A heat shock protein 90 inhibitor that modulates the immunophilins and regulates hormone receptors without inducing the heat shock response

When a cell encounters external stressors, such as lack of nutrients, elevated temperatures, changes in pH or other stressful environments, a key set of evolutionarily conserved proteins, the heat shock proteins (hsps), become overexpressed. Hsps are classified into six major families with the hsp90 family being the best understood; an increase in cell stress leads to increased levels of hsp90, which leads to cellular protection. A hallmark of hsp90 inhibitors is that they induce a cell rescue mechanism, the heat shock response. We define the unique molecular profile of a compound (SM145) that regulates hormone receptor protein levels through hsp90 inhibition without inducing the heat shock response. Modulation of the binding event between heat shock protein 90 and the immunophilins/homologs using SM145, leads to a decrease in hormone receptor protein levels. Unlike N-terminal hsp90 inhibitors, this hsp90 inhibitor does not induce a heat shock response. This work is proof of principle that controlling hormone receptor expression can occur by inhibiting hsp90 without inducing pro-survival protein heat shock protein 70 (hsp70) or other proteins associated with the heat shock response. Innovatively, we show that blocking the heat shock response, in addition to hsp90, is key to regulating hsp90-associated pathways.     

In contrast to the glucocorticoid receptor, the thyroid hormone receptor is translated in the DNA binding state and is not associated with hsp90

We have recently reported that the glucocorticoid receptor (GR) becomes bound to the 90-kDa heat shock protein (hsp90) at or near the end of receptor translation in vitro (Dalman, F. C., Bresnick, E. H., Patel, P. D., Perdew, G. H., Watson, S. J., Jr., and Pratt, W. B. (1989) J. Biol. Chem. 264, 19815-19821). In this paper we compare the hsp90 binding and DNA binding activities of the thyroid hormone receptor (TR) to those of the GR after cell-free translation of the two receptors in rabbit reticulocyte lysate. In contrast to the newly translated GR, which is bound to hsp90 and must be transformed to the DNA binding state, the TR is not bound to hsp90 and is translated in its DNA binding form without any requirement for transformation. When the GR is translated in wheat germ extract, which does not contain hsp90, it is translated in its DNA binding form in the same manner as the TR synthesized in reticulocyte lysate. These observations provide direct evidence that binding of GR to hsp90 is associated with repression of its DNA binding function. The fact that the TR does not bind to hsp90 and is translated in its DNA binding form is consistent with the different behavior of this receptor with respect to classic steroid receptors in the intact cell. We propose that binding to hsp90 may account for the fact that most of the steroid receptors are recovered in the cytosolic fraction after lysis of hormone-free cells in low salt buffer whereas the hormone-free TR is recovered in tight association with the nucleus.     

Retinoic acid receptor belongs to a subclass of nuclear receptors that do not form "docking" complexes with hsp90.

We have recently reported that, in contrast to the glucocorticoid receptor, the thyroid hormone receptor does not bind to hsp90 when the receptor is translated in rabbit reticulocyte lysate [Dalman, F. C., Koenig, R. J., Perdew, G. H., Massa, E., & Pratt, W. B. (1990) J. Biol. Chem. 265, 3615-3618]. All of the steroid receptors that are known to bind hsp90 are recovered in the cytosolic fraction when hormone-free cells are ruptured in hypotonic buffer. In contrast, unliganded thyroid hormone receptors and retinoic acid receptors are tightly associated with nuclear components. In this paper, we translated the human estrogen receptor and the human retinoic acid receptor in reticulocyte lysate and then immunoadsorbed the [35S]methionine-labeled translation products with the 8D3 monoclonal antibody against hsp90. The estrogen receptor is bound to hsp90, as indicated by coimmunoadsorption, but the retinoic acid receptor is not. Translation and immunoadsorption of chimeric proteins containing the DNA binding domain of one receptor and the N-terminal and COOH-terminal segments of the other show that the DNA binding finger region of the estrogen receptor is neither necessary nor sufficient for hsp90 binding. These observations suggest that there are two classes within the steroid receptor family. In one class (e.g., glucocorticoid, mineralocorticoid, sex hormone, and dioxin receptors), the receptors bind to hsp90 and remain in some kind of inactive "docking" mode until hormone-triggered release of hsp90 occurs. In the retinoic acid/thyroid hormone class, the unligated receptors do not bind to hsp90, and the receptors appear to proceed directly to their high-affinity nuclear acceptor sites without entering the "docking" state.     

Characterization of progesterone receptor binding to the 90- and 70-kDa heat shock proteins

In this study a model system for expression of the chicken progesterone receptor in cultured cells was developed using a quail fibroblast cell line, QT6. The chicken progesterone receptor form A expressed in QT6 cells was evaluated and determined to have a number of similarities to receptor isolated from chicken oviduct. These include hormone binding, sedimentation profile, phosphorylation pattern, heat shock protein (hsp) 70 and hsp90 associations and the ability to stimulate a reporter gene construct. Therefore, the receptor expressed in this system functioned adequately for further evaluation of the particular region (or regions) involved in hsp70 and hsp90 binding. Several receptor deletion mutants were tested for hsp70/hsp90 binding; only the d369-659 mutant, which has the entire steroid-binding domain deleted, was unable to bind hsp90 and hsp70. Three separate regions of the steroid-binding domain were found to partially restore hsp90 and hsp70 binding to the d369-659 mutant protein. However, hsp binding was not abolished when these or other regions of the steroid binding domain were deleted individually. These findings indicate that hsp90 and hsp70 both bind to the steroid-binding domain of the receptor through interactions at multiple locations or through some structural quality that is distributed throughout this region of the protein.     

The importance of ATP binding and hydrolysis by hsp90 in formation and function of protein heterocomplexes.

The chaperone hsp90 is capable of binding and hydrolyzing ATP. Using information on a related ATPase, DNA gyrase B, we selected three conserved residues in hsp90's ATP-binding domain for mutation. Two of these mutations eliminate nucleotide binding, while the third retains nucleotide binding but is apparently deficient in ATP hydrolysis. We first analyzed how these mutations affect hsp90's binding to the co-chaperones p23 and Hop, and to the hydrophobic resin, phenyl-Sepharose. These experiments showed that ATP's effects, specifically, increased affinity for p23 and decreased affinity for Hop and phenyl-Sepharose, are brought on by ATP binding alone. We also tested the ability of hsp90 mutants to assist hsp70, hsp40, and Hop in the refolding of denatured firefly luciferase. While hsp90 is capable of participating in this process in a nucleotide-independent manner, the ability to hydrolyze ATP markedly potentiates hsp90's effect. Finally, we assembled progesterone receptor heterocomplexes with hsp70, hsp40, Hop, p23, and wild type or mutant hsp90. While neither ATP binding nor hydrolysis was necessary to bind hsp90 to the receptor, mature complexes containing p23 and capable of hormone binding were only obtained with wild type hsp90.     

Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90

In this work, we examine the cellular localization and protein interactions of mouse glucocorticoid receptors that have been overexpressed in Chinese hamster ovary (CHO) cells (Hirst, M. A., Northrop, J. P., Danielsen, M., and Ringold, G. M. (1990) Mol. Endocrinol. 4, 162-170). We demonstrate that wild-type unliganded mouse glucocorticoid receptor, which is expressed in CHO cells to a level approximately 10 times that of L cells, is localized entirely to the nucleus by indirect immunofluorescence with the BuGR antireceptor monoclonal antibody. Overexpressed receptors that have either no hormone binding activity or no DNA binding activity because of point mutations also localize to the nucleus, providing genetic proof that the nuclear localization cannot reflect a steroid-mediated shift of the receptor from the cytoplasm to the nucleus and that DNA binding activity is not required for nuclear localization. Like unliganded progesterone receptors, which also associate in a loosely bound "docking" complex with the nucleus, the mouse glucocorticoid receptor overexpressed in CHO cells is associated with both hsp90 and hsp70. This is in contrast to the untransformed mouse glucocorticoid receptor in L cell cytosol, which is associated with hsp90 but not hsp70. The difference in hsp70 association between cell types could reflect overexpression of the receptor in CHO cells. However, like receptors in CHO cells selected for very high levels of overexpression, receptors in CHO cells selected for an intermediate level of receptor expression that is comparable to that of L cells are also bound to hsp70. This observation argues against an explanation of hsp70 association based purely on receptor overexpression, and we speculate that association of the unliganded glucocorticoid receptor with hsp70 might be a consequence of its nuclear localization in the CHO cells. Although there are differences between the mouse receptor in CHO cells and L cells, the nuclear localization signal of the untransformed mouse receptor reacts equivalently with the AP64 antibody against NL1 in cytosols prepared from both cell types.     

Structure of the progesterone receptor of chick oviduct. The 110 kDa B protein which binds the hormone has affinity for DNA

Purified "B" protein (MW approximately 110 kDa) that binds progesterone, and more than 90% of the activated receptor labelled with tritiated hormone in the oviduct cytosol of chicks pretreated with estrogen have the same chromatography behavior on DNA-cellulose. Conversely, neither the nonactivated "8S" receptor, that includes the heat-shock protein hsp90, nor the later bind to DNA. With other biochemical and immunological arguments, these results indicate that the hormone binding B unit of the progesterone receptor binds DNA as do all steroid hormone receptors.     

Evidence that heat shock protein-70 associated with progesterone receptors is not involved in receptor-DNA binding.

In the absence of hormone, human progesterone receptors (PR) are recovered in the cytosolic fraction of cell lysates as a multimeric complex containing the steroid-binding polypeptide, heat shock protein-90 (hsp90), and heat shock protein-70 (hsp70). Activated forms of human PR that acquire the ability to bind to DNA are dissociated from hsp90, but retain association with hsp70. The present study has examined whether associated hsp70 has a function in receptor-DNA binding. When activated PR was bound to specific target DNA in a gel shift assay, no hsp70 was detectable in the PR-DNA complex, as evidenced by the failure of several antibodies to hsp70 to affect the mobility or the amount of complexes. To determine whether hsp70 might indirectly influence DNA-binding activity, we have examined the effect of hsp70 dissociation on PR-DNA-binding activity. Dissociation was achieved either by treatment of immunoaffinity-purified immobilized PR complexes with ATP or by the binding of PR complexes to ATP-agarose, followed by elution with high salt. Under both conditions, dissociation from hsp70 neither enhanced nor impaired the ability of PR to bind to specific DNA. These results suggest that hsp70 is not involved in PR binding to DNA, either directly by participating in DNA binding or indirectly by modulating PR-DNA-binding activity. This implies that hsp70 functions at an earlier stage in the receptor activation pathway. Consistent with the known involvement of hsp70 in stabilizing unfolded states of other target proteins, we propose that hsp70 may assist in nuclear transport of PR or in assembly-disassembly of the 8-10S multimeric complex.     

Transformation of glucocorticoid and progesterone receptors to the DNA-binding state

This brief review explores some recent observations relating to the structure of untransformed glucocorticoid and progesterone receptors and the mechanism by which the receptors are transformed to the DNA-binding state. In their molybdatestabilized, untransformed state, progesterone and glucocorticoid receptors exist as a heteromeric 8-9S complex containing one unit of steroid binding phosphoprotein and one or two units of the 90 kD heat shock protein hsp90. When the receptors are transformed, the steroid-binding protein dissociates from hsp90. In cytosol preparations, temperature-mediated dissociation proceeds much more rapidly in the presence of hormone. The dissociated receptor binds to DNA with high affinity, regardless of whether it is in the hormone-bound or the hormone-free state. These observations raise the possibility that the primary, and perhaps the only, role for the hormone is to promote dissociation of the receptor-hsp90 complex. Molybdate, vanadate, and tungstate inhibit receptor transformation to the DNA-binding form, an effect that appears to reflect the ability of these transition metal oxyanions to stabilize the complex between the steroid receptor and hsp90. By promoting the formation of disulfide bonds, hydrogen peroxide also stabilizes the glucocorticoid receptor-hsp90 complex and prevents receptor transformation. A small, heat-stable factor present in all cytosol preparations inhibits receptor transformation, and, when the factor is removed, glucocorticoid receptors are rapidly transformed. This ubiquitous factor has the physical properties of a metal anion, and it is proposed that molybdate and vanadate affect steroid receptor complexes by interacting with a metal anion-binding site that is normally occupied by this endogenous receptor-stabilizing factor.     

Monovalent cation selectivity for ATP-dependent association of the glucocorticoid receptor with hsp70 and hsp90.

We have reported previously that incubation of the immunopurified transformed hormone-free glucocorticoid receptor with rabbit reticulocyte lysate reconstitutes the receptor complex with hsp90 and that reconstitution is accompanied by concomitant repression of DNA binding activity and regeneration of the steroid binding conformation (Scherrer, L. C., Dalman, F. C., Massa, E., Meshinchi, S., and Pratt, W. B. (1990) J. Biol. Chem. 265, 21397-21400). In this work we further characterize this system by defining the small M(r) components of reticulocyte lysate required for both structural and functional reconstitution of the receptor-hsp90 complex. Reconstitution is ATP-dependent and there is a direct relationship between the extent of hsp90 binding to the receptor and the number of specific steroid binding sites that are generated. Dialysis of reticulocyte lysate inactivates its reconstituting activity. Addition of an ATP-regenerating system or readdition of small M(r) lysate components (in the form of a Centricon C30 filtrate) has little effect, but the presence of both restores full reconstituting activity to dialyzed lysate, as assayed by steroid binding activity and by the binding of hsp90 and hsp70 to the receptor. The small M(r) activity is heat-stable, and it can be completely replaced by NH+4, K+, and Rb+, with K+ producing a maximal effect at the concentration normally present in undialyzed lysate. Na+ and Li+ have no reconstituting activity. This ion selectivity demonstrates that a monovalent cation binding site is involved in receptor heterocomplex reconstitution. It is intriguing that the protein unfoldase (e.g. clathrin uncoating ATPase) activity of hsp70 is known to have a similar monovalent cation dependence, and that under all conditions where hsp90 becomes bound to the receptor, we find that hsp70 is also bound.     

The binding activity of estrogen receptor to DNA and heat shock protein (Mr 90,000) is dependent on receptor-bound metal

1,10-Phenanthroline inhibited the DNA-cellulose binding of the transformed calf uterus estrogen receptor (homodimer of 66-kDa molecules: 5 S estrogen receptor) in a temperature- and concentration-dependent manner. This result appears related to the metal-chelating property of 1,10-phenanthroline, since the inhibition was decreased by addition of Zn2+ and Cd2+, but not by Ca2+, Ba2+, or Mg2+ for which the affinity of the chelator is low. Only a slight inhibition was observed in the presence of the 1,7-phenanthroline, a nonchelating analogue. After dialysis or filtration to remove free 1,10-phenanthroline, DNA binding of the 5 S estrogen receptor was still inhibited. Conversely, the chelator was unable to release prebound 5 S estrogen receptor from DNA-cellulose. The 5 S estrogen receptor DNA binding was inhibited when 1,10-phenanthroline was present during the transformation to activated receptor of the hetero-oligomeric nontransformed 9 S estrogen receptor, in which the hormone binding subunits are associated with heat shock protein, Mr 90,000 (hsp 90) molecules. In contrast, if 1,10-phenanthroline was removed before the transformation took place, only a slight inhibition was observed. Other experiments with EDTA indicated a similar inhibition of DNA-cellulose binding by the 5 S estradiol receptor, and all metal ions chelated by this agent prevented its inhibitory effect. The results indicate that 1,10-phenanthroline inhibited the DNA binding of the transformed 5 S estradiol receptor by chelating metal ion tightly bound to the receptor, which is not accessible to the chelator when the receptor is bound to DNA or to hsp 90. Therefore, they suggest that the metal ion may play a critical role in the interaction with DNA and hsp 90 by maintaining the structural integrity of the implicated receptor domain.     

Control of steroid receptor function and cytoplasmic-nuclear transport by heat shock proteins.

As targeted proteins that move within the cell, the steroid receptors have become very useful probes for understanding the linked phenomena of protein folding and transport. From the study of steroid receptor-associated proteins it has become clear over the past two years that these receptors are bound to a multiprotein complex containing at least two heat shock proteins, hsp90 and hsp56. Attachment of receptors to this complex in a cell-free system appears to require the protein unfolding/folding activity of a third heat shock protein, hsp70. Like the oncogenic tyrosine kinase pp60src, steroid receptors bind to this complex of chaperone proteins at the time of their translation. Binding of the receptor to the hsp90 component of the system occurs through the hormone binding domain and is under strict hormonal control. The hormone binding domain of the receptor acts as a transferable regulatory unit that confers both tight hormonal control and hsp90 binding onto chimaeric proteins. The model of folding and transport being developed for steroid receptors leads to some general suggestions regarding the folding and transport of targeted proteins in the cell.     

Evidence from pulse-chase labeling studies that the antiglucocorticoid hormone RU486 stabilizes the nonactivated form of the glucocorticoid receptor in mouse lymphoma cells

A pulse-chase labeling technique was used to determine the properties of glucocorticoid receptors occupied by the antiglucocorticoid hormone RU486 in S49.1 mouse lymphoma cells. Cells were pulse-labeled with [35S]methionine and then at the beginning of the chase, either no hormone (control), dexamethasone, or RU486 was added to cells. At 4 h into the chase, cytosol was prepared and receptors were immunoadsorbed to protein A-Sepharose using the BuGR2 antireceptor antibody. Immunoadsorbed proteins were resolved by gel electrophoresis and analyzed by autoradiography. The 90 kDa heat shock protein (hsp90) coimmunoadsorbed with receptors from control cells when protein A-Sepharose pellets were washed with 250 mM NaCl but not when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that hsp90-receptor complexes are disrupted by a high concentration of salt in the absence of molybdate. hsp90 coimmunoadsorbed with receptors from RU486-treated cells even when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that RU486 stabilizes the association of hsp90 with the glucocorticoid receptor. In contrast, hsp90 did not coimmunoadsorb with receptors from dexamethasone-treated cells, consistent with earlier evidence that hsp90 dissociates from the receptor when the receptor binds glucocorticoid hormone. Dexamethasone induced a rapid quantum decrease in the amount of normal receptor recovered from cytosol but did not induce a decrease in the amount of nuclear transfer deficient receptor recovered from cytosol, consistent with tight nuclear binding of normal receptors occupied by dexamethasone. In contrast, RU486 did not induce a quantum decrease in the recovery of normal receptors from cytosol, indicating that receptors occupied by RU486 are not tightly bound in the nuclear fraction. We conclude that the antiglucocorticoid hormone RU486, in contrast to the glucocorticoid hormone dexamethasone, stabilizes the association between the glucocorticoid receptor and hsp90. The decreased affinity of receptors occupied by RU486 for the nuclear fraction may be due to their association with hsp90 and may account for the failure of RU486 to exert agonist activity.     

Stoichiometry, abundance, and functional significance of the hsp90/hsp70-based multiprotein chaperone machinery in reticulocyte lysate

Rabbit reticulocyte lysate contains a multiprotein chaperone system that assembles the glucocorticoid receptor (GR) into a complex with hsp90 and converts the hormone binding domain of the receptor to its high affinity steroid binding state. This system has been resolved into five proteins, with hsp90 and hsp70 being essential and Hop, hsp40, and p23 acting as co-chaperones that optimize assembly. Hop binds independently to hsp70 and hsp90 to form an hsp90.Hop.hsp70 complex that acts as a machinery to open up the GR steroid binding site. Because purified hsp90 and hsp70 are sufficient for some activation of GR steroid binding activity, some investigators have rejected any role for Hop in GR.hsp90 heterocomplex assembly. Here, we counter that impression by showing that all of the Hop in reticulocyte lysate is present in an hsp90.Hop.hsp70 complex with a stoichiometry of 2:1:1. The complex accounts for approximately 30% of the hsp90 and approximately 9% of the hsp70 in lysate, and upon Sephacryl S-300 chromatography the GR.hsp90 assembly activity resides in the peak containing Hop-bound hsp90. Consistent with the notion that the two essential chaperones cooperate with each other to open up the steroid binding site, we also show that purified hsp90 and hsp70 interact directly with each other to form weak hsp90.hsp70 complexes with a stoichiometry of 2:1.     

Elimination and reconstitution of the requirement for hormone in promoting temperature-dependent transformation of cytosolic glucocorticoid receptors to the DNA-binding state

Cytosols contain a heat-stable, chelatable, anionic, molybdate-like factor that stabilizes glucocorticoid receptors in a heteromeric complex with hsp90 (refers to the 90-kDa heat shock protein) and inhibits their transformation to the DNA-binding state (Meshinchi, S., Grippo, J.F., Sanchez, E.R., Bresnick, E.H., and Pratt, W.B. (1988) J. Biol. Chem. 263, 16809-16817). In this work, we demonstrate that removal of this factor by passage of L cell cytosol through the metal-chelating resin Chelex-100 makes the glucocorticoid receptor unstable, thus markedly facilitating both its dissociation from hsp90 and its transformation to the DNA-binding state. In normal cytosol, both temperature-mediated dissociation of hsp90 and temperature-mediated receptor transformation are hormone-dependent events. In the Chelex-treated, metal-depleted cytosol, however, temperature-mediated dissociation of hsp90 and receptor transformation occur very rapidly in a manner that is no longer hormone-dependent. When boiled L cell cytosol is added to the metal-depleted receptor system, the hormone dependence of both temperature-mediated dissociation of receptor from hsp90 and receptor transformation to the DNA-binding state is reconstituted. Like boiled cytosol, molybdate stabilizes the receptor complex and inhibits its transformation in metal-depleted cytosol, but it does not reconstitute the hormone dependence of the system. These results support the proposal that an endogenous metal anion interacts with the glucocorticoid receptor to stabilize it in the heteromeric, inactive, non-DNA-binding state in cytosol and that binding of the hormone promotes conversion of the receptor to the DNA-binding state through an effect on this metal anion center.