During the past few years, it has become clear that several of the steroid hormone receptors are phosphoproteins containing sulfhydryl moieties that are critical for receptor function. A variety of studies have also shown that cytosols prepared from several tissues contain at least three endogenous factors that interact with steroid receptors to affect either their ligand binding or DNA binding functions. The long-term objective of this work is to study the roles played by both covalent receptor modification and by the endogenous factors in determining the steroid-binding conformation and DNA-binding activity of glucocorticoid receptors. The steroid hormones are used in the therapy of a wide variety of diseases and understanding the mechanisms by which their receptors may be regulated constitutes a fundamental problem in molecular endocrinology. The first endogenous cytosolic factor under study is a heat-stable activity that converts glucocorticoid receptors from a nonbinding to a steroid-binding state. We have recently shown that this factor is thioredoxin and that glucocorticoid receptors are maintained in their reduced, steroid-binding state by a NADPH-dependent, thioredoxin-mediated reducing system. In this proposal we intend: 1) to determine if thioredoxin binds to the receptors; and 2) to determine if thioredoxin acts solely in reduction or if it must be bound to the receptor for the receptor to function. The second endogenous factor is a small, heat-stable component that we have shown acts like molybdate to stabilize unoccupied receptors and to block transformation of the steroid-receptor complex to the DNA-binding state. In this proposal we present a series of experiments designed to: 1) identify the composition of the stabilizing factor; 2) to synthesize the factor from a cytosolic """"""""precursor""""""""; 3) to determine more completely its mechanism of action. The third endogenous factor is a heat-labile macromolecule that binds to transformed steroid-receptor complexes and prevents their binding to DNA. We have extensively purified this factor and in this proposal we present experiments designed to determine: 1) if the inhibitor is a normal component of the untransformed glucocorticoid-receptor complex; and 2) if the inhibitor interacts with other steroid receptors to limit DNA binding.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK031573-05
Application #
3230170
Study Section
Endocrinology Study Section (END)
Project Start
1982-07-01
Project End
1988-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
5
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Pratt, W B; Morishima, Y; Murphy, M et al. (2006) Chaperoning of glucocorticoid receptors. Handb Exp Pharmacol :111-38
Kovacs, Jeffrey J; Murphy, Patrick J M; Gaillard, Stephanie et al. (2005) HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor. Mol Cell 18:601-7
Morishima, Yoshihiro; Peng, Hwei-Ming; Lin, Hsia-lien et al. (2005) Regulation of cytochrome P450 2E1 by heat shock protein 90-dependent stabilization and CHIP-dependent proteasomal degradation. Biochemistry 44:16333-40
Peng, Hwei-Ming; Morishima, Yoshihiro; Jenkins, Gary J et al. (2004) Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase. J Biol Chem 279:52970-7
Billecke, Scott S; Draganov, Dragomir I; Morishima, Yoshihiro et al. (2004) The role of hsp90 in heme-dependent activation of apo-neuronal nitric-oxide synthase. J Biol Chem 279:30252-8
Pratt, William B; Galigniana, Mario D; Morishima, Yoshihiro et al. (2004) Role of molecular chaperones in steroid receptor action. Essays Biochem 40:41-58
Pratt, William B; Galigniana, Mario D; Harrell, Jennifer M et al. (2004) Role of hsp90 and the hsp90-binding immunophilins in signalling protein movement. Cell Signal 16:857-72
Harrell, Jennifer M; Murphy, Patrick J M; Morishima, Yoshihiro et al. (2004) Evidence for glucocorticoid receptor transport on microtubules by dynein. J Biol Chem 279:54647-54
Kanelakis, Kimon C; Pratt, William B (2003) Regulation of glucocorticoid receptor ligand-binding activity by the hsp90/hsp70-based chaperone machinery. Methods Enzymol 364:159-73
Pratt, William B; Toft, David O (2003) Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp Biol Med (Maywood) 228:111-33

Showing the most recent 10 out of 65 publications