TISSUE-SPECIFIC EFFECTS OF PROGESTINS

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DESCRIPTION: (Adapted from the applicant's abstract) There are two progesterone receptors (PR): 933 aa B-receptors, and 769 aa A-receptors with a 164 aa N-terminal truncation. The B:A ratio differs among tissues; transcription by B vs. A differs in ligand-, promoter- and cell-specific ways; B are transactivators when A are inhibitors. The PR N-termini containing the homologous DNA binding domain (DBD) but lacking the hormone binding domain (HBD), are independent functional domains. They are constitutive transactivators that recapitulate the functional differences of the full-length receptors. Hypothesis: structural differences at their N-termini explain functional differences of the two PR. Aim 1. Structure of A- (NT-A) and B-receptor (NT-B) N-termini. The functionally autonomous domains, NT-A and NT-B, plus full-length A- and B- receptors, will be purified to homogeneity. Biochemical function will be documented by in vitro transcription, DNA binding affinity and cooperativity will be quantified; self-association properties will be analyzed by sedimentation equilibrium; size and shape defined by sedimentation velocity. The structure of purified N-termini will be compared by limited proteolysis and spectroscopic assays. Conformational changes due to DNA binding and the HBD will be mapped. These studies will define structural differences at the N-termini of the two PR. AIM 2. Isolation and characterization of proteins that mediate the unique functional properties of the two PR N-termini. We postulate that structural differences at the N-termini lead to recruitment of unique transcriptional co-regulators to each PR isoform. NT-A and NT-B will be used as "bait" in yeast one-and two- hybrid assays and in conventional chromatographic protein isolation and sequencing that interact with both PR N-termini, or ones that interact only with one, and confer unique properties to each PR isoform. AIM 3. Mutational and transcriptional analyses of structural subdomains in NT-A vs. NT-B. Preliminary data show that NT-A is organized into 7 protease inaccessible cassettes separated by 6 solvent-exposed loops and that its activation function is a stable subunit. These domains, and newly defined structural and functional domains in NT-B, will be mutated to assess their role in constitutive transcription by NT-A vs. NT-B, and ligand-regulated transcription by full-length PR. The proposed studies address the presently unknown functions of the two PR N-termini.