title: Genetic analysis of the N-terminal end of the glucocorticoid receptor hormone binding domain.
abstract: Four site-directed missense mutations were constructed at the N-terminal end of the mouse glucocorticoid receptor (GR) hormone binding domain. This small subdomain is highly conserved among the steroid hormone receptors and is within a larger subregion believed to be important for hormone binding, transcriptional activation, and hsp90 binding. The ability of mutant and wild type GR to activate a reporter gene in response to various concentrations of dexamethasone was examined in transiently transfected COS-7 cells. Mutant GR species V544G (valine-544 changes to glycine) and V549G activated the reporter gene to approximately the same extent as wild type GR, but required approx. 7 and 23 times greater hormone concentrations, respectively. In contrast, double mutant LL541/2GG (leucines changed to glycines) could not activate transcription even at 10 microM dexamethasone or deacylcortivazol, while E543A (glutamic acid to alanine) was functionally indistinguishable from wild type GR. GR mutants LL541/2GG and V549G had reduced abilities to bind covalently to affinity label dexamethasone 21-mesylate. The partially and fully functional mutant GR species had no deficiency in transcriptional transactivation activity in the presence of saturating concentrations of agonist.
authors.affiliation: Department of Pathology, University of Southern California Health Sciences Center, Los Angeles 90033.
authors.forename: J
authors.initials: J
authors.lastname: Milhon
authors.forename: K
authors.initials: K
authors.lastname: Kohli
authors.forename: M R
authors.initials: MR
authors.lastname: Stallcup
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D003907:Dexamethasone / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation / Q000502:physiology*; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D000345:Affinity Labels; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D004247:DNA; C031710:dexamethasone 21-methanesulfonate