Heat shock protein 90 as a critical factor in maintaining glucocorticosteroid receptor in a nonfunctional state.

The 70-kDa heat shock protein (hsp70) has been shown to be an important participant in several intracellular events, including protein folding and trafficking. Hsp70 binds to many, if not all, proteins during their translation and maintains its association with some protein complexes as a subunit. We have examined the possibility that hsp70 may be associated with one or more forms of the rat hepatic glucocorticoid receptor (GR). Unliganded GR was immunoprecipitated from cytosol with the anti-GR monoclonal antibody BUGR2 and then subjected to western blotting. Both hsp70 and the 90-kDa heat shock protein (hsp90) were found to be specifically associated with the GR. Hsp70 was also bound to the liganded unactivated and activated (transformed) forms of the GR complex, while as expected, hsp90 was absent from the activated GR. Immunoprecipitation of cytosolic hsp70 with the anti-hsp70 monoclonal antibody N27 resulted in coprecipitation of GR. The components of the immunopurified GR were also analyzed by laser densitometry after polyacrylamide gel electrophoresis and Coomassie blue staining. These experiments revealed that hsp70 is bound to the GR in an approximate 1:5 ratio. Unactivated GR complexes isolated via a ligand affinity purification scheme contained hsp90 and trace amounts of hsp70. Collectively, these experiments demonstrate that hsp70 is specifically associated with several forms of the native rat hepatic GR, although its binding is substoichiometric. This is in direct contrast to hsp90, which binds as a dimeric subunit to the heteromeric unactivated GR complex.

mentioning

confidence: 99%

Heat shock protein 70 is associated in substoichiometric amounts with the rat hepatic glucocorticoid receptor

Diehl

Schmidt²

1993

“…In hormone-free cells and cytosols prepared from hormonefree cells, steroid receptors are associated with hsp90 [see Pratt (1987Pratt ( , 1990 for reviews]. Several studies of hsp90 binding by receptors deleted for various regions have established that HBD is necessary for interaction of GR or PR with hsp90 (Pratt et al, 1988;Howard et al, 1991;Dalman et al, 1991;Cadepond et al, 1991;Schowalter et al, 1991). This conclusion is supported by the demonstration that hsp90 remains bound to the HBD when receptor in the heterocomplex is cleaved by protease (Denis et al, 1988; Chakraborti & Simons, 1991).…”

mentioning

confidence: 99%

Evidence that the hormone binding domain of steroid receptors confers hormonal control on chimeric proteins by determining their hormone-regulated binding to heat-shock protein 90

Scherrer¹,

Picard²,

Massa³

et al. 1993

119

Previously, it has been shown that the hormone binding domain of the glucocorticoid receptor acts as a transferable regulatory cassette that can confer hormonal control onto chimeric proteins [Picard, D., Salser, S. J., & Yamamoto, K. R. (1988) Cell 54, 1073-1080]. The hormone binding domain of the glucocorticoid receptor contains its site of interaction with the 90-kDa heat-shock protein, hsp90 [Dalman, F. C., Scherrer, L. C., Taylor, L. P., Akil, H., & Pratt, W. B. (1991) J. Biol. Chem. 266, 3482-3490]. We have now transfected COS cells with cDNAs for fusion proteins containing beta-galactosidase and portions of the glucocorticoid receptor, and we demonstrate a correlation between hormone regulation of fusion protein localization and binding of the fusion proteins to hsp90. The hormone binding domain (residues 540-795) of the rat glucocorticoid receptor is sufficient for conferring hormone regulation onto a fusion protein and for intracellular binding of a fusion protein to hsp90. The hormone binding domain of the rat glucocorticoid or the human estrogen receptor is also sufficient to permit reticulocyte lysate-mediated refolding of a fusion protein into association with hsp90. Consistent with the results of fusion protein localization in intact cells, binding of a fusion protein to hsp90 blocks binding of antibody directed against the NL1 nuclear localization signal of the glucocorticoid receptor. These observations argue strongly that the hormone binding domain of the glucocorticoid receptor confers hormonal control of fusion proteins by conferring hormone-regulated binding to hsp90.

“…The 8-10S receptor complex is unable to bind to DNA, and activation to the DNA binding state is accompanied by receptor conversion to 4S and dissociation from hsp 90 [see Pratt (1990) and references cited therein]. This and other observations have suggested that hsp 90 functions as a repressor to maintain steroid receptors in an inactive state in the absence of hormone (Mendel et al, 1986;Sanchez et al, 1987;DeMarzo et al, 1991;Dalman et al, 1990; Cadepond et al, 1991). Other studies indicate that hsp 90 may also function to maintain the unoccupied receptor in a conformation competent to bind steroid (Bresnick et al, 1989; Dalman et al, 1989).…”

mentioning

confidence: 99%

Heat shock alters the composition of heteromeric steroid receptor complexes and enhances receptor activity in vivo

Edwards

Estes²,

Fadok³

et al. 1992

Under normal cellular conditions, human progesterone receptors (PR), immune-isolated from cytosols of T47D breast cancer cells, associate with two heat shock proteins (hsps), hsp 90 and hsp 70. Receptors activated by hormone binding in vivo and extracted from nuclei with 0.5 M NaCl no longer associate with hsp 90 but retain association with hsp 70. We have examined the effect of heat shock treatment of cells on hsp-receptor interactions and on receptor function. Heat shock resulted in a partial reduction in cellular levels of PR, but receptors that remained were functional for both steroid and DNA binding activities. By steady-state [35S]methionine labeling prior to heat shock treatment, it was determined that heat shock did not affect the composition or maintenance of preexisting cytosolic PR.hsp 90.hsp 70 complexes. By contrast, immune isolation of PR complexes from cells pulse-labeled with [35S]methionine showed that heat shock altered the composition of newly synthesized hsps associated with PR. After heat shock, both the highly inducible form of hsp 70 (72K hsp) and a 100K hsp were bound to cytosol PR, and inducible 72K hsp remained bound with the nuclear-activated PR. Neither of these hsps were associated in detectable amounts with PR under normal cellular conditions. With respect to receptor function, heat shock treatment substantially enhanced the activity of PR in vivo as determined by measuring hormone-dependent PR-mediated transcription of a target reporter gene (MMTV-CAT) that was stably transfected into T47D cells. Heat shock treatment alone, in the absence of hormone, did not stimulate MMTV-CAT expression nor did it affect transcription from a control reporter gene, pSV2-CAT, suggesting that enhanced receptor activity was due to an effect on PR-mediated processes and not to a general effect on transcription. Induction of the heat shock response by a related chemical stress (sodium arsenite) also enhanced PR activity in vivo. Interestingly, sodium arsenite produced both a greater induction of hsp 90 and hsp 70 synthesis and a greater fold enhancement of PR-mediated gene transcription than did heat shock. This suggests that enhancement of PR activity is related not only to induction of hsp synthesis but also to the severity of the stress response. The present results provide an indication that in certain cells there may exist an interrelationship between the activation pathways by which cells respond to stress and to steroid hormones. Possible mechanisms responsible for heat shock effects on PR activity are discussed.