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 recepter gene in response to various concentrations of dexamethasone was examined in transiently transfected COS-7 cells. Mutant GR species V544G (valine-544 changed 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 μM 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.