ESTROGEN-RECEPTOR RESIDUES REQUIRED FOR STEREOSPECIFIC LIGAND RECOGNITION AND ACTIVATION

The mouse estrogen receptor (mER) has been shown to exhibit stereospec ific binding of certain stilbene estrogen agonists. The region of the mER involved in the stereochemical recognition of ligands was further defined using a stilbene isomer, Indenestrol B (IB). The IB compound h as a chiral carbon bearing an ethyl substituent, and the wild type ute rine mER has been shown to bind the enantiomers, IB-S and IB-R, with s imilar affinity. The wild type mER expressed in yeast exhibited a very minor preference for IB-S in transactivation (1.5-fold lower half-max imal dose than IB-R). The IB enantiomers could then be used to determi ne whether stereochemically distinct compounds with similar transcript ional activity utilize different amino acids in AF-2 for transactivati on. Mutant mERs with glycine substitutions at Met521, His528, Met532, and Val537 were expressed in yeast and measured for IB-S- and IB-R-ind uced transactivation and ligand binding. The M532G mER showed a 124-fo ld and 50-fold reduction in transactivation induced by IB-S and IB-R, respectively, without a corresponding change in their ligand-binding a ffinities. Therefore, Met532 is required for transactivation induced b y both IB enantiomers but does not discriminate based on stereospecifi city. In contrast, the H528G mER displayed a gross change in stereospe cific ligand recognition as illustrated by a 11O-fold reduction in tra nsactivation by IB-S and only a 8.8-fold decrease in activity by IB-R. As a result, H528G mER displayed a switch in ligand preference such t hat IB-R was now 8-fold more active than IB-S in transactivation. Ther efore, His528 appears largely involved in transactivation specifically induced by IB-S but has a minimal influence in IB-S ligand binding. T he remaining mutant mERs displayed wild type ligand binding and transa ctivation properties for the IB enantiomers illustrating no stereospec ific recognition. These results imply that individual IB enantiomers b ind to the mER with similar affinity but utilize at least one differen t amino acid within the AF-2 domain for signal transduction. The bindi ng of stereochemically distinct ligands may alter the tertiary structu re of the mER and cause repositioning of the AF-2 region that mediates transcription of specific genes and/or affect the binding of receptor -associated proteins, such as coactivators, which could influence tran scription.