The long-term objectives of this research program are to elucidate the estrogen-, antiestrogen- and DNA-binding mechanisms and activities of the estrogen receptor; and the application of these basic studies to the regulation and function of reproductive tissues and the control of estrogen-dependent cancers. The immediate specific aims are: (1) to determine whether the affinity of the estrogen receptor for its estrogen responsive element (ERE), a specific nucleotide sequence of the prolactin gene, controls the selectivity of the receptor for specific genes. The in vitro affinity of the estrogen receptor for the ERE and the in vivo response to estradiol will be measured after site-directed in vitro mutagenesis of the ERE. The affinity of the receptor for the DNA will be measured by the gel mobility assay. The ERE's will be linked to the thymidine kinase promoter linked to the chloramphenicol acetyltransferase gene. The plasmid constructs will be transfected into GH3 cells and responsiveness to estradiol measured. (2) The affinity of the antiestrogen receptor complexes for the ERE and mutant EREs will be measured to determine whether the antiestrogens change the interaction and selectivity of the receptor, so that the receptor interacts with other genes. (3) Isolate and characterize DNA-binding protein(s) that bind at or near the ERE of the prolactin gene whose binding is dependent upon/or modified by the presence of the purified estrogen receptor. (4) Identify and characterize the in vivo phosphorylation sites of the estrogen receptor in calf uteri and MCF-7 cells. Determine the effects of receptor phosphorylation on the receptor's estrogen- and DNA-binding activities. Such information could provide insight into the fundamental mechanisms of steroid hormone action.
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