The mineralocorticoid receptor (MR) that mediates signals generated by aldosterone is an important receptor involved in the stress mechanism.Because of the lability of the steroid-receptor complex in tissue extracts, its properties and functions have not been well characterized.Therefore, it is proposed to overexpress the human MR in insect cells using the baculovirus system. This will facilitate the generation of milligram quantities of receptor that can be purified from Spodoptera frugiperda (Sf9) cells. In addition,the first 350 amino acid truncated MR together with the DNA binding domain but lacking the ligand binding domain will be overexpressed as will a full length GR-MR chimeric receptor.These receptors will be purified from Sf9 cells for experiments bearing on activation and transcriptional functions. The oligomeric form of the MR will be reconstituted from overexpressed and purified MR, HSP70 and other purified cytosolic factors (p59, modulator) to study the mechanism of assembly and to define the cytoplasmic activation mechanism in vitro. The specific DNA binding of the MR and its derivatives will be analyzed as will the activation of cell-free transcription. This will entail specifically phosphorylated MR and its different forms that have been overexpressed and purified from Sf9 cells.The MR responsive element will be mapped on the 5' flanking regions of the Na,K-ATPase alpha and beta subunits' DNA by footprinting. Specific transcription factors that interact with the N-terminal domain will be isolated after their binding to the overexpressed N-terminus by chemical (and reversible) cross- linking. A specific anti-MR antibody that will not cross-react with the glucocorticoid receptor will be generated from a predicted peptide sequence in the N-terminus. This antibody will be used in the DNA binding experiments and as a means to purify the MR. This information will impact on the understanding of the stress adaptation mechanism at the molecular level and will apply to human diseases of hyperaldosteronism and hypertension.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK044441-04
Application #
2414816
Study Section
General Medicine B Study Section (GMB)
Program Officer
Margolis, Ronald N
Project Start
1994-05-01
Project End
1998-10-31
Budget Start
1997-05-22
Budget End
1998-10-31
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Thomas Jefferson University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
061197161
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
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Derfoul, A; Robertson, N M; Lingrel, J B et al. (1998) Regulation of the human Na/K-ATPase beta1 gene promoter by mineralocorticoid and glucocorticoid receptors. J Biol Chem 273:20702-11
Bostanjoglo, M; Reeves, W B; Reilly, R F et al. (1998) 11Beta-hydroxysteroid dehydrogenase, mineralocorticoid receptor, and thiazide-sensitive Na-Cl cotransporter expression by distal tubules. J Am Soc Nephrol 9:1347-58
Robertson, N M; Zangrilli, J; Fernandes-Alnemri, T et al. (1997) Baculovirus P35 inhibits the glucocorticoid-mediated pathway of cell death. Cancer Res 57:43-7
Bruner, K L; Derfoul, A; Robertson, N M et al. (1997) The unliganded mineralocorticoid receptor is associated with heat shock proteins 70 and 90 and the immunophilin FKBP-52. Recept Signal Transduct 7:85-98
Schumacher, R J; Hansen, W J; Freeman, B C et al. (1996) Cooperative action of Hsp70, Hsp90, and DnaJ proteins in protein renaturation. Biochemistry 35:14889-98
Bodine, P V; Litwack, G (1995) Purification of the glucocorticoid receptor-mineralocorticoid receptor modulator-2 from rabbit liver. Receptor 5:133-43
Robertson, N M; Bodine, P V; Hsu, T C et al. (1995) Modulator inhibits nuclear translocation of the glucocorticoid receptor and inhibits glucocorticoid-induced apoptosis in the human leukemic cell line CEM C-7. Cancer Res 55:548-56