NUCLEOCYTOPLASMIC SHUTTLING OF STEROID RECEPTORS

Pioneering work by the Jensen and Gorski laboratories in the late 1960s demonstrated that ligand-bound steroid receptors were tightly associated with the nucleus^3,4. These results were instrumental in focusing attention toward the gene-regulatory function for the steroid receptors. In considering mechanisms responsible for the accumulation of ligand-bound steroid receptors within nuclei, many groups set out to establish the subcellular localization of unliganded steroid receptors. Thus, a number of questions were asked regarding the mechanism of steroid receptor trafficking within cells. In which subcellular compartment do steroid receptors first encounter ligand? Is ligand binding required for the accumulation of steroid receptors within the nucleus?

With the availability of specific antireceptor antibodies, it became possible to assess steroid receptor compartmentalization using conventional cell biologic approaches. Rather than provide definitive conclusions regarding steroid receptor subcellular localization, initial results from these studies implicated the possible existence of distinct subcellular trafficking pathways for different receptors. For example, unoccupied progesterone receptors (PRs) and estrogen receptors (ERs) appeared to be localized predominately within nuclei^5,6, while unliganded GRs^7,8,9, mineralocorticoid receptors (MRs)¹⁰, and androgen receptors (ARs)^11,12 appeared to localize predominantly within the cytoplasm. Since legitimate concerns were raised about the effects of different fixation conditions on receptor compartmentalization and the specificity of the available antibodies¹³, a consensus did not emerge concerning the localization of unliganded steroid receptors until very recently. More recent experiments using steroid receptor-green fluorescent protein (GFP) conjugates in live cells have provided definitive proof of the hormone-dependent cytoplasmic to nuclear transport of GR¹⁴ and MR¹⁵.

Prior to the development of tools to visualize protein trafficking in live cells, the dynamic nature of steroid receptor trafficking between different subcellular compartments was recognized using elegant cell fusion experiments. Thus, as first demonstrated by Guiochon-Mantel et al for rabbit PR and human ER¹⁶, and later by other investigators for chicken PR¹⁷, rat GR¹⁸, and mouse ER¹⁹, steroid receptors have the capacity to shuttle between the nuclear and cytoplasmic compartments. Thus, steroid receptors that accumulate within either the cytoplasm or nucleus are not confined to those compartments, but establish an equilibrium distribution based on the relationship between their relative rates of nuclear import versus nuclear export. Receptors will localize within the cytoplasm if their rate of nuclear import is limiting, while a limitation in the rate of receptor nuclear export would lead to their preferential accumulation within nuclei²⁰. These rates are likely to be limited not by differences in inherent rate of passage through the nuclear pore complex (NPC), but rather by the rate of receptor release from compartment-specific anchoring complexes²⁰.

ROLE OF HEAT SHOCK PROTEIN 90 IN CYTOPLASMIC TO NUCLEAR TRANSPORT OF STEROID RECEPTORS

Unliganded, cytoplasmic GR that is competent to bind hormone exists as a heteromeric complex that contains a dimer of the 90 kD heat shock protein, hsp90, an immunophilin protein of the FK506-binding family (that is, FKBP-52 or FKBP-54), and a 23 kD protein, p23²¹. Other immunophilins (for example, Cyp40) or heat shock proteins (for example, hsp70) that are found associated with unliganded steroid receptors are likely to be involved in the maturation of the receptor to its hormone-binding conformation²². Unliganded, cytoplasmic MR has also been found to be complexed with hsp90, FKBP52, and hsp70, although the stoichiometry of these complexes may be different for GR and MR²³.

While the constitutive nuclear localization of ligand-binding domain (LBD)-deleted GRs suggested that nuclear import of the receptor is restricted by their association within heteromeric complexes^9,24, this view is now recognized as being overly simplistic. For example, some unliganded steroid receptors that appear to be localize predominantly within the nucleus are also assembled into heteromeric complexes²⁵. This includes unliganded GR, which in some cells appears to accumulate within the nucleus²⁶.

How do we reconcile these results with the presumed role for steroid receptor heteromeric complexes in limiting nuclear import? An important point to consider is that the assembly of steroid receptor heteromeric complexes is a dynamic process. This was first shown for PR in vitro²⁷, in which the association with chaperones such as hsp90 is transient even in the absence of hormone binding. Thus, individual components of steroid receptor heteromeric complexes are likely to be constantly turning over. As a result, the amino acid signals encoded within steroid receptors that are required for their nuclear import^{9,12,28,29,30,31} may be transiently exposed to appropriate cytoplasmic transport proteins³², even in the absence of bound ligand. Thereafter, a productive interaction might ensue that would commit steroid receptor-nuclear transport protein complexes to associate with the NPC proteins. It follows that the stability of steroid receptor-heteromeric complexes, which probably varies for individual receptors and perhaps within different cell types, could have a direct impact on the cytoplasmic retention of unliganded receptors.

We have confirmed that the hormone dependence of GR import reflects a requirement for receptor activation or more precisely the dissociation of hsp90 from a GR-heteromeric complex³³. This was shown using a novel delivery system to enable sufficient accumulation of sodium molybdate in live cells to stabilize GR-hsp90 complexes. Stabilization of GR-hsp90 complexes led to a dramatic reduction in hormone-dependent nuclear import of the receptor in vivo³³. Since the composition of GR heteromeric complexes in sodium molybdate-treated cells was not examined, it is unclear whether this treatment led to the generation of novel GR/hsp90-containing complexes or captured a particular intermediate complex³³. Nonetheless, these results implied that the efficiency of nuclear import is governed by the relative stability of receptor heteromeric complexes. This hypothesis was also supported by the results of PR nuclear import in sodium molybdate-treated cells. In this case, both hormone-independent and -dependent nuclear import of PR was inhibited by sodium molybdate treatment³³. One interpretation of these results is that stabilization of PR heteromeric complexes also has a detrimental affect on the ability of this receptor to import into nuclei. Thus, the differential localization of unliganded GR (that is, cytoplasmic) versus PR (that is, nuclear) observed in most cell types may simply result from differences in the inherent stabilities of receptor heteromeric complexes.

An opposing view of the fate of hsp90 during steroid receptor has been proposed in which PR and GR are hypothesized to remain associated with hsp90 during their nuclear import³⁴. This conclusion was based on studies with hsp90 chimeras that possessed a linked, heterologous nuclear localization signal sequence (NLS). Coexpression of an NLS-hsp90 conjugate was required for the nuclear accumulation of PR and GR derivatives that lacked their own NLS³⁴. While these results provide a convincing demonstration of the association between hsp90 and GR or PR in vivo, they do not address whether the cotransport of hsp90 with steroid receptors occurs for native steroid receptors. Once associated with nuclear transport proteins, an hsp90/steroid receptor complex may be artificially stabilized and remain together during transit through the NPC. Such ternary complexes may not exist for native steroid receptors, which may be precluded from interacting with nuclear transport factors when stably associated with heat shock proteins.

ROLE OF HEAT SHOCK PROTEIN 70 IN CYTOPLASMIC TO NUCLEAR TRANSPORT OF STEROID RECEPTORS

Cytoplasmic hsp70 is used in many protein-folding reactions and in this capacity plays an important role in organelle trafficking, where importing substrates are reversibly unfolded to allow passage through organelle membrane-anchored channels³⁵. It therefore seemed unnecessary to invoke a role for hsp70 in nuclear protein import or export, where protein unfolding is not considered to be associated with transit through the NPC³⁶. Nonetheless, independent studies using both in vitro and in vivo assays of nuclear import appeared to implicate a role for hsp70 in nuclear import^37,38. Genetic evidence in yeast^39,40, as well as biochemical studies showing the direct binding of hsp70 to nuclear import signal sequences (NLSs)³⁷, also support a role for hsp70 in nuclear transport. While hsp70 may play a general role in presenting transporting substrates to the nuclear pore machinery, it performs this function in the absence of global unfolding of these substrates³⁹.

While various studies implicate a general requirement for hsp70 in nuclear protein import, this does not seem to be a universal property of this trafficking pathway. For example, we have found that GRs do not require hsp70 for efficient in vitro hormone-dependent nuclear import⁴¹. The fact that intracellular protein trafficking can display differential requirements for a chaperone such as hsp70 is not unique to nuclear import. In yeast, the mitochondrial hsp70 protein (mt-hsp70), a product of the Ssc1 gene, is required for in vitro mitochondrial import of some, but not all, fusion proteins tested⁴². Specifically, mitochondrial import of a chimeric protein containing a 167 amino acid segment of the yeast cytochrome b₂ protein fused to dihydrofolate reductase did not require functional mt-hsp70⁴². These results highlight how the precise context of an organelle import signal sequence can determine whether hsp70 is required to facilitate transport. Hsp70 functions during the maturation of naive GR to a hormone-binding conformation. Thus, during the hsp70-mediated maturation of GR, its NLS may be folded in the precise conformation required for subsequent interactions with the nuclear import machinery. GR, and perhaps other steroid receptors, may not be distinguished from other proteins that require hsp70 for import into nuclei. All proteins that are destined to enter the nucleus may require hsp70 for appropriate presentation of their NLS, but could differ in the precise timing during their maturation where this process occurs.

ROLE OF hsp70 AND ITS DnaJ PARTNER IN SUBNUCLEAR TRAFFICKING OF STEROID RECEPTORS

Following the appropriate interactions of NLS-protein/NLS-receptor complexes with specific NPC proteins (that is, nucleoporins)^43,44, NLS proteins must engage components of the NPC, which comprise the translocation machinery to complete the nuclear import process. The soluble GTP-binding protein Ran/TC4^45,46 is used in this process to aid in the delivery and/or release of NLS proteins to various nucleoporins that are encountered during passage through the interior 50 nm of the NPC⁴⁴. Once NLS proteins are released from the NPC, they are presumably free to proceed to their ultimate destination within distinct subnuclear compartments.

A number of specific signal sequences have been identified that target proteins to specific subnuclear compartments^47,48,49,50. In addition, mechanisms responsible for regulating alternative subnuclear compartmentalization are emerging that often use distinct post-translational modifications. For example, the cell cycle-dependent binding of the hypophosphorylated retinoblastoma (Rb) tumor suppressor protein to the nuclear matrix is brought about through the action of specific protein phosphatases⁵¹. Phosphorylation also affects the subnuclear compartmentalization of the hepatocyte nuclear factor 4⁵². In addition to phosphorylation, post-translational modification by the ubiquitin-like SUMO-1 protein is also involved in the regulation of protein compartmentalization within the nucleus⁵³. Steroid receptors are also phosphoproteins in which the phosphorylation state is altered following hormone binding⁵⁴. Steroid receptor phosphorylation does not appear to impact any aspect of its subcellular trafficking (DeFranco, unpublished observations), although definitive experiments to rule out completely this level of control have not been performed.

How is steroid receptor trafficking within the nucleus regulated? Following its hormone-dependent translocation to the nucleus, GR targets to distinct subnuclear compartments, which can be recognized both in fixed and live cells^14,41,55. However, the signals required to direct receptors to preferred sites of accumulation within the nucleus have not been defined. Nonetheless, we have found that the NLS of the rat GR, in addition to its functioning as a NPC targeting signal, has an impact on receptor targeting within the nucleus³¹. This presumed dual role of the GR NLS in nuclear import and compartmentalization is distinguished by point mutations at R496, which although transparent for nuclear import activity of the NLS, exerts dramatic effects on subnuclear targeting of the receptor³¹. Carboxyl-terminal truncated GRs with mutations at R496 accumulate within a few large nuclear foci³¹, representing a departure from the characteristic nonrandom, mottled nuclear staining pattern of wild-type GR^55,56. The fact that substitution of R496 with either another basic amino acid (lysine), or an acidic (aspartate), polar (serine), or nonpolar (isoleucine) amino acid generated the identical mistargeting of the receptor³¹ argues against the fortuitous formation of a novel subnuclear targeting signal. In addition, many other mutations within and surrounding the various components of the NLS did not lead to the accumulation of mutant receptors within large nuclear foci³¹. Thus, the presence of an arginine residue immediately following the final zinc-coordinating cysteine of the GR DNA-binding domain (DBD) appears essential for appropriate subnuclear targeting of receptors.

The effects of rat GR R496 mutations on subnuclear targeting were not autonomous, as hsp70 also accumulated within R496 mutant foci³¹. In this case, there appeared to be a redistribution of hsp70, which normally localized throughout the cell^57,58 to these nuclear foci³¹. Hsp70 has been found to colocalize within nuclear granules, which form as a result of overexpression of the E1A or myc proteins^59,60,61. While a stable hsp70/E1A complex could be immunopurified following mild extraction of nuclei⁵⁹, nuclear foci that possess GR R496S and hsp70 resist even the harshest extraction methods and biochemically partition to an insoluble nuclear compartment³¹. I hypothesize that hsp70 may play a general role in sequestering or shielding "sticky" protein surfaces within the nucleus whose exposure may increase upon overexpression. The ability of hsp70 to shuttle between the nuclear and cytoplasmic compartments provides the means for this chaperone to survey continuously the cytoplasm and nucleus for misfolded proteins.

How do rat GR/R496S foci form? Since R496 makes direct contact with the phosphate backbone at both specific and nonspecific DNA sites⁶², the lack of such a stabilizing interaction might unleash a subdomain of the GR DBD that could promote the formation of large nuclear foci. It is noteworthy that R496 is the only amino acid within an helical subdomain of the second zinc finger that makes DNA contact⁶². The loss of a single phosphate contact does not appear to be solely responsible for mistargeting of carboxyl-terminal truncated GRs, as mutations at other amino acids that make phosphate contacts (that is, R489 and K490) do not lead to receptor mistargeting³¹. R496 is a highly conserved residue in the steroid/thyroid hormone superfamily of nuclear receptors, as every member of the superfamily identified to date possesses an arginine at that corresponding position^63,64. Since this amino acid serves an identical function in making phosphate backbone contacts in the crystal structures of rat GR⁶² and other members of the nuclear receptor family^65,66, it will be interesting to examine whether the mutation of this residue within other nuclear receptors also leads to an alteration in subnuclear targeting.

An important clue relating to the mechanism of GR/R496S mistargeting was provided by the results of cotransfection experiments with an hsp70 partner derived from human cells, HDJ-2^67,68. GR/R496S mistargeting in transiently transfected cells was alleviated upon overexpression of HDJ-2³¹. Cotransfection with an HDJ-2 mutant that lacks its J domain did not relieve GR/R496S mistargeting³¹. Deletion of the J domain from the E. coli DnaJ protein eliminated its ability to mediate protein refolding in vitro, in combination with DnaK and GrpE⁶⁹. Thus, HDJ-2 may act in an analogous manner, perhaps in combination with hsp70, to refold misfolded or aggregated GR/R496S within the nucleus and restore its appropriate subnuclear targeting. The mutant HDJ-21 possesses an intact cysteine-rich domain^67,68, which analogous to its demonstrated role in binding unfolded proteins in vitro⁶⁹, may direct HDJ-2 to GR/R496S nuclear foci³¹. This could explain the colocalization of HDJ-21 with GR/R496S foci in transiently transfected cells³¹. The apparent stability of GR/R496S nuclear foci in the absence of cotransfected HDJ-2 suggests that the capacity of the normal cellular complement of DnaJ homologues to alleviate GR/R496S mistargeting must be exceeded under these conditions. I postulate that only by supplementing nuclear DnaJ levels upon the introduction of exogenous HDJ-2 is an hsp70/DnaJ chaperone system sufficiently activated to either prevent or correct GR/R496S mistargeting.

ROLE OF hsp90 IN SUBNUCLEAR TRAFFICKING OF STEROID RECEPTORS

Hormone-bound GRs that enter the nucleus rapidly locate high-affinity target sites within native chromatin. These interactions are essential for the subsequent alterations in transcriptional activity of target genes⁷⁰. Although the number of steroid receptor target genes in any given cell is limited, the majority of hormone-bound nuclear receptors are associated with high-affinity chromatin binding sites⁷¹. Thus, these interactions are unlikely to be governed strictly by target site binding and include receptor interactions with unique chromatin proteins. While there were reports of steroid receptor binding to histones⁷², more recent studies have identified unique chromatin proteins that interact with steroid receptors^73,74, which may be more relevant to receptor function.

Both bulk GRs⁷¹ and receptors associated with specific target sites are released from high-affinity chromatin binding sites upon hormone withdrawal^75,76. While the kinetics of GR chromatin release appear to correlate with the kinetics of hormone dissociation^71,77, these unliganded receptors do not rapidly export the nucleus and appear to remain within some novel subnuclear compartment while awaiting their next encounter⁷¹. Thus, nuclear export of GRs is not restricted solely by their interactions with chromatin. The alternative processing fates for unliganded nuclear receptors include degradation⁷⁸ nuclear export⁷¹ and/or recycling Figure 1.

Figure 1.

Subnuclear trafficking of glucocorticoid receptor (GR). GR that is bound to hormone (H) associates with high-affinity binding sites on chromatin or with the nuclear matrix. Chromatin and the nuclear matrix are depicted as separate and distinct compartments for simplicity alone and may, in fact, be physically linked. Although it is unclear whether hormone must dissociate from receptors for their release from the nuclear matrix, adenosine 5'-triphosphate (ATP) is required for receptor release from, but not binding to, the nuclear matrix. The chaperone activity of heat shock protein 90 (hsp90) may facilitate the release of unliganded receptor from chromatin. At least three alternative processing fates are available for unliganded GR: degradation, nuclear export, and recycling. It appears that unliganded GRs are fully competent to rebind hormone and therefore likely to be assembled into a heteromeric complex.

Full figure and legend (33K)

Steroid receptor proteins can be reused and regain their competence to respond to hormone when recycled from the nuclear to cytoplasmic compartment^79,80. Are recycled receptors required to re-enter the cytoplasm in order to regain their hormone-binding competence? Orti et al hypothesized that a nuclear bypass pathway exists that permits the reutilization of nuclear GRs without an obligatory passage through the cytoplasm⁷⁹. We have recently provided direct experimental evidence for recycling of nuclear GRs in experiments using digitonin-permeabilized cells⁷¹. Nonetheless, these results raised a number of issues regarding the mechanism of GR recycling within the nucleus. It is well established that hormone-binding competent cytoplasmic GRs exist as heteromeric complexes that include molecular chaperones, such as hsp90, immunophilins, and various chaperone partners^22,25. How do unliganded nuclear receptors regain the capacity to bind hormone? What are the requirements for the reutilization of nuclear GRs?

Through our continued exploitation of the digitonin-permeabilized cell system, where receptor exchange between the nuclear and cytoplasmic compartments is minimized, we have recently uncovered some novel mechanistic features of GR nuclear recycling⁸¹. The first question we addressed concerned the rebinding of hormone to unliganded nuclear receptors. It has been well established that cytoplasmic GRs do not require energy nor ambient temperature to associate with hormone²². Likewise, the binding of hormone to unliganded nuclear GRs in permeabilized cells is temperature independent and does not require adenosine 5'-triphosphate (ATP)⁸¹. Thus, unliganded nuclear receptors, once released from chromatin, are fully primed to respond to a hormonal signal and do not require active cellular processes to do so. Since cytoplasmic GRs gain hormone-binding competence once assembled into a heteromeric complex²², our results imply that nuclear receptors may likewise be reassembled into a heteromeric complex.

In contrast to the energy-independent hormone rebinding to nuclear receptors, ATP is required in order for these recharged nuclear receptors to rebind with high affinity to chromatin in semi-intact cells⁸¹. The in vitro binding of GR to reconstituted nucleosomes does not require ATP^82,83. Thus, ATP may be used in intact nuclei not necessarily to facilitate appropriate receptor targeting to chromatin binding sites, but to prevent inappropriate targeting of receptors to alternative subnuclear compartments (that is, the nuclear matrix)⁸⁴. ATP hydrolysis appears to be essential in the permeabilized cell system to restrict GR interactions with the nuclear matrix⁷¹. Furthermore, GTP can not substitute for ATP in this process⁷¹, suggesting that Ran^45,46 and perhaps other small GTP-binding proteins are not involved in directing GR to appropriate chromatin binding sites.

What is the composition of nuclear GR heteromeric complexes? Is the same mechanism used to mature recycled nuclear receptors as naive cytoplasmic receptors? Are the same molecular chaperones (for example, hsp90) used in this process? We have used a pharmacological approach to examine the role of hsp90 in the maturation of unliganded nuclear GRs. Geldanamyin (GA), a benzoquinone ansamycin, has been used to study hsp90-mediated reactions given its selective binding to hsp90 and the resulting disruption of hsp90 chaperone function⁸⁵. For example, GA treatment of intact cells leads to a rapid loss of GR hormone binding and accelerates GR degradation⁸⁶. In digitonin-permeabilized cells, GA also leads to the inhibition of hormone binding to recycled nuclear GRs⁸¹. These results provide strong support for the notion that hsp90 is required for nuclear receptors to gain hormone-binding competence. It will be a challenge of future studies to establish whether the same steroid receptor maturation pathway so elegantly elaborated for cytoplasmic PR and GR in vitro is utilized to mature recycled nuclear receptors.

In addition to implicating hsp90 in hormone binding of recycled nuclear GRs, we have also uncovered a potential role for this chaperone in subnuclear trafficking of nuclear receptors. Hormone withdrawal leads to rapid chromatin release of both bulk GRs⁷¹ and receptors associated with high-affinity target sites⁷⁶. However, despite the fact that chromatin-associated GRs release their bound hormone in the presence of GA, their subsequent release from chromatin was dramatically inhibited⁸⁷. Importantly, nuclear receptors in GA-treated, hormone-withdrawn cells were not associated with the nuclear matrix but loosely associated with nuclei⁸¹. Based on these results, we postulated that the chaperoning activity of hsp90 is used to facilitate GR release from high-affinity chromatin-binding sites upon hormone dissociation Figure 1. Since our studies examined the chromatin-binding properties of bulk receptors, it is unknown whether this hsp90 dependence applies to GR associated with chromatin of specific target genes. However, the techniques used to examine GR association with target gene chromatin⁷⁶ can be applied in permeabilized cells to address this issue.

Additional studies of GA effects illustrate the dynamic nature of unliganded and liganded GR interactions with chromatin. For example, GRs remained chromatin associated, if GA was added to cells following a 30-minute hormone withdrawal (DeFranco, unpublished observations). This observation suggests that unliganded nuclear receptors may also have some limited capacity to interact with chromatin. I hypothesize that as long as hsp90 function is not compromised, the release of unliganded receptors from high-affinity chromatin-binding sites is more favored than their association with these sites. This could explain why unliganded nuclear receptors, while not detected on chromatin, can be driven to high-affinity chromatin binding sites by GA treatment following hormone withdrawal. Since there was a minimal loss of chromatin-bound receptors when cells were incubated with GA in the continuous presence of hormone (DeFranco, unpublished observations), some fraction of hormone-bound receptors may release from high-affinity chromatin binding sites. Once hormone dissociates from these chromatin-released liganded receptors, their hormone and chromatin rebinding would be inhibited by GA.

Despite the fact that hsp90 has been considered to be an abundant (that is, 1 to 2%) cytoplasmic protein, a small but significant fraction (approximately 3%) of hsp90 is found within nuclei⁸⁸. Thus, there appears to be sufficient nuclear pools of hsp90 to perform biologically relevant functions. In vitro studies have revealed a role for hsp90 in DNA binding of helix-loop-helix transcription factors^89,90. Interestingly, hsp90 has been found to facilitate the release of GR⁹¹ and ER⁹² from DNA in vitro. In these studies, the ATP dependence of hsp90 effects on receptor release from DNA was not strictly addressed. GA inhibits ATP binding to hsp90 through its occupancy of the ATP-binding site of hsp90⁸⁵. Given the GA effects on GR chromatin binding that we observed in digitonin-permeabilized cells⁸¹, it will be relevant to consider the ATP requirement for hsp90 effects on steroid receptor release from chromatin and DNA.

What property of steroid receptors could account for the putative hsp90 requirements for chromatin release? Upon hormone binding, steroid receptor LBDs undergo a conformational change⁹³ that is characterized by the movement of a particular exposed helix (that is, helix 12) toward the hormone-binding pocket^87,94. As a result, hydrophobic segments of the LBD, as well as the bound hormone itself, are no longer solvent exposed. Furthermore, previously inaccessible LBD surfaces become exposed and available for interactions with appropriate coactivators and other components of the transcriptional machinery. It is unclear how the elaborately networked LBD structure responds to the release of bound hormone. Does the LBD simply "relax" to reaquire the conformation it possessed when initially unliganded? In such a scenario, the exposure of hydrophobic segments of the unliganded receptor's hormone-binding pocket might increase the propensity for receptor aggregation unless molecular chaperones such as hsp90 are present to prevent such inappropriate interactions. Unliganded nuclear receptors in GA-treated, hormone-withdrawn cells remain salt extractable and thus are unlikely to be significantly aggregated.

It is clear from the various studies described in this review that steroid receptor encounters with molecular chaperones are not restricted solely to the cytoplasm. As receptors traffic through distinct subnuclear compartments, their individual domains may adopt various folded or unfolded states. Thus, nuclear molecular chaperone complexes, which may either bear some resemblance to cytoplasmic complexes or possess unique compositions, may be called on to maintain nuclear receptors in biologically active conformations.

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