ESTROGEN-RECEPTORS - LIGAND DISCRIMINATION AND ANTIESTROGEN ACTION

We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the estrogen re ceptor (ER) important in hormone binding, ligand discrimination betwee n estrogens and antiestrogens, and transcriptional activation. Affinit y labelling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In die abs ence of a cysteine at 530 (i.e. Cys530A1a mutant), C381 becomes the si te of estrogen-competible tamoxifen aziridine labeling. Hence these tw o residues, although far apart in the primary linear sequence of the E R protein, must be close in the three-dimensional structure of the pro tein, in the ER ligand binding pocket, so that the ligand can reach ei ther site. Site-directed and region-specific chemical mutagenesis have identified a region around C530 important in discrimination between e strogens and antiestrogens, and other mutants have allowed identificat ion of residues important in hormone-dependent transcriptional activat ion. Some transcriptionally inactive ER mutants also function as poten t dominant negative ERs, suppressing the activity of wild-type ERs at low concentrations. These studies are beginning to provide a more deta iled picture of the ER hormone binding domain and amino acids importan t in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since t here is now substantial evidence for a mixture of wild-type and varian t ERs in breast Cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understandi ng of the possible role Of variant receptors in altered response or re sistance to antiestrogen and endocrine therapy in breast cancer.