title: A novel, C-terminal dominant negative mutation of the GR causes familial glucocorticoid resistance through abnormal interactions with p160 steroid receptor coactivators.
abstract: Primary cortisol resistance is a rare, inherited or sporadic form of generalized end-organ insensitivity to glucocorticoids. Here, we report a kindred in which affected members had a heterozygous T to G base substitution at nucleotide 2373 of exon 9alpha of the GR gene, causing substitution of Ile by Met at position 747. This mutation was located close to helix 12, at the C terminus of the ligand-binding domain, which has a pivotal role in the formation of activation function (AF)-2, a subdomain that interacts with p160 coactivators. The affinity of the mutant GR for dexamethasone was decreased by about 2-fold, and its transcriptional activity on the glucocorticoid-responsive mouse mammary tumor virus promoter was compromised by 20- to 30-fold. In addition, the mutant GR functioned as a dominant negative inhibitor of wild-type receptor-induced transactivation. The mutant GR through its intact AF-1 domain bound to a p160 coactivator, but failed to do so through its AF-2 domain. Overexpression of a p160 coactivator restored the transcriptional activity and reversed the negative transdominant activity of the mutant GR. Interestingly, green fluorescent protein (GFP)-fused GRalphaI747M had a slight delay in its translocation from the cytoplasm into the nucleus and formed coarser nuclear speckles than GFP-fused wild-type GRalpha. Similarly, a GFP-fused p160 coactivator had a distinctly different distribution in the nucleus in the presence of mutant vs. wild-type receptor, presenting also as coarser speckling. We conclude that the mutation at amino acid 747 of the GR causes familial, autosomal dominant glucocorticoid resistance by decreasing ligand binding affinity and transcriptional activity, and by exerting a negative transdominant effect on the wild-type receptor. The mutant receptor has an ineffective AF-2 domain, which leads to an abnormal interaction with p160 coactivators and a distinct nuclear distribution of both.
authors.affiliation: Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, 10 Center Drive, MSC 1583, Bethesda, MD 20892, USA.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Vottero
authors.forename: Tomoshige
authors.initials: T
authors.lastname: Kino
authors.forename: Herve
authors.initials: H
authors.lastname: Combe
authors.forename: Pierre
authors.initials: P
authors.lastname: Lecomte
authors.forename: George P
authors.initials: GP
authors.lastname: Chrousos
mesh_terms: D000293:Adolescent; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D019556:COS Cells; D002352:Carrier Proteins / Q000502:physiology*; D004268:DNA-Binding Proteins; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D005799:Genes, Dominant; D005938:Glucocorticoids / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000502:physiology*; D009687:Nuclear Proteins / Q000502:physiology*; D051958:Nuclear Receptor Coactivator 2; D029543:Nucleocytoplasmic Transport Proteins; D010375:Pedigree; D012333:RNA, Messenger / Q000502:physiology; D016601:RNA-Binding Proteins; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D011987:Receptors, Steroid / Q000502:physiology*; D014018:Tissue Distribution; D014157:Transcription Factors / Q000494:pharmacology / Q000502:physiology*; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D005938:Glucocorticoids; C491719:MYBBP1A protein, human; C498001:NCOA2 protein, human; D009687:Nuclear Proteins; D051958:Nuclear Receptor Coactivator 2; D029543:Nucleocytoplasmic Transport Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D014157:Transcription Factors