Steroid receptors belong to a superfamily of proteins that, in addition to the classic steroid hormones, also bind vitamin D3, retinoic acid and thyroid hormone. When occupied by hormone, receptors bind to their cognate DNA response elements and act as enhancers to regulate gene transcription. All steroid receptors studied to date are phosphoproteins but the function of phosphorylation is unknown. We have purified human progesterone receptors (PR) and have made monoclonal antibodies to them. There are two human PRs: B-receptors of 120 kDa and A-receptors of 94 kDa. By immunoaffinity purification, gel electrophoresis, immunoblotting, autoradiography, chemical cleavage, phosphotryptic peptide mapping, and reverse-phase high pressure liquid chromatography, we have shown that in cultured breast cancer cells, PRs are phosphorylated at multiple sites when unoccupied by hormone. This represents basal phosphorylation. A second, hormone-dependent phosphorylation occurs within 5 min of progestin treatment of cells, and increases the specific activity of [32P]orthophosphate labeling 5-10 fold. PR phosphorylation is on serine residues located predominately in the amino-terminal half of the receptors. In this application we plan to precisely map the serine residues involved in PR phosphorylation and determine the function of phosphorylation:
Aim 1 is to sequence the amino acids of enzymatic phosphopeptide fragments eluted from HPLC and place the peptides in context on the full-length human PR protein.
Aim 2 is to generate a series of cDNA mutants of the A- and B- receptors. First, to delete three phosphorylated clusters in the amino- terminus by mutagenesis of full-length hPR cDNAs encoding B- and A- receptors, or to delete the three major functional domains and test the mutant receptors for basal and hormone-dependent phosphorylation. Second, to use oligonucleotide-directed site specific mutagenesis to conservatively mutate key serine residues to alanine, and test the mutant receptors for phosphorylation and ability to trans-activate progestin responsive reporter genes. Third, to test the mutant receptors on simple and complex promoters to evaluate the function of phosphorylation. A number of iterative cycles of mutagenesis should converge on the residues whose mutation abrogates or modifies receptor function.
In Aim 3, DNA binding receptor mutants will be constructed to evaluate the DNA binding requirement for PR phosphorylation. These studies will delineate the function of PR phosphorylation, define the specific roles of A- and B-receptors in transactivation, and serve as a model for the role of phosphorylation of other steroid receptors and transcription factors.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA055595-03
Application #
2096719
Study Section
Reproductive Endocrinology Study Section (REN)
Project Start
1992-04-07
Project End
1996-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
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
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Horwitz, K B; Tung, L; Takimoto, G S (1996) Novel mechanisms of antiprogestin action. Acta Oncol 35:129-40
Horwitz, K B; Tung, L; Takimoto, G S (1995) Novel mechanisms of antiprogestin action. J Steroid Biochem Mol Biol 53:9-17
Horwitz, K B; Sartorius, C A; Hovland, A R et al. (1995) Surprises with antiprogestins: novel mechanisms of progesterone receptor action. Ciba Found Symp 191:235-49;discussion 250-3
Horwitz, K B (1994) How do breast cancers become hormone resistant? J Steroid Biochem Mol Biol 49:295-302
Horwitz, K B (1994) Hormone-resistant breast cancer or ""feeding the hand that bites you"". Prog Clin Biol Res 387:29-45
Sartorius, C A; Melville, M Y; Hovland, A R et al. (1994) A third transactivation function (AF3) of human progesterone receptors located in the unique N-terminal segment of the B-isoform. Mol Endocrinol 8:1347-60
Horwitz, K B (1994) Hormone 'resistance' in breast cancer: the role of normal and mutant steroid receptors. Cancer Treat Res 71:111-27
Sartorius, C A; Tung, L; Takimoto, G S et al. (1993) Antagonist-occupied human progesterone receptors bound to DNA are functionally switched to transcriptional agonists by cAMP. J Biol Chem 268:9262-6
Takimoto, G S; Tasset, D M; Eppert, A C et al. (1992) Hormone-induced progesterone receptor phosphorylation consists of sequential DNA-independent and DNA-dependent stages: analysis with zinc finger mutants and the progesterone antagonist ZK98299. Proc Natl Acad Sci U S A 89:3050-4