GENETIC-ANALYSIS OF THE N-TERMINAL END OF THE GLUCOCORTICOID RECEPTORHORMONE-BINDING DOMAIN

Four site-directed missense mutations were constructed at the N-termin al end of the mouse glucocorticoid receptor (GR) hormone binding domai n. This small subdomain is highly conserved among the steroid hormone receptors and is within a larger subregion believed to be important fo r hormone binding, transcriptional activation, and hsp90 binding. The ability of mutant and wild type GR to activate a reporter gene in resp onse to various concentrations of dexamethasone was examined in transi ently transfected COS-7 cells. Mutant GR species V544G (valine-544 cha nged to glycine) and V549G activated the reporter gene to approximatel y the same extent as wild type GR, but required approx. 7 and 23 times greater hormone concentrations, respectively. In contrast, double mut ant LL541/2GG (leucines changed to glycines) could not activate transc ription even at 10 mu M dexamethasone or deacylcortivazol, while E543A (glutamic acid to alanine) was functionally indistinguishable from wi ld type GR. GR mutants LL541/2GG and V549G had reduced abilities to bi nd covalently to affinity label dexamethasone 21-mesylate. The partial ly and fully functional mutant GR species had no deficiency in transcr iptional transactivation activity in the presence of saturating concen trations of agonist.