The glucocorticoid receptor (GR) is associated through its hormone binding domain (HBD) with the abundant and ubiquitous protein chaperone hsp90. Binding to hsp90 is required for the GR to have steroid binding activity and for hormonal control of receptor function. GR- hsp90 heterocomplexes are assembled by a multi-protein chaperone system in reticulocyte lysate that we have reconstituted from five purified components during the past seven years. This assembly system forms complexes between hsp90 and a variety of transcription factors and protein kinases involved in signal transduction, cell cycle control and oncogenesis. The components of the assembly system from pants and animals compliment each other when placed in the heterologous purified system, suggesting that this hsp90-based protein folding system serves a basic, conserved function in all eukaryotic cells. The five protein assembly machinery consists of the essential chaperones hsp90 and hsp70 and the non-essential cp-chaperones hsp40, Hop and p23. Recently, we have developed a two-step system of GR-hsp90 heterocomplex assembly in which immobilized GR is incubated with purified hsp70, washed free of unbound protein, and then incubated with the other chaperones. The first step in the assembly is the ATP- dependent and hsp40-dependent formation of a GR-hsp70 complex that is now 'primed' to bind hsp90 in a second ATP-dependent assembly step. Steroid binding activity is generated during the second incubation with gsp90.
Specific aims 1 -5 of this proposal focus on the mechanism of heterocomplex assembly by determining the stochiometry of gsp70 to GR in the 'primed' GR.hsp70 complex, the site of hsp70binding to the GR HBD, the nucleotide binding state of hsp70 hsp90 at each step of assembly, the potential for GR-bound hsp70 (and possibly hsp90) to ratchet back and forth between ATP-dependent and ADP-dependent conformations during assembly, and the role of hsp90 phosphorylation in its assembly activity. Several lines of evidence support the proposal that the hsp90/hsp70- based chaperone machinery acts to open the hydrophobic ligand binding cleft in the GR HBD to access by hormone.
Specific aim 6 is designed to further test this model and to see if it can be applied to non-receptor proteins in the opening of hydrophobic clefts to access by other ligands.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK031573-23
Application #
6843094
Study Section
Endocrinology Study Section (END)
Program Officer
Margolis, Ronald N
Project Start
1982-07-01
Project End
2006-08-31
Budget Start
2005-03-01
Budget End
2006-08-31
Support Year
23
Fiscal Year
2005
Total Cost
$256,702
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Pharmacology
Type
Schools of Medicine
DUNS #
073133571
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
Morishima, Yoshihiro; Kanelakis, Kimon C; Murphy, Patrick J M et al. (2003) The hsp90 cochaperone p23 is the limiting component of the multiprotein hsp90/hsp70-based chaperone system in vivo where it acts to stabilize the client protein: hsp90 complex. J Biol Chem 278:48754-63
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

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