Our objective is to understand the mechanism by which antiestrogens are effective as estrogen antagonists. Our studies have provided strong evidence that antiestrogens exert their antagonistic, growth suppressive effects via the estrogen receptor system and we have noted some distinct differences between the state of association of antiestrogen- and estrogen-receptor complexes.
Our specific aims are directed at 3 key points: the physical (state of association/conformational) nature of the antiestrogen receptor complex, the chemical nature (state of phosphorylation) of this complex, and the nature of its interaction with gene sites. We will determine whether the biological differences between estrogens and antiestrogens arise from differences in the degree of receptor dimerization (association state dependent) and/or the conformation of the individual ligand-monomer units (ligand-dependent). To address this issue, we will study receptor antiestrogen and estrogen complexes from MCF-7 breast cancer cells, as monomers and dimers under carefully controlled conditions, with respect to their sensitivity towards digestion by exogenous proteases, generation of characteristic peptide fragments, and interaction with specific antireceptor monoclonal antibodies. In addition, in view of the charge heterogeneity and reported differences in sensitivity of receptor-antiestrogen complexes to protein phosphatase, we will seek to obtain direct evidence for phosphorylation of the receptor in intact cells and determine whether antiestrogen and estrogen receptor complexes differ in their state of phosphorylation. We will also analyze the pattern of gene interaction of receptor-antiestrogen and receptor-estrogen complexes using a novel, in vivo approach in which receptors labeled with antiestrogen or estrogen in intact cells will be crosslinked to DNA by exposure to UV light. DNA fragments in which the receptor has attached to an estrogen regulated gene will be enriched and isolated by receptor immunopurification and gene hybridization techniques. Our goals will be to demonstrate that an estrogen regulated gene (avian apolipoprotein-II vs. control genes) and receptors are in intimate association and to determine whether the nature of the ligand (estrogen vs antiestrogen) alters the receptor-gene crosslinking efficiency. These studies should provide a significant advancement in understanding the mechanism of action of these intriguing and medically important compounds.
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