Estrogen and progesterone are the principle hormones involved in regulating growth of breast cancer cells. Their actions are mediated by specific nuclear receptors that act as hormone-dependent transcriptional regulators. An important step in steroid hormone action is receptor binding to cis- acting sequences, termed hormone response elements (HREs), in target genes. Working with progesterone receptors (PR) in T47D human breast cancer cells as a model, the overall goal of this project is to understand the molecular mechanisms that control steroid receptor binding to HREs. We propose that PR binding to DNA is controlled by a hormone-dependent multistep activation process involving release of the inhibitory molecule, heat shock protein 90 (hsp 90), dimerization, phosphorylation and interaction with other nuclear protein factor(s). Combined biochemical and INTERACTION WITH OTHER NUCLEAR PROTEIN FACTOR(S). Biochemical studies are proposed to isolate, characterize and investigate the mode of action of nuclear protein factor(s) undertaken by conventional chromatography schemes, sequence- specific DNA affinity columns and PR-protein affinity methods. Monoclonal antibodies will be generated for further molecular and chemical analysis.
AIM #2. PR DIMERIZATION. Activated PR are capable of forming stable multimers (suggesting dimers) between full length (PR-B) and truncated (PR- A) proteins in the absence of DNA. Biochemical studies are proposed with purified PR-A and PR-B, to confirm the subunit composition and stoichiometry of all possible PR dimers (AA, AB, BB), to determine the chemical nature of PR-PR interactions, and to explore the role of ligand and phosphorylation, apart from their effects on hsp 90 release, as factors controlling PR dimerization. Mutagenesis studies are proposed to study structure-function relationships. PR mutants will be expressed in mammalian cells to test hypotheses that 1) hsp 90 blocks dimerization surfaces, 2) dimerization in turn controls DNA binding, and 3) that functional DNA binding domains are bipartite, requiring the juxtaposition of identical domains from two PR proteins through dimerization.
AIM #3. DEVELOPMENT OF MOLECULAR PROBES. Deletion and site-directed substitution mutants of PR have been designed and will be cloned into appropriate vectors for expression in vitro or in mammalian cells. For studies that require quantities of purified PR a baculovirus expression system will be developed for overproduction of receptors.
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