POSITIVE AND NEGATIVE DISCRIMINATION OF ESTROGEN-RECEPTOR AGONISTS AND ANTAGONISTS USING SITE-SPECIFIC DNA RECOMBINASE FUSION PROTEINS

Activation of the estrogen receptor (ER) by hormone involves at least two steps. First, hormone binding initially relieves repression, a pro perty imposed on EW in cis by its ligand-binding domain (EBD). Subsequ ently, the derepressed ER binds specific genomic sites and regulates t ranscription. In addition to the natural hormone, ER binds a broad ran ge of ligands that evoke a spectrum of responses ranging from full ER activation by agonists to partial activation and inhibition by partial or complete antagonists. How these different ligands evoke different ER responses remains unclear. To address this issue, we have developed a nontranscriptional assay for ER ligand responsiveness based on Flp recombinase/human EBD protein chimeras. These fusion proteins transduc e the transient event of ligand binding into a permanent DNA change in a human cell line system. A fusion protein including ER D, E, and F d omains was activated by ail the ER ligands tested, demonstrating that both agonists and antagonists serve to relieve initial repression, and that differences between them lie downstream in the activation pathwa y. Mutant variants of the Flp-ER protein that distinguish between agon ists and antagonists, and a mutant EBD that selectively lost the abili ty to respond to 17 beta-estradiol but not to other ligands, were also identified. Thus, agonists and antagonists can be functionally distin guished in a nontranscriptional assay.