Background: Primary generalised glucocorticoid resistance (PGGR) is a rare condition characterised by tissue insensitivity to glucocorticoids owing to inactivating mutations of the hGR gene. A new case of PGGR was reported in a patient with an adrenal incidentaloma harboring a novel heterozygous point mutation in the hGR gene, which resulted in threonine (T) to isoleucine (I) substitution at amino acid position 556 of the receptor.
Objective and hypotheses: To elucidate the molecular mechanisms of action of the hGRαT556I.
Methods and results: Compared with the wild-type receptor (hGRαWT), the mutant receptor hGRαT556I demonstrated a 50% 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. Transrepression assays showed that the hGRαT556I displayed enhanced ability to transrepress the nuclear factor (NF)-κB signaling pathway. Dexamethasone-binding assays demonstrated that the affinity of the hGRαT556I for the ligand was 50% lower than that of the hGRαWT (Kd: 21.3±4.09 nM vs 10.8±0.99 nM, P<0.05). In vitro binding assays revealed no significant difference in the association of hGRαT556I with the glucocorticoid-response elements (GREs) following exposure to dexamethasone. In subcellular localization and nuclear translocation studies, both the hGRαWT and the hGRαT556I were predominantly localized in the cytoplasm of cells in the absence of ligand. Addition of dexamethasone resulted in slower nuclear translocation of the hGRαT556I compared with the hGRαWT (53.3±1.8 min vs 15.5±0.46 min, P<0.05). GST pull-down assays showed that the hGRαT556I interacted with the GRIP1 coactivator mostly through its AF-1 domain. Structural biology studies demonstrated that the T556I mutation affected mildly the local 3D arrangement of the receptor and the electrostatic surface of the region.
Conclusions: The mutant receptor hGRαT556I impairs glucocorticoid signal transduction through multiple molecular mechanisms.
01 - 03 Oct 2015
European Society for Paediatric Endocrinology