MUTATIONAL ANALYSIS OF THE ESTROGEN-RECEPTOR LIGAND-BINDING DOMAIN - INFLUENCE OF LIGAND STRUCTURE AND STEREOCHEMISTRY ON TRANSACTIVATION

The mouse estrogen receptor (mER) exhibits ligand stereochemical speci ficity for indenestrol A (IA), a stilbestrol estrogen. IA has a chiral C3 methyl group, and the mER preferentially binds the S-enantiomer (I A-S). resulting in elevated biological activity when compared with the IA-R enantiomer. To elucidate the mechanisms for this stereochemical recognition, we have constructed a series of mERs with individual amin o acid substitutions at Met521, His528, Met532, and Val537. The abilit ies of yeast-expressed wild-type and mutant mERs to transactivate an e strogen-responsive reporter gene construct were measured in the presen ce of diethylstilbestrol (DES) and IA enantiomers. The concentration o f IA-S required to induce half-maximal transactivation by wild-type mE R was 10-fold lower than IA-R, which is attributed to the 15-fold grea ter binding affinity for IA-S. Wild-type mER displayed similar dose-re sponse curves for IA-R and demethyl IA, which lacks a C3 methyl group, demonstrating that the presence and correct orientation of the C3 met hyl group on the IA compound is required for high-affinity ligand bind ing and transcriptional activity. Each mutant exhibited a reduced pref erence for IA-S enantiomer with respect to transactivation, suggesting that this region of the mER functions in ligand stereochemical recogn ition and activation. A mutation at Met532 diminished DES- and IA-S-in duced transactivation by 7.5-fold and 40-fold respectively, with minim al change on their binding affinity. These data suggest that Met532 is required for transactivation induced by the potent agonist, IA-S, and the M532G mutation effectively uncouples IA-S ligand binding from tra nsactivation. Use of these stereochemically different ligands in combi nation with mutagenesis of the mER demonstrates that ligand structure could influence transactivation by specifically altering the conformat ion of the mER AF-2 region.