The long term objective of this proposal is to study the mechanism of steroid hormone action at the molecular level. After hormone binding occurs, steroid-recptor complexes are transformed in a temperature-dependent manner from a form that does not bind to DNA to a form that bind to DNA with affinity. This transformation of the receptor is a necessary step in the pathway by which these hormones regulate gene expression. The goal of this work is to understand the nature of this transformation process as it occurs with the glucocorticoid-receptor complex. Steroids of the glucocorticoid class are used in the therapy of a wide variety of diseases and understanding the mechanism by which these hormones cause their receptor-mediated effects constitutes a fundamental problem in molecular endocrinology. During the past three years we developed methods to directly demonstrate that the glucocorticoid receptor in mouse L cells is a phosphoprotein. In this proposal, I present a series of experiments utilizing covalent labeling of the L cell receptor protein with 32P or with (3H)dexamethasone 21-mesylate followed by specific immunoadsorption with anti-receptor antibody to study the structure of the transformed and untransformed receptor and the way in which covalent modification may be involved in defining the DNA-binding state.
Specific aims are: 1) to determine how many phosphates are present on the 98K steriod-binding protein and to map the phosphopeptides produced by complete protease digestion; 2) to determine whether the association of the steriod-receptor complex with DNA following cell-free transformation and the association of the receptor with nuclei after transformation in the intact cell is related (as our preliminary studies suggest) to the extent of receptor phosphorylation; 3) to determine if protein phosphatases and an endogenous membrane-bound, receptor-inactivating enzyme are able to convert non-DNA-binding phosphorylated receptors to a DNA-binding state; 4) to determine by direct assay if phosphatase-mediated inactivation of steroid-binding capacity in L cell cytosol is accompanied by receptor dephosphorylation; 5) to purify a protein kinase from L cell cytosol that phosphorylates the receptor and determine if phosphorylation of the DNA-binding form of the receptor converts it to a non-DNA-binding form.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA028010-09
Application #
3167923
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1980-06-01
Project End
1989-05-31
Budget Start
1988-06-01
Budget End
1989-05-31
Support Year
9
Fiscal Year
1988
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
Thomas, Monzy; Harrell, Jennifer M; Morishima, Yoshihiro et al. (2006) Pharmacologic and genetic inhibition of hsp90-dependent trafficking reduces aggregation and promotes degradation of the expanded glutamine androgen receptor without stress protein induction. Hum Mol Genet 15:1876-83
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
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
Galigniana, Mario D; Harrell, Jennifer M; Housley, Paul R et al. (2004) Retrograde transport of the glucocorticoid receptor in neurites requires dynamic assembly of complexes with the protein chaperone hsp90 and is linked to the CHIP component of the machinery for proteasomal degradation. Brain Res Mol Brain Res 123:27-36
Gerges, Nashaat Z; Tran, Irwin C; Backos, Donald S et al. (2004) Independent functions of hsp90 in neurotransmitter release and in the continuous synaptic cycling of AMPA receptors. J Neurosci 24:4758-66
Galigniana, Mario D; Morishima, Yoshihiro; Gallay, Philippe A et al. (2004) Cyclophilin-A is bound through its peptidylprolyl isomerase domain to the cytoplasmic dynein motor protein complex. J Biol Chem 279:55754-9
Murphy, Patrick J M; Galigniana, Mario D; Morishima, Yoshihiro et al. (2004) Pifithrin-alpha inhibits p53 signaling after interaction of the tumor suppressor protein with hsp90 and its nuclear translocation. J Biol Chem 279:30195-201

Showing the most recent 10 out of 83 publications