The major goal of this investigation is to elucidate the role of specific intracellular receptor proteins in the estrogenic regulation of cell proliferation and function in hormone-responsive tissues and cancers derived from these tissues. The proposal focuses on the use of biochemical, immunochemical and recombinant DNA techniques to determine the structure, composition and intracellular interactions and dynamics of the estrogen receptor and progesterone receptor from human or rodent sources. We are especially interested in the molecular mechanisms by which activity and expression of these receptors are regulated by estrogens, progestins and their antagonists in breast cancer cell lines. This work is facilitated by the availability in our laboratory of purified, or partially purified receptor proteins, monoclonal antibodies specific for both receptors, and cloned cDNAs that correspond to all of the translated mRNA for human ER from MCF-7 breast cancer cells and 90% of the translated mRNA for human PR from T47D breast cancer cells.
Specific aims i nclude: 1) To isolate, sequence and characterize cDNA clones corresponding to the entire mRNA for T47D human PR; 2) to isolate, sequence and characterize genomic clones for human ER and PR, including upstream regulatory sequences; 3) to characterize the regulation of ER and PR expression in breast cancer cells and rat uterus by estrogens, progestins and their antagonists; 4) to locate and characterize ER and any PR binding sites (responses elements) on the chromosomal genes for both receptors; 5) to dissect and characterize the functional domains of both receptors by in vitro mutagenesis, including domain switching between ER and PR, coupled with transient expression assays in heterologous cells containing estrogen- or progestin-responsive reporter genes; 6) to isolate mutant fragments either from transfected cells or from in vitro translations of transcripts and study the steroid-binding and specific DNA-binding properties of these fragments; 7) to overexpress human ER and PR cDNAs in suitable cell lines, purify expressed receptor and carry out detailed structural and biochemical studies on full length receptor or genetically engineered receptor fragments; 8) to determine the amino acids involved in the binding of both agonists and antagonists, and the location, role and hormonal regulation of phosphorylation sites in rat and human ER and PR.
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