title: 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.
abstract: The syndrome of familial or sporadic glucocorticoid resistance is characterized by hypercortisolism without the clinical stigmata of Cushing syndrome. This condition is usually caused by mutations of the human GR, a ligand-activated transcription factor that shuttles between the cytoplasm and the nucleus. A pathological human mutant receptor, in which Ile was replaced by Asn at position 559, had negligible ligand binding, was transcriptionally extremely weak, and exerted a transdominant negative effect on the transactivational activity of the wild-type GR, causing severe glucocorticoid resistance in the heterozygous state. To understand the mechanism of this mutant's trans-dominance, we constructed several N-terminal GR fusion chimeras to green fluorescent protein (GFP) and demonstrated that their transactivational activities were similar to those of the original proteins. The GFP-human (h) GRalphaI559N chimera was predominantly localized in the cytoplasm, and only high doses or prolonged glucocorticoid treatment triggered complete nuclear import that took 180 vs. 12 min for GFP-hGRalpha. Furthermore, hGRalphaI559N inhibited nuclear import of the wild-type GFP-hGRalpha, suggesting that its trans-dominant activity on the wild-type receptor is probably exerted at the process of nuclear translocation. As the ligand-binding domain (LBD) of the GR appears to play an important role in its nucleocytoplasmic shuttling, we also examined two additional GR-related fusion proteins. The natural hGR isoform beta (GFP-hGRbeta), containing a unique LBD, was transactivation-inactive, moderately trans-dominant, and localized instantaneously and predominantly in the nucleus; glucocorticoid addition did not change its localization. Similarly, GFP-hGR514, lacking the entire LBD, was instantaneously and predominantly localized in the nucleus regardless of presence of glucocorticoids. Using a cell fusion system we demonstrated that nuclear export of GFP-hGRalphaI559N (250 min) and GFP-hGRbeta (300 min) was drastically impaired compared with that of GFP-hGRalpha (50 min) and GFP-hGR514 (50 min), suggesting that an altered LBD may impede the exit of the 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 transcription.
authors.affiliation: Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1583, USA. kinot@mail.nih.gov
authors.forename: T
authors.initials: T
authors.lastname: Kino
authors.forename: R H
authors.initials: RH
authors.lastname: Stauber
authors.forename: J H
authors.initials: JH
authors.lastname: Resau
authors.forename: G N
authors.initials: GN
authors.lastname: Pavlakis
authors.forename: G P
authors.initials: GP
authors.lastname: Chrousos
mesh_terms: D002459:Cell Fusion; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism / Q000502:physiology*; D003907:Dexamethasone / Q000494:pharmacology; D005799:Genes, Dominant / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D009154:Mutation / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection; D014178:Translocation, Genetic / Q000235:genetics*
chemical_list: D008024:Ligands; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone