of the receptor. 45 In vitro functional studies showed that …”

“…45 He harbored a novel heterozygous mutation in the NR3C1 gene that resulted in substitution of valine (V) by alanine (A) at amino acid position 423 in the LBD of the receptor. 45 In vitro functional studies showed that …”

“…45 Structural biology studies highlighted the critical role of the hydrophobic valine at this position within the first zinc finger of the DBD of the receptor. The hydrophobic nature of valine at amino acid position 423 protects the four zinc-binding cysteines (C421, C424, C438, and C441) from the destructive diffusion of water molecules.…”