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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK048238-07
Application #
6380886
Study Section
Reproductive Endocrinology Study Section (REN)
Program Officer
Margolis, Ronald N
Project Start
1994-09-01
Project End
2004-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
7
Fiscal Year
2001
Total Cost
$293,642
Indirect Cost

  Institution

Name
University of Colorado Denver
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045

  Publications

Hopp, Torsten A; Weiss, Heidi L; Hilsenbeck, Susan G et al. (2004) Breast cancer patients with progesterone receptor PR-A-rich tumors have poorer disease-free survival rates. Clin Cancer Res 10:2751-60
Sartorius, Carol A; Shen, Tianjie; Horwitz, Kathryn B (2003) Progesterone receptors A and B differentially affect the growth of estrogen-dependent human breast tumor xenografts. Breast Cancer Res Treat 79:287-99
Takimoto, Glenn S; Tung, Lin; Abdel-Hafiz, Hany et al. (2003) Functional properties of the N-terminal region of progesterone receptors and their mechanistic relationship to structure. J Steroid Biochem Mol Biol 85:209-19
Richer, Jennifer K; Jacobsen, Britta M; Manning, Nicole G et al. (2002) Differential gene regulation by the two progesterone receptor isoforms in human breast cancer cells. J Biol Chem 277:5209-18
Abdel-Hafiz, Hany; Takimoto, Glenn S; Tung, Lin et al. (2002) The inhibitory function in human progesterone receptor N termini binds SUMO-1 protein to regulate autoinhibition and transrepression. J Biol Chem 277:33950-6
Jacobsen, Britta M; Richer, Jennifer K; Schittone, Stephanie A et al. (2002) New human breast cancer cells to study progesterone receptor isoform ratio effects and ligand-independent gene regulation. J Biol Chem 277:27793-800
Tung, L; Shen, T; Abel, M G et al. (2001) Mapping the unique activation function 3 in the progesterone B-receptor upstream segment. Two LXXLL motifs and a tryptophan residue are required for activity. J Biol Chem 276:39843-51
Shen, T; Horwitz, K B; Lange, C A (2001) Transcriptional hyperactivity of human progesterone receptors is coupled to their ligand-dependent down-regulation by mitogen-activated protein kinase-dependent phosphorylation of serine 294. Mol Cell Biol 21:6122-31
Bain, D L; Franden, M A; McManaman, J L et al. (2001) The N-terminal region of human progesterone B-receptors: biophysical and biochemical comparison to A-receptors. J Biol Chem 276:23825-31
Sartorius, C A; Takimoto, G S; Richer, J K et al. (2000) Association of the Ku autoantigen/DNA-dependent protein kinase holoenzyme and poly(ADP-ribose) polymerase with the DNA binding domain of progesterone receptors. J Mol Endocrinol 24:165-82

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