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Heat Shock Protein Interactions with the Glucocorticoid Receptor
Pulmonary Pharmacology & Therapeutics (1998) 11, 7–12 Article No. pu980119
PULMONARY PHARMACOLOGY & THERAPEUTICS
Review Heat Shock Protein Interactions with the Glucocorticoid Receptor G. Bertorelli∗, V. Bocchino, D. Olivieri Department of Respiratory Disease, University of Parma, Italy based on their capacity to associate with other proteins in a way which modifies the destiny and function of the latter. Recent interest has focused on the cytoprotective properties of the stress response in diverse forms of cellular and tissue injury. Hsp has also been reported to prevent cell death by apoptosis. Although at first sight this antiapoptotic effect might appear protective, it actually could promote persistence rather than resolution of inflammation, by preventing the physiological removal of inflammatory cells by apoptosis, an essential component of the healing process. Finally, we know that hsp also serve many important physiological functions and have a key physiological role in hormone receptors.
INTRODUCTION
‘. . . In contrast to such “shocks” for which the genome is unprepared, are those a genome must face repeatedly, and for which it is prepared to respond in a programmed manner. Examples are the “heat shock” responses in eukaryotic organisms, and the “SOS” responses in bacteria. Each of these initiates a highly programmed sequence of events within the cell that serves to cushion the effects of the shock. Some sensing mechanism must be present in this instance to alert the cell to imminent danger, and to set in motion the orderly sequence of events that will mitigate this danger. The responses of genomes to anticipated challenges are not so precisely programmed. Nevertheless, these are sensed, and the genome responds in a discernible but initially unforeseen manner.’ Barbara McClintock – Nobel lecture, 8 December 1983
THE GLUCOCORTICOID RECEPTOR These prophetic words by Barbara McClintock eloquently capture the essence of the heat shock response, which was described over 30 years ago when Ferruccio Ritossa,1 noted that a temperature shift from 25 to 30°C induced a new puffing pattern in specific regions of the giant chromosome from the salivary glands of Drosophila. The products of this phenomenon were identified as a specific group of proteins termed heat shock proteins (hsp). Although initially a temperaturedependent pattern of gene expression in Drosophila, this was subsequently found to occur in virtually all organisms from prokaryotes to humans and there is now a greater understanding of the stress response to thermal or non-thermal cytotoxic stimuli. The stress response is a highly conserved cellular defence mechanism defined by the rapid and specific expression of stress proteins, with concomitant transient inhibition of non-stress protein gene expression. It is now clear that the importance of many hsp is
Steroid hormones are essential constituents of the intercellular communication system that maintains homeostasis in higher organisms. Glucocorticoids, a major subclass of steroid hormones, modulate a large number of metabolic, cardiovascular, immune, and behavioral functions.2,3 At the cellular level, most known effects of glucocorticoids are mediated by a >94 kDa intracellular protein, the glucocorticoid receptor (GR). GR belongs to the phylogenetically conserved superfamily of nuclear hormone receptor, which includes receptors for mineralocorticoids, androgens, progestins, estrogens, vitamin D, thyroid hormone, retinoic acid, and a growing number of socalled orphan receptors for which no specific ligand has yet been identified.4,5 All members of the nuclear hormone receptor family share a characteristic three-domain structure: (a) the N-terminal domain contains sequences responsible for activation of target genes and interacts with components of the basal transcription machinery and/or other transcription factors;6–8 (b) two highly conserved ‘zinc fingers’ in the central part of the receptor
∗ Author for correspondence. Present address: Department of Respiratory Diseases, Rasori Hospital, Via Rasori 10, 43100, Parma, Italy. E-mail: [email protected] 1094–5539/98/011007+06 $30.00/0
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molecule constitute the DNA-binding domain,6,9,10 which also participate in receptor dimerization,11 nuclear translocation,12 and transactivation;7,13 (c) the Cterminal or ligand-binding domain, which binds the hormonal ligand,6,14 contains sequences important for hsp binding,15–17 nuclear translocation,12 dimerization,18 and transactivation,19–22 as well as silencing the receptor in the absence of hormone.7,23 The GR is transformed from a silent to an active transcription factor in response to glucocorticoids.24–27 The non-ligand GR is part of a multiprotein complex that consists of the receptor, two molecules of hsp90, and one molecule each of hsp70, and hsp56, an immunophilin of the FK506- and rapamycin-binding class.16,17,28–31 In addition, other less well characterized proteins have occasionally been found to participate in this complex.28 In the absence of hormone, this complex most likely undergoes constant cycles of dissociation and ATP- and hsp70-dependent reassociation.30,32 Intracellular localization of GR/hsp70 complex also seems to be less static than previously thought. Experimental evidence supports a model of constant bidirectional movement of the complex between the cytosol and the nucleus.17,33 The exact composition of this complex may determine the predominant direction of this movement; for example, the presence of hsp56 may direct the complex to the cell nucleus.17,34,35 However, it has not been shown conclusively whether any component of the hsp complex remains bound to the receptor during nucleocytoplasmic transfer. Quantitative differences between cell types with regard to GR transfer may account for the apparently contradictory reports as to whether the GR is a cytoplasmic or a nuclear protein.36–41 Independent of its intracellular localization, the main function of the GR/hsp complex is to keep the receptor protein in an inactive, yet potential ligand-active state.16,17,28–31,42,43 As lipophilic substances, glucocorticoids are able to cross the cell membrane readily to interact with the intracellular GR. Ligand binding induces an as yet undetermined conformational change in the GR molecule with a number of functional consequences: the hormone-bound GR dissociates from the hsp complex and is no longer able to reassociate with it.16,24,25,32 Furthermore, the partially phosphorylated receptor protein becomes hyperphosphorylated.32,44,45 Finally, hormone binding may cause nuclear translocation of cytoplasmic GR molecules.39 GR are recovered from cells in large (9S) heterocomplexes that contain both hsp and immunophilins. Some components of the receptor heterocomplexes are proteins with established chaperone functions. One critical function of the hsp is to facilitate the folding of the hormone-binding domain (HBD) of the receptor into a high-affinity steroidbinding conformation. These hsp appear to act as
protein-folding machines that assemble heterocomplexes between the hsp and the receptors.46 Hsp90 interacts directly with the HBD of the nuclear receptors, an association which appears to account for a repression of receptor function relieved upon subsequent binding of hormone. This ability of the hormone to control HBD-chaperone interaction is now regarded as the earliest event in the molecular pathway of steroid hormone action (Fig. 1). HSP90 FAMILY The hsp90 family is a group of highly conserved stress proteins found ubiquitously in eukaryotes. Hsp90 is the most abundant constitutive hsp in eukaryotic cells, accounting for 1–2% of cytosolic proteins. Its exact function in the cell has not yet been defined but, only recently, hsp90 has been clearly shown to function as a molecular chaperone that facilitates the folding of various proteins. Whether or not hsp90 has enzyme activity is controversial. In unstressed cells, hsp90 is localized predominantly in the cytoplasm, but is also present in the nucleus (Fig. 2). Under conditions of primary stress or restress, hsp90 moves from the cytoplasm into the nucleus, a redistribution seen in some cell types but not in others. The presence of some hsp90 in nuclei of unstressed cells is important because it has previously been reported that there is no nuclear hsp90 and that steroid receptor and hsp90 cannot exist in a complex with each other in nuclei. It is not known whether hsp90 performs a chaperone function in the nucleus or whether the low levels present reflect hsp90 that has been brought into the nucleus with other molecules, such as steroid receptor. GR are in heterocomplex with hsp90 in the absence of an appropriate steroid agonist. This heterocomplex contains two molecules of hsp90 and, when studies of receptor-hsp90 complexes began, there was concern that they might be formed upon cell homogenization, thus not reflecting a native state of receptors in cells. Both metabolic labelling studies and studies in which steroid receptors were cross-linked to hsp90 in intact cells have demonstrated that receptors are bound to hsp90 in vivo. As ligand-regulated transcription factors, the steroid receptors move through the cytoplasm, traverse the nuclear pores, and subsequently move within the nucleus to their site of action. Their nuclear localization is mediated by nuclear localization signal (NLS) sequences in the receptor themselves and constant bidirectional transfer of receptors into and out of the nucleus. The GR may be cytoplasmic because the NLS in the untransformed GR is not readily accessible to the NLS-binding, and when it does become accessible upon transformation, nuclear translocation occurs. The receptor-hsp90 complexes
Heat Shock Protein Interactions
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GC
GR
hsp90 GR
hsp90
hsp90 GR
transcription
GR
GR
GRE
hsp90
GR
GR Fos
GR jun
jun
transcription
AP-1 site
Fig. 1 Simplified model of GR-mediated transcriptional modulation. Hormone binding causes dissociation of the GR/hsp complex and nuclear translocation (modified from C. M. Bamberger et al, Endocr Rev 1996, 17: 245–261).
Fig. 2 BAL cytospin of an asthmatic patient. Positive cells are stained in red by immunocytochemistry method using a monoclonal antibody to human hsp90 (StressGen). In alveolar macrophages, hsp90 is localized predominantly in the cytoplasm, but is also present in the nucleus.
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are in dynamic state, in that they are constantly dissociating and being reformed in the cell; thus, the proposed ‘piggyback’ movement might involve chaperone-mediated association of the receptor with several hsp90-NSL molecules.47 HSP90-GR INTERACTION Hsp90 interacts with the HBD of the GR but there is no clearly demarcated region within the HBD responsible for this binding. Also, no physical properties of the HBD contribute to an explanation of highaffinity interaction of hsp90 with this domain and not with many other proteins. Although ‘minimal’ hsp90binding segments of the HBD have been defined, it has been suggested that almost the whole HBD is involved in GR association with hsp90.48 After the discovery of receptor binding to hsp90, it was thought that dissociation of hsp90 from the receptor might account for loss of steroid-binding activity as well as acquisition of DNA-binding activity. Consistent with this theory was the discovery that 4S unliganded GR recovered from sucrose gradients had no steroid-binding activity, whereas GR recovered in the 9S peak had normal steroid-binding activity. Also, transformed liganded GR was found to be unable to rebind steroid after ligand dissociation. There was a direct relationship between the amount of GRassociated hsp90 and the glucocorticoid-binding capacity of immunopurified receptors. The GR HBD alone was sufficient to bind steroid only when it was bound to hsp90. There was no binding of steroid by the intact GR in the absence of hsp90.49 Because the 9S receptor–hsp90 heterocomplexes can be physically stabilized by molybdate, the protein composition of heterocomplexes can be readily studied. Receptor transformation measured either by 9S to 4S conversion or by acquisition of DNA-binding activity is both hormone-dependent and temperaturedependent, and inhibited by molybdate. Exposure of intact cells to glucocorticoid causes dissociation of the GR from hsp90 and binding of steroid to cytosolic GR promotes temperature-dependent dissociation of the receptor from hsp90 and generation of the DNAbinding state. Artifactual conditions causing receptor transformation in a steroid-independent manner also dissociate the GR–hsp90 complex to yield the DNAbinding state. Importantly, the rate of hsp90 dissociation from cytosolic GR-hsp90 heterocomplexes was shown to be the same as the rate of receptor acquisition of DNA-binding activity.50 The receptor–hsp90 heterocomplex represents a normal transition state in a general protein-folding process. In the majority of chaperone proteins, this intermediate is transient, but, in the case of steroid receptor, the complex between HBD and hsp90 is
particularly stable. Hsp90 is conceived as trapping the receptor HBD in a partially unfolded conformation, which for the GR is the only state in which there is an accessible steroid-binding site. Binding of steroid in the steroid-binding pocket then favours continued folding of the HBD, destabilizing its interaction with hsp90. Untransformed steroid receptors are unable to dimerize and unable to bind to DNA. Because the major dimerization site is located in a region of the HBD that is part of, or adjacent to, the hsp90-binding region, it is easy to conceive how binding of hsp90 might abrogate receptor dimerization.51 In contrast to molybdate, which permits receptor heterocomplex assembly but impedes GR–hsp90 dissociation, geldanamycin allows (and even may facilitate) heterocomplex dissociation, but blocks reassembly. Recent studies with geldanamycin suggest that association with hsp90 may inhibit receptor degradation. Geldanamycin causes destabilization of the GR. Because geldanamycin accelerates the degradation of these proteins, it has been suggested that hsp90 binding stabilized proteins to degradation in vivo.52 The Raf-1-MEK-MAPK pathway plays an important role in transducing extracellular growth factor signalling into altered nuclear transcription factor function. The benzoquinone ansamycin, geldanamycin, specifically binds to hsp90 and alters its complex with Raf-1. This leads to a decrease in Raf1 levels and disruption of the Raf-1 MEK-MAPK signalling pathway. The enhanced degradation of Raf1 protein is prevented by inhibitors of the proteasome, while inhibition of lysosomal or other proteases is ineffective. Raf-1, protected from geldanamycin-induced degradation, is of higher molecular weight and shows a laddering pattern consistent with its polyubiquitination. Unlike Raf-1 in untreated cells, the protein is insoluble in Triton ×100- or NP40-based buffers. Signalling through this pathway is inhibited by geldanamycin (concomitant with loss of Raf-1 protein) but restored if Raf-1 was protected from geldanamycin-induced degradation by proteasome inhibitors.53 p53 is a tumour suppressor protein causing the nucleus to effect cell cycle arrest and apoptosis. In some cells p53 is located in the cytoplasm and may downregulate its activity. Hsp90 forms a complex with the cytoplasmically localized mutant p53 (TSp53wall35) with transformed cells and binds both a native and a denatured form of p53, as determined by conformation-specific antibodies. Hsp90 does not bind p53 in a spatial-specific manner since it remains bound to p53 when induced to translocate to the nucleus by the protein synthesis-inhibitor cycloheximide. Treatment of transformed cells with geldanamycin causes a rapid destabilization of p53 by
Heat Shock Protein Interactions
50% and residual p53 surviving geldanamycin treatment is incapable of translocating to the nucleus. Geldanamycin does not destabilize p53 in cells where this protein is genotypically wild-type. Although geldanamycin appears to dramatically alter the translocating properties of mutant p53 it does not dissociate the p53-hsp90 complex. It is possible that a second chaperone protein, p23, which also binds p53, may play an important role in these geldanamycin-mediated effects.54 By the late 1980s, it was clear that other proteins were present in receptor heterocomplexes in addition to hsp90. Two of these proteins, hsp70 and hsp23, are required for assembly of stable receptor-hsp90 heterocomplexes. Another essential member of the chaperone system, p60, is only present in receptor heterocomplexes as a transient participant during their assembly and is not recovered in native receptor heterocomplexes immunoadsorbed from the cytosol. Other potential assembly components are p56, p48 and hsp40 and immunophilins. The receptor forms an initial ATP-dependent tight complex with hsp90hsp60-hsp70 (foldosome) which is unstable in the absence of p23 or molybdate. When p60 and variable amounts of the hsp70 leave the intermediate complex, this site is free to associate with the p23.55 In conclusion, the native, hormone-free state of GR is a large multiprotein complex. The untransformed 9S state of the steroid receptor has provided a unique system for studying the function of the ubiquitous, abundant and conserved heat shock protein, hsp90. It is not yet clear how far the discovery of this hsp90 heterocomplex assembly system will be extended to the development of a general understanding of protein processing in the cell but, because this assembly system is apparently present in all eukaryotic cells, it probably performs an obligatory function for many proteins. The bacterial homologue of hsp90 is not an essential protein,56 but hsp90 is essential in eukaryotes, and recent studies indicate that the development of the cell nucleus from prokaryotic progenitors was accompanied by the duplication of genes for hsp90 and hsp70.57 Because chaperone function of hsp90 is nonessential in bacteria, this function has been used to perform additional fundamental duties in eukaryotic cells, such as trafficking of proteins to and from the nucleus. It is likely that, like hsp70, hsp90 binds to unfolded regions of proteins, giving it the power to interact with hundreds or thousands of proteins regardless of structural features or binding motifs. Because the steroid receptors have evolved a persistent interaction with the ‘substrate’ site of hsp90, they have generated interest in a fundamental heterocomplex assembly process normally so dynamic that it might not otherwise have been visualized. Thus, just as the description of steroid receptor genes led to the discovery of a superfamily of transcription factors,
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the ultimate legacy of the study of untransformed steroid receptors may be more fundamental and farreaching than an understanding of how the hormones transform their receptors.
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Date received: 17 March. Date revised: 12 August. Date accepted: 24 August.
PULMONARY PHARMACOLOGY & THERAPEUTICS
Review Heat Shock Protein Interactions with the Glucocorticoid Receptor G. Bertorelli∗, V. Bocchino, D. Olivieri Department of Respiratory Disease, University of Parma, Italy based on their capacity to associate with other proteins in a way which modifies the destiny and function of the latter. Recent interest has focused on the cytoprotective properties of the stress response in diverse forms of cellular and tissue injury. Hsp has also been reported to prevent cell death by apoptosis. Although at first sight this antiapoptotic effect might appear protective, it actually could promote persistence rather than resolution of inflammation, by preventing the physiological removal of inflammatory cells by apoptosis, an essential component of the healing process. Finally, we know that hsp also serve many important physiological functions and have a key physiological role in hormone receptors.
INTRODUCTION
‘. . . In contrast to such “shocks” for which the genome is unprepared, are those a genome must face repeatedly, and for which it is prepared to respond in a programmed manner. Examples are the “heat shock” responses in eukaryotic organisms, and the “SOS” responses in bacteria. Each of these initiates a highly programmed sequence of events within the cell that serves to cushion the effects of the shock. Some sensing mechanism must be present in this instance to alert the cell to imminent danger, and to set in motion the orderly sequence of events that will mitigate this danger. The responses of genomes to anticipated challenges are not so precisely programmed. Nevertheless, these are sensed, and the genome responds in a discernible but initially unforeseen manner.’ Barbara McClintock – Nobel lecture, 8 December 1983
THE GLUCOCORTICOID RECEPTOR These prophetic words by Barbara McClintock eloquently capture the essence of the heat shock response, which was described over 30 years ago when Ferruccio Ritossa,1 noted that a temperature shift from 25 to 30°C induced a new puffing pattern in specific regions of the giant chromosome from the salivary glands of Drosophila. The products of this phenomenon were identified as a specific group of proteins termed heat shock proteins (hsp). Although initially a temperaturedependent pattern of gene expression in Drosophila, this was subsequently found to occur in virtually all organisms from prokaryotes to humans and there is now a greater understanding of the stress response to thermal or non-thermal cytotoxic stimuli. The stress response is a highly conserved cellular defence mechanism defined by the rapid and specific expression of stress proteins, with concomitant transient inhibition of non-stress protein gene expression. It is now clear that the importance of many hsp is
Steroid hormones are essential constituents of the intercellular communication system that maintains homeostasis in higher organisms. Glucocorticoids, a major subclass of steroid hormones, modulate a large number of metabolic, cardiovascular, immune, and behavioral functions.2,3 At the cellular level, most known effects of glucocorticoids are mediated by a >94 kDa intracellular protein, the glucocorticoid receptor (GR). GR belongs to the phylogenetically conserved superfamily of nuclear hormone receptor, which includes receptors for mineralocorticoids, androgens, progestins, estrogens, vitamin D, thyroid hormone, retinoic acid, and a growing number of socalled orphan receptors for which no specific ligand has yet been identified.4,5 All members of the nuclear hormone receptor family share a characteristic three-domain structure: (a) the N-terminal domain contains sequences responsible for activation of target genes and interacts with components of the basal transcription machinery and/or other transcription factors;6–8 (b) two highly conserved ‘zinc fingers’ in the central part of the receptor
∗ Author for correspondence. Present address: Department of Respiratory Diseases, Rasori Hospital, Via Rasori 10, 43100, Parma, Italy. E-mail: [email protected] 1094–5539/98/011007+06 $30.00/0
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1998 Academic Press
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molecule constitute the DNA-binding domain,6,9,10 which also participate in receptor dimerization,11 nuclear translocation,12 and transactivation;7,13 (c) the Cterminal or ligand-binding domain, which binds the hormonal ligand,6,14 contains sequences important for hsp binding,15–17 nuclear translocation,12 dimerization,18 and transactivation,19–22 as well as silencing the receptor in the absence of hormone.7,23 The GR is transformed from a silent to an active transcription factor in response to glucocorticoids.24–27 The non-ligand GR is part of a multiprotein complex that consists of the receptor, two molecules of hsp90, and one molecule each of hsp70, and hsp56, an immunophilin of the FK506- and rapamycin-binding class.16,17,28–31 In addition, other less well characterized proteins have occasionally been found to participate in this complex.28 In the absence of hormone, this complex most likely undergoes constant cycles of dissociation and ATP- and hsp70-dependent reassociation.30,32 Intracellular localization of GR/hsp70 complex also seems to be less static than previously thought. Experimental evidence supports a model of constant bidirectional movement of the complex between the cytosol and the nucleus.17,33 The exact composition of this complex may determine the predominant direction of this movement; for example, the presence of hsp56 may direct the complex to the cell nucleus.17,34,35 However, it has not been shown conclusively whether any component of the hsp complex remains bound to the receptor during nucleocytoplasmic transfer. Quantitative differences between cell types with regard to GR transfer may account for the apparently contradictory reports as to whether the GR is a cytoplasmic or a nuclear protein.36–41 Independent of its intracellular localization, the main function of the GR/hsp complex is to keep the receptor protein in an inactive, yet potential ligand-active state.16,17,28–31,42,43 As lipophilic substances, glucocorticoids are able to cross the cell membrane readily to interact with the intracellular GR. Ligand binding induces an as yet undetermined conformational change in the GR molecule with a number of functional consequences: the hormone-bound GR dissociates from the hsp complex and is no longer able to reassociate with it.16,24,25,32 Furthermore, the partially phosphorylated receptor protein becomes hyperphosphorylated.32,44,45 Finally, hormone binding may cause nuclear translocation of cytoplasmic GR molecules.39 GR are recovered from cells in large (9S) heterocomplexes that contain both hsp and immunophilins. Some components of the receptor heterocomplexes are proteins with established chaperone functions. One critical function of the hsp is to facilitate the folding of the hormone-binding domain (HBD) of the receptor into a high-affinity steroidbinding conformation. These hsp appear to act as
protein-folding machines that assemble heterocomplexes between the hsp and the receptors.46 Hsp90 interacts directly with the HBD of the nuclear receptors, an association which appears to account for a repression of receptor function relieved upon subsequent binding of hormone. This ability of the hormone to control HBD-chaperone interaction is now regarded as the earliest event in the molecular pathway of steroid hormone action (Fig. 1). HSP90 FAMILY The hsp90 family is a group of highly conserved stress proteins found ubiquitously in eukaryotes. Hsp90 is the most abundant constitutive hsp in eukaryotic cells, accounting for 1–2% of cytosolic proteins. Its exact function in the cell has not yet been defined but, only recently, hsp90 has been clearly shown to function as a molecular chaperone that facilitates the folding of various proteins. Whether or not hsp90 has enzyme activity is controversial. In unstressed cells, hsp90 is localized predominantly in the cytoplasm, but is also present in the nucleus (Fig. 2). Under conditions of primary stress or restress, hsp90 moves from the cytoplasm into the nucleus, a redistribution seen in some cell types but not in others. The presence of some hsp90 in nuclei of unstressed cells is important because it has previously been reported that there is no nuclear hsp90 and that steroid receptor and hsp90 cannot exist in a complex with each other in nuclei. It is not known whether hsp90 performs a chaperone function in the nucleus or whether the low levels present reflect hsp90 that has been brought into the nucleus with other molecules, such as steroid receptor. GR are in heterocomplex with hsp90 in the absence of an appropriate steroid agonist. This heterocomplex contains two molecules of hsp90 and, when studies of receptor-hsp90 complexes began, there was concern that they might be formed upon cell homogenization, thus not reflecting a native state of receptors in cells. Both metabolic labelling studies and studies in which steroid receptors were cross-linked to hsp90 in intact cells have demonstrated that receptors are bound to hsp90 in vivo. As ligand-regulated transcription factors, the steroid receptors move through the cytoplasm, traverse the nuclear pores, and subsequently move within the nucleus to their site of action. Their nuclear localization is mediated by nuclear localization signal (NLS) sequences in the receptor themselves and constant bidirectional transfer of receptors into and out of the nucleus. The GR may be cytoplasmic because the NLS in the untransformed GR is not readily accessible to the NLS-binding, and when it does become accessible upon transformation, nuclear translocation occurs. The receptor-hsp90 complexes
Heat Shock Protein Interactions
9
GC
GR
hsp90 GR
hsp90
hsp90 GR
transcription
GR
GR
GRE
hsp90
GR
GR Fos
GR jun
jun
transcription
AP-1 site
Fig. 1 Simplified model of GR-mediated transcriptional modulation. Hormone binding causes dissociation of the GR/hsp complex and nuclear translocation (modified from C. M. Bamberger et al, Endocr Rev 1996, 17: 245–261).
Fig. 2 BAL cytospin of an asthmatic patient. Positive cells are stained in red by immunocytochemistry method using a monoclonal antibody to human hsp90 (StressGen). In alveolar macrophages, hsp90 is localized predominantly in the cytoplasm, but is also present in the nucleus.
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are in dynamic state, in that they are constantly dissociating and being reformed in the cell; thus, the proposed ‘piggyback’ movement might involve chaperone-mediated association of the receptor with several hsp90-NSL molecules.47 HSP90-GR INTERACTION Hsp90 interacts with the HBD of the GR but there is no clearly demarcated region within the HBD responsible for this binding. Also, no physical properties of the HBD contribute to an explanation of highaffinity interaction of hsp90 with this domain and not with many other proteins. Although ‘minimal’ hsp90binding segments of the HBD have been defined, it has been suggested that almost the whole HBD is involved in GR association with hsp90.48 After the discovery of receptor binding to hsp90, it was thought that dissociation of hsp90 from the receptor might account for loss of steroid-binding activity as well as acquisition of DNA-binding activity. Consistent with this theory was the discovery that 4S unliganded GR recovered from sucrose gradients had no steroid-binding activity, whereas GR recovered in the 9S peak had normal steroid-binding activity. Also, transformed liganded GR was found to be unable to rebind steroid after ligand dissociation. There was a direct relationship between the amount of GRassociated hsp90 and the glucocorticoid-binding capacity of immunopurified receptors. The GR HBD alone was sufficient to bind steroid only when it was bound to hsp90. There was no binding of steroid by the intact GR in the absence of hsp90.49 Because the 9S receptor–hsp90 heterocomplexes can be physically stabilized by molybdate, the protein composition of heterocomplexes can be readily studied. Receptor transformation measured either by 9S to 4S conversion or by acquisition of DNA-binding activity is both hormone-dependent and temperaturedependent, and inhibited by molybdate. Exposure of intact cells to glucocorticoid causes dissociation of the GR from hsp90 and binding of steroid to cytosolic GR promotes temperature-dependent dissociation of the receptor from hsp90 and generation of the DNAbinding state. Artifactual conditions causing receptor transformation in a steroid-independent manner also dissociate the GR–hsp90 complex to yield the DNAbinding state. Importantly, the rate of hsp90 dissociation from cytosolic GR-hsp90 heterocomplexes was shown to be the same as the rate of receptor acquisition of DNA-binding activity.50 The receptor–hsp90 heterocomplex represents a normal transition state in a general protein-folding process. In the majority of chaperone proteins, this intermediate is transient, but, in the case of steroid receptor, the complex between HBD and hsp90 is
particularly stable. Hsp90 is conceived as trapping the receptor HBD in a partially unfolded conformation, which for the GR is the only state in which there is an accessible steroid-binding site. Binding of steroid in the steroid-binding pocket then favours continued folding of the HBD, destabilizing its interaction with hsp90. Untransformed steroid receptors are unable to dimerize and unable to bind to DNA. Because the major dimerization site is located in a region of the HBD that is part of, or adjacent to, the hsp90-binding region, it is easy to conceive how binding of hsp90 might abrogate receptor dimerization.51 In contrast to molybdate, which permits receptor heterocomplex assembly but impedes GR–hsp90 dissociation, geldanamycin allows (and even may facilitate) heterocomplex dissociation, but blocks reassembly. Recent studies with geldanamycin suggest that association with hsp90 may inhibit receptor degradation. Geldanamycin causes destabilization of the GR. Because geldanamycin accelerates the degradation of these proteins, it has been suggested that hsp90 binding stabilized proteins to degradation in vivo.52 The Raf-1-MEK-MAPK pathway plays an important role in transducing extracellular growth factor signalling into altered nuclear transcription factor function. The benzoquinone ansamycin, geldanamycin, specifically binds to hsp90 and alters its complex with Raf-1. This leads to a decrease in Raf1 levels and disruption of the Raf-1 MEK-MAPK signalling pathway. The enhanced degradation of Raf1 protein is prevented by inhibitors of the proteasome, while inhibition of lysosomal or other proteases is ineffective. Raf-1, protected from geldanamycin-induced degradation, is of higher molecular weight and shows a laddering pattern consistent with its polyubiquitination. Unlike Raf-1 in untreated cells, the protein is insoluble in Triton ×100- or NP40-based buffers. Signalling through this pathway is inhibited by geldanamycin (concomitant with loss of Raf-1 protein) but restored if Raf-1 was protected from geldanamycin-induced degradation by proteasome inhibitors.53 p53 is a tumour suppressor protein causing the nucleus to effect cell cycle arrest and apoptosis. In some cells p53 is located in the cytoplasm and may downregulate its activity. Hsp90 forms a complex with the cytoplasmically localized mutant p53 (TSp53wall35) with transformed cells and binds both a native and a denatured form of p53, as determined by conformation-specific antibodies. Hsp90 does not bind p53 in a spatial-specific manner since it remains bound to p53 when induced to translocate to the nucleus by the protein synthesis-inhibitor cycloheximide. Treatment of transformed cells with geldanamycin causes a rapid destabilization of p53 by
Heat Shock Protein Interactions
50% and residual p53 surviving geldanamycin treatment is incapable of translocating to the nucleus. Geldanamycin does not destabilize p53 in cells where this protein is genotypically wild-type. Although geldanamycin appears to dramatically alter the translocating properties of mutant p53 it does not dissociate the p53-hsp90 complex. It is possible that a second chaperone protein, p23, which also binds p53, may play an important role in these geldanamycin-mediated effects.54 By the late 1980s, it was clear that other proteins were present in receptor heterocomplexes in addition to hsp90. Two of these proteins, hsp70 and hsp23, are required for assembly of stable receptor-hsp90 heterocomplexes. Another essential member of the chaperone system, p60, is only present in receptor heterocomplexes as a transient participant during their assembly and is not recovered in native receptor heterocomplexes immunoadsorbed from the cytosol. Other potential assembly components are p56, p48 and hsp40 and immunophilins. The receptor forms an initial ATP-dependent tight complex with hsp90hsp60-hsp70 (foldosome) which is unstable in the absence of p23 or molybdate. When p60 and variable amounts of the hsp70 leave the intermediate complex, this site is free to associate with the p23.55 In conclusion, the native, hormone-free state of GR is a large multiprotein complex. The untransformed 9S state of the steroid receptor has provided a unique system for studying the function of the ubiquitous, abundant and conserved heat shock protein, hsp90. It is not yet clear how far the discovery of this hsp90 heterocomplex assembly system will be extended to the development of a general understanding of protein processing in the cell but, because this assembly system is apparently present in all eukaryotic cells, it probably performs an obligatory function for many proteins. The bacterial homologue of hsp90 is not an essential protein,56 but hsp90 is essential in eukaryotes, and recent studies indicate that the development of the cell nucleus from prokaryotic progenitors was accompanied by the duplication of genes for hsp90 and hsp70.57 Because chaperone function of hsp90 is nonessential in bacteria, this function has been used to perform additional fundamental duties in eukaryotic cells, such as trafficking of proteins to and from the nucleus. It is likely that, like hsp70, hsp90 binds to unfolded regions of proteins, giving it the power to interact with hundreds or thousands of proteins regardless of structural features or binding motifs. Because the steroid receptors have evolved a persistent interaction with the ‘substrate’ site of hsp90, they have generated interest in a fundamental heterocomplex assembly process normally so dynamic that it might not otherwise have been visualized. Thus, just as the description of steroid receptor genes led to the discovery of a superfamily of transcription factors,
11
the ultimate legacy of the study of untransformed steroid receptors may be more fundamental and farreaching than an understanding of how the hormones transform their receptors.
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Date received: 17 March. Date revised: 12 August. Date accepted: 24 August.