Background: Generalized glucocorticoid resistance is a rare condition characterized by partial, end-organ insensitivity to glucocorticoids, compensatory elevations in ACTH and cortisol secretion, and increased production of adrenal steroids with androgenic and/or mineralocorticoid activity. We identified a new case of glucocorticoid resistance caused by a novel, heterozygous point mutation at nucleotide position 2209 (T → C) of the hGRα gene, resulting in phenylalanine (F) to leucine (L) substitution at amino acid 737 within helix 11 in the ligand-binding domain of the protein. We studied the molecular mechanisms through which the mutant receptor hGRαF737L impairs glucocorticoid signal transduction.
Methods-Results: In transient transfection assays, the mutant receptor hGRαF737L demonstrated a 1.8-fold reduction in its ability to transactivate the glucocorticoid-inducible MMTV promoter in response to dexamethasone compared to the wild-type hGRα. Coexpression of a constant amount of hGRα and progressively increasing concentrations of hGRαF737L did not result in a dose-dependent decrease in the transcriptional activity of hGRα, suggesting that the mutant receptor does not exert a dominant negative effect upon the wild-type receptor. Dexamethasone-binding assays showed that hGRαF737L displayed a 1.9-fold reduction in the affinity for ligand compared to the wild-type receptor (Kd: 13.9±1.7 vs. 7.8±0.3 nM, P=0.006). Western blot analyses demonstrated no differences in the expression of hGRα and hGRαF737L proteins. In nuclear translocation studies, the mutant receptor displayed an 11.9-fold delay in nuclear translocation compared to the wild-type hGRα. Finally, the mutant receptor hGRαF737L preserved its ability to bind to DNA and displayed an abnormal interaction with the GRIP1 coactivator in vitro.
Conclusions: The mutant receptor hGRαF737L causes generalized glucocorticoid resistance because of decreased affinity for ligand, marked delay in nuclear translocation and abnormal interaction with the p160 coactivators. These findings confirm the importance of the C-terminus of the ligand-binding domain of the receptor in conferring transactivational activity.