Antiestrogens (AEs), intriguing compounds that are able to antagonize many of the actions of estrogens, are of particular medical importance because of their efficacy In controlling the growth and spread of hormone-responsive breast and uterine tumors. Our objective is to understand the mechanism by which AEs interact with the estrogen receptor and are effective as estrogen antagonists. Our studies address the question of estrogen antagonist action at two levels: ligand-receptor interaction and receptor-receptor interaction (dimerization).
Our specific aims are to identify the ligand discriminating site in the estrogen receptor through site-directed and random mutagenesis and biocharacter screening, and to investigate the role of receptor dimerization in AE action through the use of bivalent ligands and a dimer contact peptide. Based upon results from our affinity labeling studies, we will prepare a set of mutant estrogen receptors in which carefully controlled substitutions of specific amino acids are made in those regions In the hormone binding domain-E thought to be the most critically involved in distinguishing between estrogen agonists and antagonists, a site we term the ligand discriminating site. Conservative amino acid substitutions will be made, so as to maintain estrogen receptor structure and ligand binding but to change the interaction that the altered domain might have with the basic and polar side chains of AEs. Additional affinity labeling studies using new derivatives of AEs will be conducted to obtain further information on the contact sites between estrogen receptor and the basic side chains of the AEs. In complementary studies, we will use random mutagenesis of selected portions of estrogen receptor cDNA and biocharacter screening in yeast to identify further the residues important in determining agonist/ antagonist character. We will prepare and evaluate the agonist/antagonist potency of bivalent AE and estrogen ligands and their effects on the monomer-dimer equilibrium of receptor. We will also synthesize a dimer contact oligopeptide and determine if it blocks receptor dimerization and alters transactivation. The implications of progress in this area include the development of improved AEs, a greater understanding of the .effectiveness of AEs in treatment of hormone-responsive breast cancer, and elucidation of aspects of ligand character recognition and the regulation of transcription by AEs and estrogens.
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