Background: Primary generalized glucocorticoid resistance (PGGR) or Chrousos syndrome is a rare familial or sporadic condition caused by mutations in the hGR gene, which reduce tissue sensitivity to glucocorticoids. A new case of PGGR caused by a novel heterozygous point mutation in the hGR gene, which resulted in threonine (T) to isoleucine (I) substitution at amino acid position 556 in the ligand-binding domain of the receptor, was recently reported in a patient with an adrenal incidentaloma.
Objective and Hypotheses: To delineate the molecular mechanisms of action of the natural mutant receptor hGRαT556I.
Methods and results: Compared with the WT receptor (hGRαWT), the mutant receptor hGRαT556I demonstrated a 22% reduction in its ability to transactivate the glucocorticoid-inducible MMTV promoter in response to dexamethasone, and did not exert a dominant negative effect upon the hGRαWT. Western blot analyses showed equal protein expression of hGRαWT and hGRαT556I. Dexamethasone-binding assays showed that the affinity of the mutant receptor hGRαT556I for the ligand was 50% lower than that of the hGRαWT (21.3±4.09 vs 10.8±0.99 nM). In subcellular localization and nuclear translocation studies, both the hGRαWT and hGRαT556I were predominantly localized in the cytoplasm of cells in the absence of ligand. Addition of dexamethasone resulted in slower translocation of the hGRαT556I into the nucleus (50 min), compared to the hGRαWT (15 min). The 3D molecular modeling study of the T556I mutation revealed that the OH moiety of T556 established strong hydrogen bonding interactions with the O group of P637 backbone. The T556I mutation led to the disruption of the hydrogen bond and significant relocation of the P637 bearing loop, thus affecting mildly the local 3D arrangement of the receptor and hence the electrostatic surface of the region.
Conclusions: The natural mutant receptor hGRαT556I alters glucocorticoid signal transduction through multiple molecular mechanisms.
20 - 22 Sep 2014
European Society for Paediatric Endocrinology