The long-term objective of this research program is to determine how progestins modulate gene expression with emphasis on the mechanism of action of progesterone receptors (PRs) in regulating gene transcription. Two isoforms of PR have previously been identified: A- and B-receptors. We have recently identified a third progesterone receptor, termed the C- receptor. Based on mRNA mapping studies, the C-receptor lacks the N- terminus and the first DNA binding finger of A- and B-receptors, but contains the second DNA-binding finger, the hormone binding domain, the nuclear localization signal, and sequences for dimerization. Since the C-receptor lacks the first DNA-binding finger, we predict it does not bind DNA and, by itself, would be transcriptionally inactive. Our working hypothesis is that C-receptors negatively modulate progestin action by dimerization with active A- and B-receptors.
Aim #1 is to determine if C-transcripts are translated and if utilization of the start-site (Met 595) in the human PR cDNA results in the synthesis of C- receptors. This will be accomplished by using hybrid-selection and generating synthetic C-receptors in an in vitro transcription-translation system.
Aim #2 is to determine the interactions of C-receptors with A- or B-proteins and assess its effects on the transcriptional activity of A- and B-receptors. This will be done by analyzing the different isoforms contained in dimers identified using antibody co-precipitation techniques and gel retardation assays. The transactivational capacity of A-and B-receptors in the presence of C-receptors will be assessed in transfection studies.
Aim #3 is to determine the presence of C-receptors in progestin-responsive cells and tissues. this will be accomplished by analyzing cells or tissues for the presence of C-specific mRNA transcripts and C-receptor proteins. We will also determine whether levels of the C-receptor in breast cancer tumors are of clinical value and if so, develop a C-receptor assay.
Aim #4 will determine if the antiprogestins, RU486, ZR112, and ZR98, alter C-receptor properties.
Aim #5 is to determine if C-receptors are hormonally regulated, if C- receptors are phosphorylated (as A- and B-receptors) and determine the role of phosphorylation in C-receptor function. These studies should provide valuable information on mechanisms affecting progestin regulation of breast cancer cell growth and may lead to development of a useful marker in the treatment of breast cancer.
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