Pathologic human GR mutant has a transdominant negative effect on the wild-type GR by inhibiting its translocation into the nucleus: Importance of the ligand-binding domain for intracellular GR trafficking

The syndrome of familial or sporadic glucocorticoid resistance is character ized by hypercortisolism without the clinical stigmata of Cushing syndrome. This condition is usually caused by mutations of the human GR, a ligand-ac tivated transcription factor that shuttles between the cytoplasm and the nu cleus. A pathological human mutant receptor, in which Ile was replaced by A sn at position 559, had negligible ligand binding, was transcriptionally ex tremely weak, and exerted a transdominant negative effect on the transactiv ational activity of the wild-type GR, causing severe glucocorticoid resista nce in the heterozygous state. To understand the mechanism of this mutant's trans-dominance, we constructed several N-terminal GR fusion chimeras to g reen fluorescent protein (GFP) and demonstrated that their transactivationa l activities were similar to those of the original proteins. The GFP-human (h) GR alpha I559N chimera was predominantly localized in the cytoplasm, an d only high doses or prolonged glucocorticoid treatment triggered complete nuclear import that took 180 vs. 12 min for GFP-hGR alpha. Furthermore, hGR alpha I559N inhibited nuclear import of the wild-type GFP-hGR alpha, sugge sting that its trans-dominant activity on the wild-type receptor is probabl y exerted at the process of nuclear translocation. As the ligand-binding do main (LBD) of the GR appears to play an important role in its nucleocytopla smic shuttling, we also examined two additional GR-related fusion proteins. The natural hGRisoform beta (GFP-hGR beta), containing a unique LBD, was t ransactivation-inactive, moderately trans-dominant, and localized instantan eously and predominantly in the nucleus; glucocorticoid addition did not ch ange its localization. Similarly, GFP-hGR514, lacking the entire LBD, was i nstantaneously and predominantly localized in the nucleus regardless of pre sence of glucocorticoids. Using a cell fusion system we demonstrated that n uclear export of GFP-hGR alpha I559N (250 min) and GFP-hGR beta (300 min) w as drastically impaired compared with that of GFP-hGR alpha (50 min) and GF P-hGR514 (50 min), suggesting that an altered LBD may impede the exit of th e GR from the nucleus. We conclude that the trans-dominant negative effect of the pathological mutant is exerted primarily at the translocation step, whereas that of the natural isoform beta is exerted at the level of transcr iption.