Glucocorticoid Receptor Heterocomplex Research Articles

The transportosome system as a model for the retrotransport of soluble proteins

The classic model of action of the glucocorticoid receptor (GR) sustains that its associated heat-shock protein of 90-kDa (HSP90) favours the cytoplasmic retention of the unliganded GR, whereas the binding of steroid triggers the dissociation of HSP90 allowing the passive nuclear accumulation of GR. In recent years, it was described a molecular machinery called transportosome that is responsible for the active retrograde transport of GR. The transportosome heterocomplex includes a dimer of HSP90, the stabilizer co-chaperone p23, and FKBP52 (FK506-binding protein of 52-kDa), an immunophilin that binds dynein/dynactin motor proteins. The model shows that upon steroid binding, FKBP52 is recruited to the GR allowing its active retrograde transport on cytoskeletal tracks. Then, the entire GR heterocomplex translocates through the nuclear pore complex. The HSP90-based heterocomplex is released in the nucleoplasm followed by receptor dimerization. Subsequent findings demonstrated that the transportosome is also responsible for the retrotransport of other soluble proteins. Importantly, the disruption of this molecular oligomer leads to several diseases. In this article, we discuss the relevance of this transport machinery in health and disease.

Pattern of heat shock factor and heat shock protein expression in lymphocytes of bipolar patients: Increased HSP70-glucocorticoid receptor heterocomplex

Bipolar disorder (BD), a stress-related disease, is characterized by altered glucocorticoid receptor (GR) signalling. Stress response includes activation of heat shock factor (HSF) and subsequent heat shock protein (HSP) synthesis which regulate GR folding and function. The objective of this study was to investigate the possible role of HSFs, HSPs and their interaction with GR in BD. We applied immunoprecipitation, SDS-PAGE/Western blot analysis and electrophoretic mobility shift assay (EMSA) in lymphocytes (whole cell or nuclear extracts) from BD patients and healthy subjects and determined the HSPs (HSP90 and HSP70), the heterocomplexes HSP90-GR and HSP70-GR, the HSFs (HSF1 and HSF4) as well as the HSF-DNA binding. The HSP70-GR heterocomplex was elevated (p < 0.05) in BD patients vs healthy subjects, and nuclear HSP70 was reduced (p ≤ 0.01) in bipolar manic patients. Protein levels of HSF1, HSF4, HSP90, HSP90-GR heterocomplex, and HSF-DNA binding remained unaltered in BD patients vs healthy subjects. The corresponding effect sizes (ES) indicated a large ES for HSP70-GR, HSP70, HSF-DNA binding and HSF4, and a medium ES for HSP90, HSF1 and HSP90-GR between healthy subjects and bipolar patients. Significant correlations among HSFs, HSPs, GR and HSP70-GR heterocomplex were observed in healthy subjects, which were abrogated in bipolar patients. The higher interaction between GR and HSP70 and the disturbances in the relations among heat shock response parameters and GR as observed in our BD patients may provide novel insights into the contribution of these factors in BD aetiopathogenesis.

HDAC6 Regulates Glucocorticoid Receptor Signaling in Serotonin Pathways with Critical Impact on Stress Resilience

Genetic variations in certain components of the glucocorticoid receptor (GR) chaperone complex have been associated with the development of stress-related affective disorders and individual variability in therapeutic responses to antidepressants. Mechanisms that link GR chaperoning and stress susceptibility are not well understood. Here, we show that the effects of glucocorticoid hormones on socioaffective behaviors are critically regulated via reversible acetylation of Hsp90, a key component of the GR chaperone complex. We provide pharmacological and genetic evidence indicating that the cytoplasmic lysine deacetylase HDAC6 controls Hsp90 acetylation in the brain, and thereby modulates Hsp90-GR protein-protein interactions, as well as hormone- and stress-induced GR translocation, with a critical impact on GR downstream signaling and behavior. Pet1-Cre-driven deletion of HDAC6 in serotonin neurons, the densest HDAC6-expressing cell group in the mouse brain, dramatically reduced acute anxiogenic effects of the glucocorticoid hormone corticosterone in the open-field, elevated plus maze, and social interaction tests. Serotonin-selective depletion of HDAC6 also blocked the expression of social avoidance in mice exposed to chronic social defeat and concurrently prevented the electrophysiological and morphological changes induced, in serotonin neurons, by this murine model of traumatic stress. Together, these results identify HDAC6 inhibition as a potential new strategy for proresilience and antidepressant interventions through regulation of the Hsp90-GR heterocomplex and focal prevention of GR signaling in serotonin pathways. Our data thus uncover an alternate mechanism by which pan-HDAC inhibitors may regulate stress-related behaviors independently of their action on histones.

Increased hsp70 of glucocorticoid receptor complex induced by scald and heat stress and its possible effect on the affinity of glucocorticoid receptor

Background Glucocorticoid (GC) insensitivity/GC resistance is an important etiological and prognostic factor in multiple diseases and pathophysiological processes such as scald, shock and asthma. The function of GC was mediated by glucocorticoid receptor (GR). Scald not only decreased the expression of GR but also reduced the affinity of GR, which played an important role in GC resistance in scalded rats. Whereas the molecular mechanism responsible for the decrease of GR affinity resulted from scald remains unclear. Recent studies showed that the changes of heat shock proteins (hsp) especially hsp90 and hsp70 of GR heterocomplex were associated with GR low affinity in vitro. Methods The affinity of GR in hepatic cytosols and in the cytosols of SMMC-7721 cells were determined by radioligand binding assay and scatchard plot. GR heterocomplex in cytosols were captured by coimmunoprecipation and the levels of hsp90 and hsp70 of GR complex were detected by quantitative Western blotting. Results Similar with that of hepatic cytosol of scalded rats, a remarkable decrease of GR affinity was also found in the cytosol of heat stressed SMMC-7721 cells. The level of hsp70 of GR complex in hepatic cytosol of scalded rats (30% total body surface area immersion scald) and in cytosol of heat stressed human hepatocarcinoma cell line SMMC-7721 were both increased by 1.5 fold, whereas no change of hsp90 in GR heterocomplex was found. According to the correlation analysis, there may be a positive relationship between increased hsp70 of GR complex and decreased GR affinity in the cytosols. Conclusions The primary results indicated that the level of hsp70 of GR heterocomplex was increased in the hepatic cytosol of scalded rats and the cytosol of heat stressed SMMC-7721 cells. The increase of hsp70 of GR complex might be associated with the decrease of GR affinity. Chin Med J 2010;123(13):1780–1785

Gender-related differences in the effects of antidepressant imipramine on glucocorticoid receptor binding properties and association with heat shock proteins in the rat liver and kidney

Gender-related differences in susceptibility to stress and stress-related disorders such as depression, and in response to treatment with antidepressants have been observed, but the underlying molecular mechanisms are still unknown. Considering the role of glucocorticoid hormones in the systemic reaction against stress and in pathogenesis of depression, the aim of the present work was to study gender-related differences in glucocorticoid signaling and in response of this system to a typical antidepressant drug, imipramine. Gender-related differences in glucocorticoid receptor functional properties were assessed using hepatic and renal whole cell extracts of female and male rats before and after long-term imipramine treatment. The receptor's hormone-binding parameters, B max and K D, were determined by radioligand binding assay, the glucocorticoid receptor and heat shock proteins (Hsp70 and Hsp90) levels by quantitative immunoblotting, and the interaction of these proteins within glucocorticoid receptor heterocomplex by co-immunoprecipitation. Glucocorticoid receptor binding potency ( B max/ K D ratio) was significantly higher in males than females both before and after treatment with imipramine. Gender-specific changes in the glucocorticoid receptor binding parameters in the examined tissues were observed in response to imipramine, and were found to be associated with alterations in the receptor interaction with Hsp70 and Hsp90. The results of the study point to sexual dimorphism in the glucocorticoid signaling and imply that glucocorticoid receptor functional alterations contribute to gender-related differences in vulnerability to stress and stress-related disorders, and in response to antidepressant drugs.

The glucocorticoid receptor heterocomplex gene STIP1 is associated with improved lung function in asthmatic subjects treated with inhaled corticosteroids

Corticosteroids exert their anti-inflammatory action by binding and activating the intracellular glucocorticoid receptor heterocomplex. We sought to evaluate the genes HSPCB, HSPCA, STIP1, HSPA8, DNAJB1, PTGES3, FKBP5, and FKBP4 on corticosteroid response. White asthmatic subjects (n = 382) randomized to once-daily flunisolide or conventional inhaled corticosteroid therapy were genotyped. Outcome measures were baseline FEV1, percent predicted FEV1, and percent change in FEV1 after corticosteroid treatment. Multivariable analyses adjusted for age, sex, and height were performed, fitting the most appropriate genetic model based on the quantitative mean derived from ANOVA models to determine whether there was an independent effect of polymorphisms on change in FEV1 independent of baseline level. Positive recessive model correlations for STIP1 single nucleotide polymorphisms were observed for baseline FEV1 (rs4980524, P = .009; rs6591838, P = .0045; rs2236647, P = .002; and rs2236648; P = .013), baseline percent predicted FEV1 (rs4980524, P = .002; rs6591838, P = .017; rs2236647, P = .003; and rs2236648, P = .008), and percent change in FEV1 at 4 weeks (rs4980524, P = .044; rs6591838, P = .016; and rs2236647, P = .01) and 8 weeks (rs4980524, P = .044; rs6591838, P = .016; and rs2236647; P = .01) or therapy. Haplotypic associations were observed for baseline FEV1 and percent change in FEV1 at 4 weeks of therapy (P = .05 and P = .01, respectively). Significant trends toward association were observed for baseline percent predicted FEV1 and percent change in FEV1 at 8 weeks of therapy. Positive correlations between haplotypes and percent change in FEV1 were also observed. STIP1 genetic variations might play a role in regulating corticosteroid response in asthmatic subjects with reduced lung function. Replication in a second asthmatic population is required to confirm these observations.

Targeted ablation reveals a novel role of FKBP52 in gene-specific regulation of glucocorticoid receptor transcriptional activity

FKBP52 is a tetratricopeptide repeat (TPR) protein with peptidyl-prolyl isomerase activity and is found in steroid receptor complexes, including glucocorticoid receptor (GR). It is generally accepted that FKBP52 has a stimulatory effect on GR transcriptional activity. However, the mechanism by which FKBP52 controls GR is not yet clear, with reports showing effects on GR hormone-binding affinity and/or hormone-induced nuclear translocation. To address this issue, we have generated mice with targeted ablation of the FKBP52 gene. To date, no overt defects of GR-regulated physiology have been found in these animals, demonstrating that FKBP52 is not an essential regulator of global GR activity. To better assess the impact of FKBP52 on GR, mouse embryonic fibroblasts (MEFs) were generated from wild-type (WT) and FKBP52-deficient (KO) animals. Analysis of GR activity at reporter genes showed an approximate 70% reduction of activity in 52KO MEF cells, with no effect of FKBP52 loss on thyroid receptor. Interestingly, GR activity at endogenous genes was not globally affected in 52KO cells, with reduced activity at GILZ and FKBP51, but not at SGK and p21. Thus, FKBP52 appears to be a gene-specific modulator of GR. To investigate the mechanism of this action, analyses of GR heterocomplex composition, hormone-binding affinity, and ability to undergo hormone-induced nuclear translocation and DNA-binding were performed. Interestingly, no effect of FKBP52 loss was found for any of these GR properties, suggesting that the main function of FKBP52 is a heretofore-unknown ability to control GR activity at target genes. Lastly, loss of FKBP52 did not affect the ability of GR to undergo hormone-induced autologous down-regulation, showing that FKBP52 does not contribute to all branches of GR signaling. The implications of these results to the potential actions of FKBP52 on GR activity in vivo are discussed.

Interaction of rat renal glucocorticoid receptor with Hsp90 and Hsp70 upon stress provoked by mercury

The influence of mercury on the association of rat kidney glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70 was investigated. The GR heterocomplexes with Hsp90 and Hsp70 were immunopurified from the renal cytosol of rats administered different doses of mercury (1, 2 and 3 mg Hg kg(-1) b.w.). A quantitative immunoblotting procedure was applied to determine the levels of GR, Hsp90 and two nucleocytoplasmic Hsp70 isoforms (constitutive Hsp73 and inducible Hsp72) in the renal cytosol, as well as the amounts of these proteins within GR heterocomplexes immunoprecipitated by anti-GR antibody. Mercury was found to stimulate GR association with all the examined Hsps. The most prominent effect of the metal was stimulation of Hsp72 interaction with GR. On the other hand, the metal administration led to an increase of Hsp90 level in the cytosol, while the cytosolic levels of Hsp70 isoforms remained unaltered. These findings suggest that association of Hsps, at least Hsp70, with the GR might be ascribed to changes in the affinity of their interaction rather than to changes in the Hsp availability in the cytosol. Therefore, GR heterocomplex assembly seems to be a controlled process enabling chaperoning and functioning of the GR to be in concert with physiological demands.

Mercury stimulates rat liver glucocorticoid receptor association with Hsp90 and Hsp70

The subject of the present study is the influence of mercury on association of rat liver glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70. The glucocorticoid receptor heterocomplexes with Hsp90 and Hsp70 were immunopurified from the liver cytosol of rats administered with different doses of mercury. The amounts of co-immunopurified apo-receptor, Hsp90 and Hsp70 were then determined by quantitative Western blotting. The ratio between the amount of heat shock protein Hsp90 or Hsp70 and the amount of apo-receptor within immunopurified heterocomplexes was found to increase in response to mercury administration. On the other hand, the levels of Hsp90 and Hsp70 in hepatic cytosol remained unaltered. The finding that mercury stimulates association of the two heat shock proteins with the glucocorticoid receptor, rendering the cytosolic heat shock protein levels unchanged, suggests that mercury affects the mechanisms controlling the assembly of the receptor heterocomplexes.

Pifithrin-α Inhibits p53 Signaling after Interaction of the Tumor Suppressor Protein with hsp90 and Its Nuclear Translocation

Pifithrin-alpha (PFTalpha) was originally thought to be a specific inhibitor of signaling by the tumor suppressor protein p53. However, the laboratory that discovered pifithrin recently reported that the compound also inhibits heat shock and glucocorticoid receptor (GR) signaling, and they suggested that PFTalpha targets a factor common to all three signal transduction pathways, such as the hsp90/hsp70-based chaperone machinery (Komarova, E. A., Neznanov, N., Komarov, P. G., Chernov, M. V., Wang, K., and Gudkov, A. V. (2003) J. Biol. Chem. 278, 15465-15468). Because it is important for the mechanistic study of this machinery to identify unique inhibitors of chaperone action, we have examined the effect of PFTalpha on transcriptional activation, the hsp90 heterocomplex assembly, and hsp90-dependent nuclear translocation for both p53 and the GR. At concentrations where PFTalpha blocks p53-mediated induction of p21/Waf-1 in human embryonic kidney cells, we observed no inhibition of GR-mediated induction of a chloramphenicol acetyl transferase reporter in LMCAT cells. PFTalpha did, however, cause a left shift in the dexamethasone dose response curve by increasing intracellular dexamethasone concentration, apparently by competing for dexamethasone efflux from the cell. The assembly of p53 or GR heterocomplexes with hsp90 and immunophilins was not affected by PFTalpha either in vivo or in vitro and did not affect the nuclear translocation of either transcription factor. Thus, we conclude that PFTalpha does not inhibit GR-mediated induction or the function of the chaperone machinery, and, as originally thought, it may specifically inhibit p53 signaling by acting at a stage after p53 translocation to the nucleus.

Hyperthermic stress stimulates the association of both constitutive and inducible isoforms of 70 kDa heat shock protein with rat liver glucocorticoid receptor

Glucocorticoid hormone receptor exists in the cytoplasm of target cells in the form of dynamic multiprotein heterocomplexes with heat shock proteins Hsp90 and Hsp70, and additional components of the molecular chaperone machinery. Whole body hyperthermic stress was previously shown to induce alterations in protein composition of these complexes increasing the share of Hsp70, but participation of individual Hsp70 family members was not investigated. In the present study the association of glucocorticoid receptor with constitutive and inducible forms of Hsp70 in the liver cytosol of rats exposed to 41 °C whole body hyperthermic stress was examined. Immunoprecipitation of glucocorticoid receptor heterocomplexes by monoclonal anti-receptor antibody (BuGR2) followed by quantitative immunoblotting revealed the presence of both nucleocytoplasmic Hsp70 family members, constitutive—Hsc70 and inducible—Hsp72, within the complexes. Immediately after the stress only Hsc70 was found in association with glucocorticoid receptor. However, after the induction of Hsp72 by stress, its appearance within the glucocorticoid receptor heterocomplexes was also recorded and the presence of both Hsp70 forms within the heterocomplexes was evident by the end of examined 24 h period after the stress. This study confirms that heat stress affects protein composition of rat liver glucocorticoid receptor heterocomplexes increasing the share of Hsp70 and shows that this increase could be equally ascribed to constitutive and inducible forms of Hsp70.

A New First Step in Activation of Steroid Receptors: HORMONE-INDUCED SWITCHING OF FKBP51 AND FKBP52 IMMUNOPHILINS

We have identified a new first step in the hormonal activation of the glucocorticoid receptor (GR). Rather than causing immediate dissociation of the cytoplasmic GR heterocomplex, binding of hormone-induced substitution of one immunophilin (FKBP51) for another (FKBP52), and concomitant recruitment of the transport protein dynein while leaving Hsp90 unchanged. Immunofluorescence and fractionation revealed hormone-induced translocation of the hormone-generated GR-Hsp90-FKBP52-dynein complex from cytoplasm to nucleus, a step that precedes dissociation of the complex within the nucleus and conversion of GR to the DNA-binding form. Taken as a whole, these studies identify immunophilin interchange as the earliest known event in steroid receptor signaling and provide the first evidence of differential roles for FKBP51 and FKBP52 immunophilins in the control of steroid receptor subcellular localization and transport.

Glucocorticoid receptor-Hsp90 interaction in the liver cytosol of cadmium-intoxicated rats.

The influence of cadmium on the rat liver glucocorticoid receptor (GR) binding capacity, on the cytosolic level of 90 kDa heat shock protein (Hsp90), and on the association of the two proteins was investigated. The results showed that the mode of metal application led to diverse alterations in hormone binding to the GR. Reduction of the GR binding capacity observed after in vitro treatment was proportional to the applied metal concentrations. In animals administered different doses of cadmium, GR binding capacity was not reduced, except in those that received the highest dose. A concomitant elevation of Hsp90 level was detected both in the cytosol and within the GR untransformed heterocomplexes. The results suggest that cadmium-induced reduction of the GR binding capacity seen in vitro was prevented in intact animals by the elevated level of Hsp90 within the GR heterocomplexes.

Somatostatin Increases Glucocorticoid Binding and Signaling in Macrophages by Blocking the Calpain-specific Cleavage of Hsp 90

Somatostatin has direct anti-inflammatory actions and participates in the anti-inflammatory actions of glucocorticoids, but the mechanisms underlying this regulation remain poorly understood. The objective of this study was to evaluate whether somatostatin increases glucocorticoid responsiveness by up-regulating glucocorticoid receptor (GR) expression and signaling. Somatostatin promoted a time- and dose-dependent increase in [(3)H]dexamethasone binding to RAW 264.7 macrophages. Cell exposure to 10 nM somatostatin for 18 h promoted a 2-fold increase in the number of GR sites per cell without significant modification of the affinity. Analysis of GR heterocomplex components demonstrated that somatostatin increased the level of heat shock protein (Hsp) 90, whereas the level of GR remained almost unchanged. The increase in Hsp 90 was associated with a decrease in the cleavage of its carboxyl-terminal domain. Evidence for the involvement of calpain inhibition in this process was obtained by the demonstration that 1) somatostatin induced a dose-dependent decrease in calpain activity and 2) calpain inhibitors, calpain inhibitor I and calpeptin, both abolished the cleavage of Hsp 90 and induced a dose-dependent increase in [(3)H]dexamethasone binding. Increases in glucocorticoid binding after somatostatin treatment were associated with similar increases in the ability of GR to transactivate a minimal promoter containing two glucocorticoid response elements (GRE) and to interfere with the activation of nuclear factor-kappaB (NF-kappaB). Thus, the present findings indicate that somatostatin increases glucocorticoid binding and signaling by limiting the calpain-specific cleavage of GR-associated Hsp 90. This mechanism may represent a novel target for intervention to increase glucocorticoid responsiveness.

The glucocorticoid properties of the synthetic steroid pregna-1,4-diene-11 β-ol-3,20-dione (ΔHOP) are not entirely correlated with the steroid binding to the glucocorticoid receptor

The natural steroid 11 β-hydroxyprogesterone is not only a modulator of 11 β-hydroxy-steroid dehydrogenase activity, but also an efficient inducer of tyrosine aminotransferase activity in hepatocytes. In contrast with the low affinity for the mineralocorticoid receptor, 11 β-hydroxyprogesterone binds well to both the glucocorticoid receptor and the carrier protein transcortin. It is accepted that the introduction of a 1:ene double bond into 3-keto 4:ene steroids increases the glucocorticoid potency, so that 3-keto-1,4:diene steroids show improved chemical stability and are more potent glucocorticoids than their respective 4:ene analogs. The steroid pregna-1,4-diene-11 β-ol-3,20-dione (ΔHOP) had previously been described as an anti-inflamatory compound and an inhibitor of macromolecular biosynthesis in thymocytes and lymphocytes. In such studies, ΔHOP also exhibited some particular glucocorticoid properties which made it attractive as a tool for the study of the mechanism of action of glucocorticoids. In the present paper we show that ΔHOP possesses some classical biological actions of glucocorticoids such as deposition of glycogen in rat liver, induction of TAT activity in hepatocytes, and inhibition of the uptake of leucine and thymidine by thymocytes. It also exhibits minimal sodium-retaining properties. Consistent with these biological effects, ΔHOP shows a 70 times lower relative binding affinity for the mineralocortioid receptor than aldosterone, but a reasonable affinity for the glucocorticoid receptor, and is as efficient as dexamethasone in dissociating the 90 kDa heat shock protein from the glucocorticoid receptor heterocomplex. However, the inhibition of the uptake of amino acids and nucleotides observed in the presence of ΔHOP is not efficiently blocked when thymocytes are coincubated in the presence of steroids with known antiglucocorticoid activity. ΔHOP is similarly inefficient in inducing chloramphenicol–acetyl transferase activity in cells transfected with a plasmid that possesses two canonical glucocorticoid-responsive elements. Unlike most glucocorticoids, ΔHOP does not induce the fragmentation of DNA in a regular pattern characteristic of apoptosis and it does not reduce thymus weight. This unusual dissociation of glucocorticoid parameters makes ΔHOP a useful tool to discriminate between mechanisms of action by which steroids can exert their biological effects.

Association of the rat liver glucocorticoid receptor with Hsp90 and Hsp70 upon whole body hyperthermic stress

The influence of whole body hyperthermic stress (41°C, 15 min) on association of the glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70 was followed in rat liver cytosol during a 24 h period after the stress. Total cytosolic concentration of the GR, Hsp90 and Hsp70 and the amounts of Hsp90 and Hsp70 co-immunopurified with the GR were determined by a quantitative Western blotting using appropriate monoclonal antibodies. A significant decrease in the cytosolic GR level in response to the stress was noticed. The ratio of the amount of the GR to Hsp90 recruited by the GR was found to be unaltered by hyperthermia, in spite of the stress-induced increase in the total Hsp90 concentration in the cytosol. Hsp70 was also found in association with the GR and its 2.5-fold induction by the stress was accompanied by about 3-fold increase in its relative amount that co-immunopurified with the GR. The results suggest that heat stress influences the interaction of the GR with Hsp70 through the mechanisms controlling the untransformed rat liver GR heterocomplexes assembly process.

Folding of the Glucocorticoid Receptor by the Heat Shock Protein (hsp) 90-based Chaperone Machinery: THE ROLE OF p23 IS TO STABILIZE RECEPTOR·hsp90 HETEROCOMPLEXES FORMED BY hsp90·p60·hsp70

In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor.hsp90 heterocomplexes. We have reconstituted a minimal receptor.hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833-12839). We have shown that hsp90, p60, and hsp70 are sufficient for carrying out the folding change that converts the glucocorticoid receptor (GR) hormone binding domain (HBD) from a non-steroid binding to a steroid binding conformation, but to form stable GR.hsp90 heterocomplexes, p23 must also be present in the incubation mix (Dittmar, K. D., and Pratt, W. B. (1997) J. Biol. Chem. 272, 13047-13054). In this work, we show that addition of p23 to native GR.hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR.hsp90 heterocomplexes prepared with the minimal (hsp90.p60.hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR.hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90.p60.hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR.hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90.p60.hsp70 forms a GR.hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR.hsp90 heterocomplex to inactivation and disassembly.

Protein Phosphatase 5 Is a Major Component of Glucocorticoid Receptor·hsp90 Complexes with Properties of an FK506-binding Immunophilin

Steroid receptors are recovered from hormone-free cells in multiprotein complexes containing hsp90, p23, an immunophilin, and often some hsp70. The immunophilin, which can be of the FK506- or cyclosporin A-binding class, binds to hsp90 via its tetratricopeptide repeat (TPR) domain, and different receptor heterocomplexes exist depending upon which immunophilin occupies the TPR-binding region of hsp90. We have recently reported that a protein serine/threonine phosphatase that is designated PP5 and contains four TPRs binds to hsp90 and is co-purified with the glucocorticoid receptor (GR) (Chen, M.-S., Silverstein, A. M., Pratt, W. B., and Chinkers, M. (1996) J. Biol. Chem. 271, 32315-32320). In this work, we show that PP5 is recovered with both GR that is nuclear and GR that is cytoplasmic in hormone-free cells. Approximately one-half of the GR.hsp90 heterocomplexes in L cell cytosol contains an immunophilin with high affinity FK506 binding activity, such as FKBP51 or FKBP52, and approximately 35% contains PP5. Only a small (but undetermined) fraction of the native GR.hsp90 heterocomplexes contain the cyclosporin A-binding immunophilin CyP-40. PP5, FKBP52, and CyP-40 exist in separate heterocomplexes with hsp90, and competition binding experiments with the PP5 TPR domain suggest that the three proteins occupy a common binding site on hsp90. A 55-residue connecting region between the N-terminal TPR domain of human PP5 and its C-terminal phosphatase domain has 50% amino acid homology and 22% identity with the central portion of the peptidylprolyl isomerase domain of human FKBP52. Of the 9 residues in this portion of FKBP52 involved in high affinity interactions with FK506, 3 residues are retained and 4 have homologous substitutions in PP5. Although immunoadsorbed PP5 did not bind [3H]FK506, we found that both rabbit PP5 in reticulocyte lysate and purified rat PP5 were specifically retained by an FK506 affinity matrix. Thus, we propose that PP5 possesses properties of an immunophilin with low affinity FK506 binding activity and that it determines a major portion of the native GR heterocomplexes in L cell cytosol.

The Tetratricopeptide Repeat Domain of Protein Phosphatase 5 Mediates Binding to Glucocorticoid Receptor Heterocomplexes and Acts as a Dominant Negative Mutant

We previously identified a protein-serine phosphatase designated PP5, based on the binding of its tetratricopeptide repeat (TPR) domain to the atrial natriuretic peptide receptor (Chinkers, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11075-11079). We have now identified another protein complex to which PP5 is targeted through its TPR domain. A 90-kDa protein, identified as heat shock protein 90 (hsp90) by immunoblotting, specifically co-immunoprecipitated from COS-7 cell lysates with the FLAG-tagged TPR domain of PP5. hsp90 also co-immunoprecipitated with full-length FLAG-tagged PP5 overexpressed in COS-7 cells and with endogenous PP5 from untransfected COS-7 cells or rat brain. During gel filtration, PP5 and hsp90 comigrated in a high molecular weight complex. Since glucocorticoid receptors (GR) exist as large heterocomplexes containing hsp90 bound to TPR proteins, we hypothesized that PP5 might be associated with these complexes. Consistent with this hypothesis, PP5 specifically co-immunoprecipitated with GR from mouse L cell lysates. To test the functional importance of this TPR-mediated association in living cells, we used a dominant negative PP5 mutant consisting only of its TPR domain. The mutant inhibited GR-mediated transactivation by approximately 70% in transfected CV-1 cells. This is the first evidence that the TPR proteins in steroid receptor heterocomplexes may be required for signaling in vivo.

Evidence that the FK506-binding immunophilin heat shock protein 56 is required for trafficking of the glucocorticoid receptor from the cytoplasm to the nucleus.

The FK506-binding immunophilin hsp56 (FKBP52) is one of several chaperone proteins associated with untrasformed steroid receptors in a multiprotein heterocomplex. The function of heat shock protein 56 (hsp56) with respect to receptor action is unknown. hsp56 is not required for glucocorticoid receptor heterocomplex assembly or for proper folding of the receptor hormone-binding domain into a high affinity steroid-binding conformation. In intact cells, the majority of the hsp56 is located in the nucleus, with a minority colocalizing with microtubules in the cytoplasm. hsp56 contains a conserved negatively charged domain that we speculate might serve as a nuclear localization signal recognition sequence. Here we show that injection of an antibody raised against this negative sequence into intact L cells impedes subsequent dexamethasone-mediated shift of the glucocorticoid receptor into the nucleus. Nonimmune rabbit serum and an antibody raised against another site on hsp56 do not affect receptor movement. Inhibition of receptor movement by the 419 antibody against the negative sequence is blocked by preincubation with purified hsp56, but not by preincubation with purified hsp90, hsp70, or BSA. These observations are consistent with the possibility that hsp56 is involved in receptor trafficking to the nucleus, possibly functioning as the nuclear localization signal recognition protein. Receptor trafficking to the nucleus is not affected by FK506, indicating that the peptidylprolyl isomerase activity of hsp56 is not involved.