HORMONE-BINDING AND TRANSCRIPTION ACTIVATION BY ESTROGEN-RECEPTORS - ANALYSES USING MAMMALIAN AND YEAST SYSTEMS

We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the human estro gen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. A ffinity labeling studies with the antiestrogen, tamoxifen aziridine an d the estrogen, ketononestrol azirdine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at a 530 (i.e. C530A mutant), C381 becomes the site of estrogen-competible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the p rotein in the ER ligand binding pocket, so that the ligand can reach e ither site. Site-directed mutagenesis of selected residues in the ER a nd region-specific chemical mutagenesis of the ER hormone binding doma in with initial phenotypic screening in yeast have enabled the identif ication of aregion near C530 important in discrimination between estro gens and antiestrogens and of other residues important in hormone-depe ndent transcriptional activation. Some ER mutants with alterations in the carboxy-terminal portion of the hormone binding domain are transcr iptionally inactive yet bind hormone and also function as potent domin ant negative ERs, suppressing the activity of wild-type ER at low conc entrations. These studies reveal a separation of the hormone binding a nd transcription activation functions of the ER. They are also beginni ng to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination betwee n different categories of agonist and antagonist ligands. Such informa tion will be important in the design of maximally effective antiestrog ens. In addition, since there is now substantial evidence for a mixtur e of wild-type and variant ERs in breast cancers, our studies should p rovide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lea d to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therap y in breast cancer. In addition, some dominant negative receptors may prove useful in examining ER mechanisms of action and in suppressing t he estrogen-dependent growth of breast cancer cells.