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The Heat Shock Protein 70 Cochaperone Hip Enhances Functional Maturation of Glucocorticoid Receptor

S.C. Johnson Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Multiple molecular chaperones interact with steroid receptors to promote functional maturation and stability of receptor complexes. The heat shock protein (Hsp)70 cochaperone Hip has been identified in conjunction with Hsp70, Hsp90, and the Hsp70/Hsp90 cochaperone Hop/Sti1p in receptor complexes during an intermediate stage of receptor assembly, but a functional requirement for Hip in the receptor assembly process has not been established. Because the budding yeast Saccharomyces cerevisiae contains orthologs for most of the receptor-associated chaperones yet lacks an orthologous Hip gene, we exploited the well-established yeast model for steroid receptor function to ask whether Hip can alter steroid receptor function in vivo. Introducing human Hip into yeast enhances hormone-dependent activation of a reporter gene by glucocorticoid receptor (GR). Because Hip does not similarly enhance signaling by mineralocorticoid, progesterone, or estrogen receptors, a general effect on transcription can be excluded. Instead, Hip promotes functional maturation of GR without increasing steady-state levels of GR protein. Unexpectedly, Hip binding to Hsp70 is not critical for boosting GR responsiveness to hormone. In conclusion, Hip functions by a previously unrecognized mechanism to promote the efficiency of GR maturation in cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
STEROID RECEPTORS RELY on heat shock protein (Hsp)90, Hsp70, and other components of the cellular chaperone machinery to establish and maintain functional competence (reviewed in Ref. 1). In the absence of hormone, steroid receptors are typically recovered from cell lysates as heteromeric complexes consisting of a receptor monomer, a dimer of Hsp90, the Hsp90 cochaperone p23, and one of the Hsp90-binding proteins, FKBP52, FKBP51, PP5, or CyP40, each of which contains a tetratricopeptide repeat (TPR) domain that targets binding to Hsp90. Receptor complexes also contain Hsp40, Hsp70, and the cochaperones Hop and Hip. Cell-free approaches have been used to demonstrate that receptors for progesterone (PRs) and glucocorticoids (GRs) assemble with chaperones in a multistep, ordered pathway. Among the chaperones observed in receptor complexes, Hsp40, Hsp70, Hsp90, Hop, and p23 are minimally required for efficient maturation of receptor hormone binding ability (reviewed in Ref. 1).

Hip is an Hsp70-binding cochaperone first observed as a component in PR complexes (2) and independently identified through its interaction with the ATP-binding domain of Hsp70 (3). Hip stabilizes the ADP-bound conformation of Hsp70, thus promoting Hsp70 binding to and refolding of misfolded model substrates in vitro (3).

Hop and Hip both bind Hsp70 and appear concomitantly in intermediate PR complexes in an Hsp70-dependent manner (2). Both Hop and Hip have a TPR domain that targets the cochaperone to Hsp70, although each binds independently to a separate domain of Hsp70 (4). Unlike Hip, Hop has a second TPR domain that separately targets Hop binding to Hsp90 (5, 6), a linkage that appears to be important for efficiently recruiting Hsp90 to Hsp70-PR complexes (7). In addition to being TPR proteins, Hip and Hop share a C-terminal DP domain (8) that stabilizes Hop binding to Hsp70 (7) yet destabilizes Hip binding to Hsp70 (9). In vitro, Hip DP domain mutants can arrest PR assembly at an intermediate stage, apparently blocking recruitment of Hop-Hsp90 to the growing complex (9). Hop DP mutants also arrest PR assembly in vitro by failing to bind Hsp70 in PR complexes and thus inhibiting recruitment of Hsp90 (7). Whereas these findings suggest a functional linkage between Hip and Hop in the receptor assembly process, Hip, unlike Hop, does not alter the rate of receptor assembly and maturation in a minimal in vitro assembly assay (10, 11).

Resolution of conflicting evidence for the importance of Hip in steroid receptor function may lie in cellular experiments that better address the potential physiological role of Hip. In one set of experiments (11) overexpression of Hip in a mammalian cell model had little effect on GR function, but this may reflect that endogenous Hip levels are above the rate-limiting level for GR maturation. Hip overexpression was able to reverse inhibition of GR function caused by overexpression of BAG1, an Hsp70 cochaperone that inhibits Hsp70 binding to substrates (12), but this experimental phenomenon does not exclude that Hip plays an active role in GR maturation apart from countering BAG1 actions.

In lieu of a specific, direct inhibitor of Hip function or a Hip gene knockout model, excluding a potential physiological role for Hip in vertebrate cells has been problematic because these cells typically express Hip at relatively high levels and also express multiple BAG1 isoforms. As an alternative to vertebrate cells, the yeast Saccharomyces cerevisiae lacks a gene with significant homology to Hip, and yeast lack a clear ortholog for cytoplasmic BAG1, thus providing a cellular background in which Hip can be introduced de novo and interactions with BAG1 excluded. Yeast also lack steroid receptor genes; nonetheless, Yamamoto and colleagues (13) demonstrated that a glucocorticoid-responsive system could be generated by transforming yeast with plasmids expressing GR and a reporter gene under control of glucocorticoid response elements. The yeast model for steroid signaling has been widely exploited, especially by investigators exploring the function of receptor-associated chaperones. As one example, Sti1p, the yeast ortholog for Hop, was first shown in the yeast model to be important for steroid receptor function in vivo (14). Although GR will function in a hormone-dependent manner in yeast, indicating that minimal chaperone and transcriptional factors required for function are present, GR function is not maximal. We have recently demonstrated (15) that GR function is significantly enhanced in yeast that express human FKBP52, a receptor-associated cochaperone that has no counterpart in the yeast genome. FKBP52-dependent potentiation was traced to an increase in GR hormone binding affinity as opposed to an increase in receptor number, a general enhancement of transcription, or other possible mechanisms. In this report, we employed a similar rationale to functionally test human Hip in the yeast GR-reporter model.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Human Hip Does Not Alter Yeast Growth at Moderate Temperatures
Before testing whether human Hip alters GR function in the yeast background, we first examined what effect exogenous Hip would have on the general growth properties of transformed yeast. Serial dilutions of independent yeast isolates, two for yeast transformed with an empty vector and two transformed with a plasmid that constitutively expresses human Hip (pHip), were plated and grown at 18, 30, or 37 C (Fig. 1). No differences in growth were observed at the lower temperatures; however, at the more stringent 37 C condition, cells expressing Hip displayed somewhat greater temperature sensitivity than wild-type cells; the reason for this modest growth restriction is unknown. For all following experiments, cells were grown either at 25 or 30 C, temperatures at which Hip expression does not impede growth.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Growth Properties of Yeast Expressing Hip Duplicate isolates of strains containing either an empty vector (Vector) or a Hip-expressing plasmid (pHip) were grown in selective broth at 30 C to an OD600 of approximately 1 U (2 x 107 cells/ml). Successive 5-fold serial dilutions were made of each culture and 5 µl spotted on selective plates. Plates were incubated in humidified chambers at 18 C or 30 C for 7 d or at 37 C for 3 d.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Hormone Dose-Response Curves for Yeast Reporter Strains Lacking or Containing Human Hip Yeast strains expressing one of the mammalian steroid receptors (GR, MR, PR, or ER) and the appropriate reporter gene were transformed with either an empty control vector (open circles) or the pHip expression plasmid (solid circles). Reporter gene activity was stimulated by addition of the appropriate hormonal agonist (DOC, progesterone, or 17ß-estradiol) over a range of concentrations, as indicated. Each data point represents the average (±SD) of three separate experiments. The data set for each receptor was normalized to the maximum reporter activity observed in strains lacking Hip.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Comparison of FKBP52 and Hip Effects on GR Signaling The GR reporter strain was cotransformed with two empty vectors (open circles), an FKBP52 expression plasmid plus control vector (solid circles), pHip plus control vector (open squares), or pHip plus the FKBP52 plasmid (solid squares). A DOC dose-response curve was generated for each strain. The vertical arrow highlights increased efficacy, and the horizontal arrow highlights increased potency. The results shown are representative of three independent trials.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Hip Influence on Number and Activity of Wild-Type and Mutant GR A, Whole-cell extracts were prepared from yeast reporter strains expressing no GR (first lane), wild-type GR (wtGR), or a GR point mutant (F620S or C656G) in cells cotransfected with an empty vector (–) or pHip (+). Aliquots of each extract were separated by SDS-PAGE and immunostained for GR, Hip, and, as a loading control, Sti1p. B, Hormone-induced reporter activity was measured in cells expressing wild-type or mutant GR in the presence or absence of pHip. Cells were treated with 50 nM DOC. Each bar represents the average of three replicate assays (±SD).

In a further characterization of GR maturation, we examined the GR C656G mutant, which, as demonstrated by Simons and associates (18), functions as a super GR in mammalian cells but which functions very weakly in the yeast background (S. S. Simons, Jr., personal communication). As seen in Fig. 4A, GR C656G expressed at levels comparable to other GR forms; however, reporter activity in yeast lacking Hip was near baseline (Fig. 4B). Coexpression of GR C656G and Hip elevated reporter activity by 12-fold, above the level observed with wild-type GR alone, but less than the level obtained with wild-type GR plus Hip. As with other GR forms, Hip did not alter the steady-state level of GR C656G protein. We think these observations point to an enhanced functional maturation of GR that is mediated by Hip. The interaction of Hip with other chaperone components involved in receptor assembly and functional maturation will be examined next.

Cooperation between Hip and Sti1p/Hop
Yeast Sti1p and the vertebrate ortholog Hop are cochaperone proteins that bind both Hsp70 and Hsp90. Because Hip binds the N-terminal ATPase domain of Hsp70 and Hop binds the C-terminal region of Hsp70, both cochaperones can coexist in Hsp70 complexes. Hip and Hop both participate transiently during a common intermediate stage of receptor assembly (2). Additionally, Hip and Hop share a unique structural feature termed the DP domain (8), and mutations in the DP domain of either Hip or Hop arrest assembly of steroid receptor complexes at an intermediate stage (7, 9). Collectively, these observations support the possibility that Hip coordinates with Hop to promote GR maturation in cells. Disruption of the yeast gene for Sti1p impairs steroid receptor function (14), and Sti1p will fully substitute for Hop during assembly of receptor complexes in vitro (7). As shown in Fig. 5, we genetically tested for interaction between Hip and Hop/Sti1p in the GR reporter model.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Genetic Evidence for Interaction between Hip and Sti1p/Hop A, Whole-cell extracts from wild-type (STI1) and Sti1p-minus (sti1::loxP) cells were Western immunostained for the antigen indicated on the left. Cells were transformed with an empty vector (Vec.), pHip, or a human Hop expression plasmid (pHop), as indicated above each lane. B, Reporter activity in STI1 and sti1::loxP (sti1) strains expressing Hip and/or Hop, as indicated below each plot, was measured in cultures lacking hormone (open bar) or in cultures treated with 50 nM DOC (solid bars). Values were normalized to the basal activity observed in the STI plus vector strain lacking hormone. Each bar represents the average (±SD) for three replicate samples.

GR activity was compared in each of the strains (Fig. 5B). The basal, uninduced expression of reporter was minimal (STI1, Vec., open bar) whereas reporter activity in the wild-type background was stimulated 6-fold by addition of hormone (STI1, Vec., solid bar). As seen previously, addition of pHip to the system (STI1, pHip) enhanced hormone-dependent signaling—in this case by approximately 10-fold over cells lacking pHip. In wild-type STI1 cells expressing Hop (STI1, pHop), GR signaling was marginally enhanced, probably reflecting the adequate function of endogenous Sti1p in these cells. When both Hip and Hop were expressed in wild-type cells (STI1, pHip+pHop), GR activity was enhanced by approximately 6-fold compared with vector alone (STI1, Vec., closed bar), somewhat less than the 10-fold enhancement observed with Hip alone.

In the sti1::loxP background (sti1, Vec.), hormone-dependent signaling was virtually abolished. This is a greater reduction in GR activity than originally reported for Sti1-minus yeast (14). The difference is likely due to greater sensitivity of the ß-galactosidase assay used here, which permits reporter activity measurements at a lower hormone concentration and sooner after induction, conditions that might better reflect the physiological importance of Hop/Sti1p in GR signaling. Expression of human Hop in sti1::loxP cells (sti1, pHop) restored GR signaling to levels seen in wild-type yeast, indicating that human Hop can fully substitute for Sti1p in yeast-based GR signaling. In the absence of Sti1p, Hip can still enhance signaling (sti1, pHip), but only a 3-fold increase in reporter expression is observed compared with a 10-fold increase in the presence of Sti1p (STI1, pHip). However, when Hip and Hop are coexpressed (sti1, pHip+pHop), GR activity is restored to the level observed with similar coexpression in the wild-type background (STI1, pHip+pHop). Thus, Hip and Sti1p/Hop act synergistically to promote GR activity.

Relevance of Hip Binding to Hsp70
Hip binds directly to the ATPase domain of Hsp70, and Hsp70 is a critical factor for assembly and maturation of vertebrate steroid receptor complexes. To assess the importance of Hsp70 interactions in Hip-mediated enhancement of GR function, a series of Hip mutants were examined. In Fig. 6A, a coimmunoprecipitation approach was used to characterize the ability of Hip forms to bind Hsp70 in vitro. Consistent with earlier reports (19, 20), deletion of the TPR or adjacent charge domain disrupts Hsp70 binding. Truncation of the first 14 amino acids from Hip, which disrupts homooligomerization (19), does not disrupt Hsp70 binding. Two newly generated Hip point mutants either disrupt Hsp70 binding (K118A) or enhance binding to Hsp70 (I149A).

View larger version (53K): [in this window] [in a new window]   Fig. 6. Functional Comparison of Hip Mutants A, The abilities of Hip forms to bind Hsp70 were assessed by a coimmunoprecipitation approach. The forms tested are full-length wild-type Hip (wtHip), an N-terminal truncation of 14 amino acids that is unable to oligomerize (oligo), deletions of the TPR domain (TPR) or adjacent charge region (charge), and TPR domain point mutants (K118A and I149A). Radiolabeled proteins were generated by in vitro expression in rabbit RL, and an equimolar amount of each radiolabeled Hip product was separately added to aliquots of normal RL from which Hsp70 complexes were immunoprecipitated using an anti-Hsp70 monoclonal antibody. Samples were separated by SDS-PAGE and Coomassie stained for total protein in the immunoprecipitates (top panel). The major stained bands are Hsp70, Hsp90, and Hop, which form an abundant complex in most tissues, and heavy chain (HC) of the anti-Hsp70 antibody used to isolate complexes. As indicated above the lanes, one radiolabeled Hip form was included in each sample. Hip forms recovered in Hsp70 complexes were detected by autoradiography (middle panel, [35S]-Hip Bound) of the Coomassie-stained gel. A separate gel and autoradiograph (bottom panel, [35S]-Hip Input) were prepared to illustrate the relative amounts of each radiolabeled form included in each sample. B, The steady-state level of GR protein was measured in yeast strains expressing Hip forms. Separate strains lacking GR (first lane) or expressing GR were cotransformed with empty vector (vector) or plasmid expressing one Hip form, as indicated above each lane. Whole-cell extracts were separated by SDS-PAGE and immunostained for GR (upper panel) or Hip (middle panel). As a loading control, samples were also immunostained for Sti1p (bottom panel). C, GR reporter strains were assayed for hormone-independent (*) and hormone-dependent expression of the lacZ reporter. Cells were transformed with empty vector or with plasmid expressing wild-type or mutant Hip, and treated with 50 nM DOC. The Hip point mutants K118A and I149A were compared with wild-type Hip in a separate experiment. Each data bar represents the average (±SD) of reporter read-out from three replicate samples.

As seen in Fig. 7A, Hip fails to coimmunoprecipitate with Ssa1p, one of the most abundant Hsp70 forms in yeast, which has been implicated in many common folding processes as well as in specific interactions with Sti1p (21). We also tested for Hip binding to Ssb2p, which assembles on ribosomes and participates in nascent chain folding (22); again, no interaction with Hip was detected. Because there are more than a dozen Hsp70-related genes expressed in yeast, we cannot exclude the possibility that Hip interacts with other family members.

View larger version (55K): [in this window] [in a new window]   Fig. 7. Limited Role of Hip-Hsp70 Interactions in Hip Function A, Similar to the approach described for Fig. 6A, in vitro coimmunoprecipitation assays were performed. In this case, Hip complexes were precipitated from RL that was spiked with radiolabled rat Hsc70 or yeast Hsp70 (Ssa1p or Ssb2p). A Coomassie-stained gel (upper panel) shows major proteins in samples immunoprecipitated with anti-Hip (Hip IP) and with a negative control antibody (Ctrl IP). The final three lanes are aliquots of the radiolabeled synthesis mixture for each Hsp70 (Input). The major protein components in RL Hip complexes are identified on the left along with heavy chain (HC) of the anti-Hip antibody. An autoradiograph of this gel (lower panel, [35S]) was generated to reveal radiolabeled Hsp70 forms recovered in the IP samples or Input into IP reactions. B, Hormone-dependent lacZ expression was measured in GR reporter strains transformed with an empty vector or plasmids expressing Hip or Hip point mutant, either alone or in combination with rat Hsc70. An additional strain lacking Hip plasmid expressed Hsc70 alone. Cells were treated with 50 nM DOC. Expression of rat Hsc70 in yeast was verified by immunostaining of whole-cell extracts with an antibody specific for mammalian Hsp70 (inset). Each data bar represents the average (±SD) of reporter read-out from three replicate samples.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
In this report we find that Hip can significantly boost hormone-dependent activation of reporter gene expression mediated by GR. For several years Hip has been recognized as a transient component of native steroid receptor complexes, yet there has been limited prior evidence that Hip alters steroid receptor activity, either in vitro or in vivo. In a minimal GR assembly system using purified chaperone proteins, i.e. Hsp40, Hsp70, Hsp90, Hop, and p23, inclusion of Hip neither enhanced nor inhibited restoration of GR hormone-binding ability (11). However, Hip was found to offset the inhibitory actions of BAG1 toward GR assembly in the minimal system, which might simply reflect competitive displacement of BAG1 by Hip from Hsp70 complexes. Whereas the minimal assembly system will restore GR hormone binding ability, Nordeen and colleagues (23) have recently observed that GR assembled in a minimal system fails to activate transcription in a cell-free assay, although addition of rabbit reticulocyte lysate (RL) to the system could recapitulate hormone-dependent transcriptional activation. Hip is perhaps one of the missing factors provided by RL.

Knockdown of Hip expression in mammalian cells could help reveal the physiological requirement for Hip in cellular processes. Even though RNA interference-mediated knockdown of Hip is feasible, a drawback to this approach is that reducing Hip would effectively increase the ratio of BAG1 in cells. Because there are multiple BAG-related genes expressed in mammalian cells, it would not be practical to simultaneously knockdown BAG-like activity to discriminate between BAG-dependent and BAG-independent functions of Hip. For this reason, the yeast model is a particularly attractive alternative because S. cerevisiae do not contain a Hip-related gene and the only two BAG-related genes are highly divergent and code for a membrane protein or an RNA polymerase subunit, neither of which is likely to participate in steroid receptor assembly. With the established yeast model for steroid receptor function, we found that de novo introduction of Hip enhances GR function in a BAG-independent manner.

Basis for Change in GR Activity
There are several possible ways in which Hip might enhance GR function. Because Hip does not similarly enhance MR, PR, or ER responses (Fig. 2), it is unlikely that Hip acts as a general transcription factor. Furthermore, the same yeast reporter plasmid was used for GR, MR, and PR assays because the steroid response elements in the promoter are recognized in common by each receptor. Therefore, Hip is more likely to act at an upstream step that is specific for GR.

We have shown recently that human FKBP52 will specifically enhance GR activity in yeast by elevating GR affinity for hormone (15). Hip does not have a similar effect on GR, as is apparent from the direct comparison of dose-response curves in the presence of Hip, FKBP52, or both cochaperones (Fig. 3). The Hip-dependent increase in hormonal efficacy suggests that Hip stimulates an increase in GR number; however, the steady-state level of GR protein in yeast is unaffected by the addition of Hip (Figs. 4A and 6B).

We conclude that the most likely effect of Hip on GR is to enhance functional maturation of GR through more efficient and/or stable folding of the GR ligand-binding domain. Ideally, Scatchard analysis of GR hormone-binding data, either in intact cells or cell lysates, would further confirm an increase in hormone-binding sites in the presence of Hip, but there are multiple technical difficulties to obtaining these data in yeast, including the poor bioavailability in yeast of commonly used high-affinity ligands such as dexamethasone, a much higher level of nonspecific hormone binding to yeast cell wall components, and the lability of receptor complexes in the harsh conditions required for yeast cell disruption.

It is known that GR folds less efficiently in bacterial and yeast backgrounds than in mammalian cells, and the point mutant GR F620S has been exploited to overcome folding difficulties in heterologous backgrounds. In our yeast assays, this mutant alone functioned as well as wild-type GR in the presence of Hip (Fig. 4B), yet Hip could further boost activity of GR F620S, suggesting that this mutant is still not maximally adapted for folding in the yeast background. Hip could restore significant activity from another GR mutant, C656G, which functions as a superreceptor in mammalian cells, yet is barely functional in yeast cells. We tested a GR mutant in the yeast model that combines the F620S and C656G mutations. The combined mutant had enhanced activity that was intermediate between wild-type GR and GR F620S (results not shown), so F620S could largely compensate for the folding defect in C656G. As with each GR form, Hip could further enhance activity of the double mutant, but only approximately 2-fold. It is apparent that the cellular environment of yeast lacks factors present in mammalian cells that promote efficient folding and maturation of GR. We propose that one of those factors is Hip.

Potential Mechanisms for Hip Function
The mechanism by which Hip promotes functional maturation of GR is unknown, but several presumptive mechanisms can be excluded. Hip is not simply displacing BAG1 from Hsp70 because yeast lack BAG1 counterparts. Moreover, our results suggest that Hip functions in an Hsp70-independent manner in yeast as we were unable to detect interactions between Hip and common yeast Hsp70 forms (Fig. 7A), mutations in Hip that eliminate Hsp70 binding retain activity in GR reporter assays (Fig. 6C), and coexpression of rat Hsc70 with Hip inhibited rather than enhanced Hip function (Fig. 7B).

Hip and Hop appear simultaneously at intermediate stages of steroid receptor assembly, so it is reasonable to speculate that these cochaperones function in a cooperative manner. The Hop ortholog Sti1p is required for GR maturation (Ref. 14 and Fig. 5B), and human Hop can fully complement loss of Sti1p in yeast. In a test for genetic interactions, Hip could partially rescue GR defects in the sti1::loxP strain, supporting a shared role for Hip and Hop in GR maturation. However, because Hip does not require Hsp70 for its function in yeast, the interaction between Hip and Hop/Sti1p is likely not through a common complex with Hsp70. Perhaps relevant to this discussion is our recent observation that a Hop point mutation that disrupts Hsp70 binding will partially, but not completely, rescue GR function in the sti1::loxP strain (24). Therefore, Hip and Hop may both have Hsp70-independent roles in GR maturation.

The Hip mutant with most dramatic loss of GR-enhancing ability is an N-terminal truncation that disrupts Hip homooligomerization (Fig. 6C). Free Hip typically exists as a trimer or larger homooligomer (19, 20). Truncation of as few as 14 amino acids from the N terminus of Hip completely blocks oligomer formation without disrupting Hsp70 binding (19). On the other hand, N-terminal truncation prevents Hip-mediated stimulation of Hsp70-dependent luciferase refolding in a mammalian cellular model (25). Our results further underscore the importance of Hip oligomerization, except in our case oligomerization may not relate directly to Hsp70 interactions.

Neither we nor others have observed that Hip directly binds steroid receptor; all previous indications have been that Hip indirectly associates with receptor complexes through Hsp70. There is a precedent, however, for direct Hip interactions with another receptor class. Hip binds the cytoplasmic C-terminal region of the membrane chemokine receptor CXCR2 and regulates receptor activity (26). Moreover, the Hip TPR mutant could still interact with CXCR2 and perform some of the regulatory functions of full-length Hip. In the GR pathway, Hip could transiently interact with GR in a productive manner that is too unstable for easy detection. For example, Hip has been shown to have passive chaperoning activity in vitro toward a model misfolded protein (3), and such activity may be relevant to enhancing GR maturation. On the other hand, there appears to be no limit on the general chaperoning of GR in yeast because expression of rat Hsc70 in addition to endogenous yeast chaperones failed to boost GR activity (Fig. 7B). Other possibilities are that Hip interacts with receptor only when receptor is assembled with certain chaperones or that Hip interacts with a receptor-associated chaperone to indirectly promote receptor maturation. We have minimized the likelihood that Hsp70 is a relevant target for Hip binding, and Hop/Sti1p is unlikely to be such a target because Hip can still enhance GR function in the absence of Sti1p. Further study will be required to identify Hip interaction partners that are most relevant to enhancement of GR function in vivo.

Distinctive Requirements of GR for Specific Chaperones
It appears that Hip, directly or indirectly, lowers the barrier for some step(s) along the folding pathway toward full maturation of the GR ligand-binding domain. That Hip does not similarly enhance folding of other steroid receptors suggests that a more limited set of chaperones suffices for efficient folding of some receptors. We also found that FKBP52 can greatly potentiate GR function, although by a mechanism distinct from Hip, and that FKBP52 does not stimulate other steroid receptors. Why should GR apparently require a greater diversity of chaperone activities for efficient maturation than other steroid receptors? One possibility is that structures within the ligand-binding domain that confer specificity for glucocorticoid ligands may present greater hurdles for efficient folding of the ligand-binding domain. If this were the case, evolution would seem to favor adaptations that retain ligand specificity while removing barriers to folding. For instance, why does vertebrate GR retain phenylalanine at position 620 when mutation to serine and perhaps other small amino acids would seem to enhance folding without deleteriously affecting GR function?

An intriguing possibility is that GR is adapted to be more sensitive to folding challenges than other steroid receptors. FKBP52, which is heat shock inducible (27), seems to function in opposition to FKBP51, a glucocorticoid-inducible inhibitor of GR hormone binding; the interplay between FKBP52 and FKBP51 levels could modulate cellular responsiveness to glucocorticoids (15). Sanchez and colleagues (28, 29, 30, 31) have reported cross-regulation between GR and the heat shock transcription factor pathways. Whether stress-induced expression of FKBP52 is one basis for stimulation of GR activity has not been determined. Hip is another potential stress-related regulator of GR function although cellular mechanisms for transcriptional or posttranscriptional regulation of Hip activity have been poorly characterized. Because GR mediates many aspects of vertebrate responses to physiological and psychological stress, linking its activity to select components of the molecular chaperone machinery may provide lines for cross-talk and coordination between cellular and physiological stress pathways.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Yeast Methods and Strains
The parent of all strains was W303a (MATa leu2–112 ura3–1 trp1–1 his3–11, 15 ade2–1 can1–100 GAL SUC2). As recently described (24), a yeast strain lacking Sti1p (sti1::loxP) was generated by replacing the entire coding region of STI1 with the Saccharomyces pombe his5 gene flanked by loxP sites, and then recombining out the marker by transformation with a Cre-encoding plasmid (32). STI1 deletion was confirmed by yeast colony PCR and Western blot analysis. Plasmids were introduced into yeast using a lithium acetate/polyethylene glycol protocol and propagated as described elsewhere (24). Two or more independent transformants of each strain construction were analyzed for consistent properties.

Plasmids
Wild-type rat GR was expressed from pG/N795 (13). The GR C656G point mutant was generated by site-directed mutagenesis (QuikChange kit, Stratagene, La Jolla, CA) on plasmid pG/N795. The GR F620S point mutant was expressed from pG1/F620S (16). Rat MR was expressed from pH2-rMR (33). ER was expressed from pG/ER(G) and PR was expressed from pG/cPR (34). The separate reporter plasmid used for some GR and all MR and PR experiments was pUCSS-26X (35), and the reporter for ER was pUCSS-ERE (36). For some experiments, the genes for wild-type rat GR and the reporter were combined on plasmid p2A/GRGZ (37).

Plasmid expressing human FKBP52 has been described previously (15). Human Hop cDNA was introduced into p425GPD for constitutive expression in yeast. Hop expression in transformed cells was verified by colony PCR and immunoblotting with anti-Hop antibody F5. Plasmids expressing Hip were generated by ligating the human Hip cDNA into the yeast expression vectors p423GPD, p425GPD, or p426GPD. Hip deletion mutants oligo (truncation of first 14 amino acids), TPR (deletion of amino acids 117–225), and charge (deletion of amino acids 227–281), as previously generated (19) in the in vitro expression plasmid pSPUTK (Stratagene), were subcloned into p423GPD or p425GPD. Hip point mutants K118A and I149A were generated by site-directed mutagenesis of the Hip cDNA in either pSPUTK or yeast expression vectors. Plasmids expressing Hsc70 were generated by ligating the rat Hsc70 cDNA into the yeast expression vector p425GPD or into pSPUTK. The DNA for S. cerevisiae SSA1 and SSB2 genes (provided by Jill Johnson and Elizabeth Craig, respectively) were subcloned into pSPUTK. In all instances above where novel constructs were generated by PCR or site-directed mutagenesis, the desired sequence was verified by automated sequencing.

Hormone Reporter Assays
Hormone induction assays were done as previously reported (15). Briefly, yeast strains were grown in selective media at 25 C to OD₆₀₀ of 0.05–0.12 U. Growth was monitored spectrophotometrically for at least 90 min before hormone addition to ensure that the culture was in exponential phase. Hormone (DOC, 50 nM final concentration unless otherwise noted; 17ß-estradiol; or progesterone) was added, and samples were withdrawn at 10-min intervals (typically from 70–120 min after hormone addition) for ß-galactosidase assays. Samples of 100 µl were immediately added to 100 µl of a chemiluminescent ß-galactosidase assay reagent (Gal-Screen, Tropix, Bedford, MA) in 96-well microtiter plates at room temperature. The entire plate was read in a luminometer 2 h after the last sample was collected.

To determine the rate of reporter expression, ß-galactosidase induction curves were first generated by plotting relative light units against the OD₆₀₀ of the culture sample. Regression analysis of this linear portion of each data set yielded a best-fit line (typically, R² > 0.98) the slope of which is the growth rate-normalized rate of ß-galactosidase expression. For convenience the reporter expression units are defined as the slope/1000.

In Vitro Coimmunoprecipitation
The abilities of Hip forms to bind Hsp70 immobilized on an immunoaffinity resin were assessed essentially as described previously (19). Briefly, anti-Hsp70 monoclonal antibody BB70 was adsorbed to protein G-Sepharose (Pharmacia Biotech, Piscataway, NJ) and used to immunoprecipitate Hsp70 from rabbit RL (1:1; from Green Hectares, Oregon, WI) that was supplemented with a radiolabeled Hip form. Each sample contained the same molar equivalent of radiolabeled Hip or Hip mutant in 100 µl RL with 10 µg BB70 on a 10-µl resin pellet. All samples were incubated at 30 C for 30 min with brief vortexing every 5 min to resuspend resin. Samples were centrifuged and resin pellets were washed three times in 1 ml cold wash buffer (20 mM Tris, pH 7.4; 50 mM NaCl; 10 mM monothioglycerol; 0.5% Tween 20). Proteins adsorbed to resin were separated by SDS-PAGE and visualized by Coomassie blue staining and autoradiography of the dried gel. In a similar manner radiolabeled Hsp70 forms were tested for coimmunoprecipitation from rabbit RL with anti-Hip monoclonal antibody 2G6 adsorbed to protein G-Sepharose. Control antibody used to compare background binding for the Hsp70 forms was anti-PR monoclonal antibody PR22.

Western Immunoblots
To prepare whole-cell extracts, washed yeast cell pellets were resuspended in cracking buffer [40 mM Tris-HCl (pH 7.5), 8 M urea, 5% wt/vol sodium dodecyl sulfate, 10% ß-mercaptoethanol, 0.1 mM EDTA, and 0.04% (wt/vol) bromophenol blue] at a ratio of 5 ml buffer/g cells. Cracking buffer was supplemented with phenylmethylsulfonyl fluoride (1 mM final concentration) and a protease inhibitor cocktail (Complete Protease Inhibitor Mini-Tablet; Roche Clinical Laboratories, Indianapolis, IN) per 7 ml suspension. Cells were homogenized once for 1 min with glass beads in a Mini Bead Beater (Biospec Products, Bartlesville, OK). Samples were centrifuged to remove insoluble material followed by heating in a boiling water bath. The resulting cell lysates were fractionated by SDS-PAGE and transferred to polyvinylidene fluoride membrane for immunoblot analysis. Mouse monoclonal antibodies used were BuGR2 (anti-GR MA1-510, Affinity Bioreagents, Golden CO; 1:1,000 dilution), St-2 (anti-Sti1p; 1:2,000 dilution), F5 (anti-Hop; 1:2,000 dilution), 2G6 (anti-Hip; 1:5,000 dilution), and 13D3-Ivb49-EME (anti-Hsp73, Abcam, Cambridge, UK; 1:1,000 dilution).

	ACKNOWLEDGMENTS

 
We thank Brian Freeman, Didier Picard, David Toft, Jill Johnson, and Elizabeth Craig for generously providing reagents used in these studies.

	FOOTNOTES

 
Address all correspondence and requests for reprints to: David F. Smith, S.C. Johnson Research Building, Mayo Clinic Scottsdale, 13400 East Shea Boulevard, Scottsdale, Arizona 85259. E-mail: smith.david26{at}mayo.edu.

Current address for G.M.N.: Edwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.

This work was supported by National Institutes of Health Grants DK44923 and DK48218 and by funds from the Mayo Foundation.

Abbreviations: DOC, Deoxycorticosterone; ER, estrogen receptor; GR, glucocorticoid receptor; Hsp, heat shock protein; MR, mineralocorticoid receptor; PR, progesterone receptor; RL, reticulocyte lysates; TPR, tetratricopeptide repeat.

Received for publication February 6, 2004. Accepted for publication March 29, 2004.

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