Members of the steroid and thyroid hormone receptor gene family are ligand activated transcriptional modulators. As such they play a crucial role in the control of differentiation and development, in the growth of both normal and transformed cells and in the ability of an organism to respond to external stimuli. The glucocorticoid receptor (GR) was the first steroid receptor to be cloned and it has since been studied intensively by a number of laboratories. This has led to an understanding of the overall domain structure and function of the GR. However the details of hormone binding, activation, DNA binding and transcriptional activation are far from being solved. This proposal focuses on the hormone binding domain of the GR and is designed to shed light on the following questions. 1) What are the structural determinants of the hormone binding site? 2) How does agonist binding activate the receptor, and why do antagonists bind to the receptor but fail to activate it? The problem will be approached in two ways. First, hybrid receptors will be constructed in which small regions of the GR hormone binding domain have been replaced by the corresponding sequence from either the progesterone or estrogen receptors. These receptors will be analyzed for their ability to bind to glucocorticoids, to various glucocorticoid antagonists (including progesterone) and to estrogen. When binding occurs, the ability of the hormone to activate receptor to a specific DNA binding form will be studied. Thus using this technique, which has been termed homolog-scanning mutagenesis, we should be able to study the structural characteristics of the hormone binding domain which are required for recognition of specific steroids and for receptor activation. As a long term project, the hormone binding domain of the GR and selected hybrids will be expressed in E. coli in order to purify enough protein for structural studies. The second approach is to select for cells in tissue culture that contain mutant receptors that have a) become hormone independent, or b) require reduced levels of hormone, or c) can be activated efficiently by compounds that are usually partial agonists and/or antagonists. These receptors will be characterized, the cDNAs cloned and the sequences analyzed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK042552-03
Application #
3243685
Study Section
Endocrinology Study Section (END)
Project Start
1990-04-01
Project End
1995-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
3
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Georgetown University
Department
Type
Schools of Dentistry
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
Zhang, Shimin; Jonklaas, Jacqueline; Danielsen, Mark (2007) The glucocorticoid agonist activities of mifepristone (RU486) and progesterone are dependent on glucocorticoid receptor levels but not on EC50 values. Steroids 72:600-8
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Bonovich, M T; List, H J; Zhang, S et al. (1998) Identification of glucocorticoid receptor domains necessary for transcriptional activation of the mouse mammary tumor virus promoter integrated in the genome. Exp Cell Res 239:454-62
Lu, J; Danielsen, M (1996) A Stu I polymorphism in the human androgen receptor gene (AR). Clin Genet 49:323-4
Zhang, S; Liang, X; Danielsen, M (1996) Role of the C terminus of the glucocorticoid receptor in hormone binding and agonist/antagonist discrimination. Mol Endocrinol 10:24-34
Csikos, T; Tay, J; Danielsen, M (1995) Expression of the Xenopus laevis mineralocorticoid receptor during metamorphosis. Recent Prog Horm Res 50:393-6
Martin, M B; Garcia-Morales, P; Stoica, A et al. (1995) Effects of 12-O-tetradecanoylphorbol-13-acetate on estrogen receptor activity in MCF-7 cells. J Biol Chem 270:25244-51
Zhang, S; Danielsen, M (1995) 8-Br-cAMP does not convert antagonists of the glucocorticoid receptor into agonists. Recent Prog Horm Res 50:429-35
Rosenthal, D; Hong, T; Cherney, B et al. (1994) Expression and characterization of a fusion protein between the catalytic domain of poly(ADP-ribose) polymerase and the DNA binding domain of the glucocorticoid receptor. Biochem Biophys Res Commun 202:880-7
Chen, D; Kohli, K; Zhang, S et al. (1994) Phenylalanine-780 near the C-terminus of the mouse glucocorticoid receptor is important for ligand binding affinity and specificity. Mol Endocrinol 8:422-30

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