Analysis of FKBP51/FKBP52 Chimeras and Mutants for Hsp90 Binding and Association with Progesterone Receptor Complexes

Richard L. Barent, Satish C. Nair, Damon C. Carr, Ying Ruan, Ronald A. Rimerman, Jennifer Fulton, Yan Zhang and David F. Smith

Department of Pharmacology University of Nebraska Medical Center Omaha, Nebraska 68198-6260


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FKBP51, FKBP52, and Cyp40 bind competitively to Hsp90 through their respective tetratricopeptide repeat (TPR) domains, and any one of the three immunophilins can be isolated in mature steroid receptor complexes. During cell-free assembly reactions, FKBP51 associates preferentially with progesterone and glucocorticoid receptors, but less preference is observed in FKBP51 association with estrogen receptor. A number of mutant FKBP forms were generated to map sequences responsible for FKBP51’s preferred association with progesterone receptor. A double-point mutation in the peptidyl prolyl isomerase domain of FKBP51 that reduces enzymatic activity by greater than 90% had no observed effect on FKBP51 interactions with progesterone receptor or Hsp90. Coprecipitation of FKBP51 and FKBP52 truncation mutants with Hsp90 indicated that sequences both upstream and downstream of the TPR domain are necessary for Hsp90 binding. FKBP chimeric constructs were also generated and tested for Hsp90 binding and receptor association. The TPR domain of FKBP51 required appropriate downstream sequences for Hsp90 binding, but FKBP52’s TPR domain did not. The C-terminal region of FKBP51 that functionally interacts with the TPR domain to permit Hsp90 binding also conferred preferential association with PR. In conclusion, despite the overall similarity of FKBP51 and FKBP52, these two immunophilins associate differentially with steroid receptors, and the difference relates to both the Hsp90-binding TPR domain and to poorly conserved C-terminal sequences.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Unliganded steroid receptors assemble with a number of molecular chaperone components in an ordered, dynamic pathway (1). In a previous time course study on the de novo cell-free assembly of progesterone receptor (PR) complexes (2), it was found that chaperone components associated with PR in two basic patterns. One is an early transient peak dropping to a lower steady state level, as observed for Hsp70, Hip, and Hop; the other is a more prolonged rise to a steady state plateau, as seen for Hsp90 and p23. At steady state most receptor exists in the mature complex with an Hsp90 dimer, the Hsp90-binding protein p23, and any one of three Hsp90-associated immunophilins, FKBP51, FKBP52, or Cyp40, but there is a constant disassembly and reassembly through this pathway such that early and intermediate complexes are continuously present. An important physiological significance to maturation of PR and glucocorticoid receptor (GR) complexes is that only the mature receptor complexes are capable of binding hormone with high efficiency and affinity. On the other hand, estrogen receptor (ER) has similar chaperone interactions, but these are not required to maintain the hormone-binding conformation (3).

Hsp90 and p23 play important roles in establishing and stabilizing the high-affinity hormone-binding conformation of GR and PR, but the functional requirement for immunophilins in mature receptor complexes has been difficult to define. Perhaps the best in vivo evidence of a role for Hsp90-associated immunophilins in steroid receptor function comes from a heterologous model in which a vertebrate steroid receptor is expressed in Saccharomyces cerevisiae. In the S. cerevisiae genomic sequence, there is no clear homolog for the large FKBPs, but there are two Cyp40 homologs, termed Cpr6 and Cpr7 (4). Both Cpr6 (5) and Cpr7 (6) bind to Hsp90, but only Cpr7 genetically interacts with GR. Deletion of the yeast gene for Cpr7, but not the Cpr6 gene, caused an 80% reduction in GR-dependent activation of a reporter gene (6).

FKBP52, FKBP51 and Cyp40 have peptidylprolyl isomerase (PPIase) activity, although the functional importance of this activity is an open question. The immunosuppressant FK506 binds the PPIase active site and blocks enzymatic activity, but FK506 has no apparent effect on the composition of PR complexes (7) or on the composition and in vitro assembly of GR complexes (8). FKBP52 in GR complexes will bind an FK506 affinity resin without dissociating from the receptor complex (9), but FKBP51 binds well to an FK506 resin only when it is dissociated from Hsp90 and other proteins (10).

A number of cellular studies examining the effects of immunosuppressant drugs on steroid hormone action have reported potentiation or attenuation of steroid responses (11, 12, 13, 14). These findings have been interpreted to support a role for receptor-associated immunophilins in normal receptor function; however, there are multiple immunophilin and nonimmunophilin target sites for these drugs in cells, and it has not been demonstrated that receptor-associated immunophilins are directly responsible for the reported pharmacological effects. Pratt and colleagues (15, 16) have succeeded in assembling GR into functionally mature complexes in a reconstituted system that lacks immunophilins, arguing against a role for immunophilins in hormone binding. The Pratt group (17) has proposed that FKBP52 may participate in the nuclear translocation of GR complexes. Evidence was presented that FKBP52 may bind a nuclear localization sequence (NLS) of GR, thus serving as an NLS receptor (18, 19), but this purported function for FKBP52 has not been conclusively demonstrated.

Human FKBP51 shares 60% amino acid sequence identity and 75% similarity with human FKBP52, and both are expressed constitutively in many mammalian cell and tissue types. FKPB51 is up-regulated during adipogenesis (20) and in response to glucocorticoids (21), and expression of FKBP52 is stimulated by several mitogenic growth factors (22). We have observed a dexamethasone-dependent increase of FKBP51 protein levels, but not FKBP52 levels, in rat L929 fibroblasts and HTC19.G11 cells (2-fold and 5-fold increases, respectively; Y. Ruan and D. F. Smith, unpublished). A potentially relevant correlation between FKBP expression levels and GR activity has recently been made. In B-lymphoblast extracts from glucocorticoid-resistant squirrel monkeys, where a cytosolic factor appears to be responsible for GR’s lower hormone-binding affinity (23), FKBP51 is overexpressed and FKBP52 is greatly underexpressed relative to type-matched human cells (24).

The best characterized interactions of FKBP52 and FKBP51 are with Hsp90 and steroid receptor complexes. In rabbit reticulocyte lysate (RL), a medium used for cell-free assembly studies of steroid receptor complexes, FKBP51 is present at approximately 20 nM as compared with 100, 200, and 1,000 nM concentrations for, respectively, FKBP52, Cyp40, and Hsp90 (25). Despite its lower concentration, FKBP51 was preferentially recovered in PR complexes (25), suggesting a potential functional difference in FKBP51 and FKBP52. Previous observations have also distinguished the behaviors of FKBP51 and FKBP52. In native chicken PR complexes, FKBP51’s association is uniquely sensitive to hormone binding and to sulfhydryl-modifying agents (26, 27). Unlike FKBP52, FKBP51 in heteromeric complexes binds poorly to an FK506 affinity matrix, suggesting that FKBP51’s PPIase domain is less accessible. In the present report, we have more closely examined FKBP51’s preferential association with steroid receptor complexes and have mapped out the region of FKBP51 responsible for its preference.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Interactions of Wild-Type Immunophilins with Steroid Receptor Complexes
It was recently discovered that FKBP51, relative to FKBP52 and Cyp40, associates preferentially with PR (25). The preference of FKBP51 for PR complexes had not been previously noted, primarily because it was not appreciated that FKBP51 was limiting in RL assembly mixtures. When the time course for in vitro assembly of immunophilins and other chaperone components with PR was reexamined (Fig. 1Go), it differed from an earlier analysis (2) by having a greater ratio of RL to PR in the assembly mixture. Recombinant PR-A was bound to an immunoaffinity resin and placed in RL supplemented with radiolabeled chaperone components. Aliquots were removed from the assembly mixture over a 60-min time course and immediately quenched in cold wash buffer. Resin complexes were separated by SDS-PAGE, and the gels were Coomassie-stained for total protein levels and autoradiographed to visualize radiolabeled proteins. The absorbance values for bands on x-ray film were normalized to the amount of Coomassie-stained receptor in each lane, and the values were plotted as a percentage of the maximum recovery for each component.



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Figure 1. Time Course for Assembly of PR Complexes

De novo association of chaperone components with recombinant chicken PR-A was followed in a cell-free assembly mixture. Rabbit reticulocyte lysate containing an ATP-regenerating system was supplemented with radiolabeled markers for chaperone components involved at various assembly stages: markers for chicken Hsp90-{alpha}, rat Hsc70, human Hop, and human Hip were included in one mixture, and markers for the human immunophilins FKBP52, FKBP51, and Cyp40 were included in a separate assembly mixture. After initiation of assembly reactions at 30 C, aliquots of the assembly mixtures were removed at the times indicated. Components in receptor complexes were separated by SDS-PAGE and visualized by Coomassie staining and autoradiography. Bands were quantitated by densitometry and normalized to the amount of stained PR in each lane. Values for each chaperone component are plotted as a percent of the maximum recovery level of that component. The inset in the upper panel is an expansion of the 0- 5-min time points.

 
As seen in the upper panel of Fig. 1Go, the recoveries of Hsp70, Hip, and Hop with PR complexes all attain a maximum in the first 5–10 min and then drop to lower levels. Hsp90 recovery contrasts by reaching a sustained plateau of maximal binding in 10–15 min. These patterns are consistent with an earlier analysis (2). Expansion of the scale to better resolve changes over the first 5 min (inset) reveals that Hsp70 binding precedes the binding of Hip and Hop, reflecting the initial formation of an early PR complex with Hsp70, followed by intermediate complexes containing Hip, Hop, and Hsp90. The delayed plateau of Hsp90 binding reflects accumulation of relatively stable mature complexes, which lack Hsp70, Hip, and Hop, as assembly reaches steady state conditions.

Immunophilins, which are restricted to mature PR complexes, display two patterns of recovery with PR complexes (Fig. 1Go, lower panel). Cyp40 and FKBP52 achieve maximal binding levels over 5–15 min but decrease thereafter. In contrast, FKBP51 levels do not reach a maximum until 30 min after initiation of assembly. These changes are consistent with FKBP51 being competitively preferred in mature PR complexes but also being present at a lower concentration in RL than FKBP52 and Cyp40.

It was shown qualitatively that ER complexes assembled in vitro contain FKBP51 (3), but FKBP51’s binding was not quantitated relative to FKBP52 and Cyp40. To our knowledge, GR complexes have not been directly tested for FKBP51. In Fig. 2Go, the immunophilin compositions of in vitro-assembled PR, GR, and ER complexes were compared. In preliminary characterizations, immunoprecipitation levels for each of the three receptors were quantitated by densitometry of Coomassie-stained receptor bands on one-dimensional SDS gels (not shown). For compositional determinations, approximately 0.5 µg of receptor was incubated with 500 µl RL under maximal assembly conditions for 45 min. PR complexes contained the typical proportions of immunophilins with FKBP51 > Cyp40 > FKBP52. Surprisingly, GR complexes contained an even higher proportion of FKBP51, apparently at the expense of Cyp40. ER complexes had relatively higher proportions of Cyp40 and FKBP52 than observed in PR and GR complexes. The relative association levels seen in Fig. 2Go were consistently observed in replicate receptor assemblies.



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Figure 2. Comparison of the Composition of Steroid Receptor Complexes Assembled in Vitro

Recombinant PR and ER were immunoadsorbed from Sf9 cell extracts, and native GR was immunoadsorbed from L cell extracts. Each resin pellet (~0.5 µg receptor) was incubated with 500 µl RL at 30 C for 45 min. Protein components were separated by two-dimensional gel electrophoresis and visualized by silver staining. The region of each gel containing Hsp90, Hsp70, and immunophilin spots are shown. Most of the unmarked spots are antibody heavy chain components (in the 50-kDa region) or RL proteins binding nonspecifically to immunoaffinity resins, but some may be minor, unidentified proteins associated with receptor complexes. The heavy chain components of anti-PR antibody resolved to the basic side of the gel region shown. Proteins with more acidic isoelectric points are to the right in each panel.

 
Mutagenic Analysis of FKBPs
FKBP51 and FKBP52 are homologous proteins with highly conserved domain structures, as illustrated by their sequence alignment (Fig. 3AGo). The PPIase domain indicated on the alignment (single underline) corresponds to the region of homology to FKBP12. The tetratricopeptide repeat (TPR) region contains three TPR units (double underlines), and there is a consensus calmodulin-binding motif (CaMBC; dashed underline) downstream from the TPRs. The putative NLS recognition sequence of FKBP52 (amino acids 143–147) is indicated by a gray bar. Interestingly, the corresponding sequence of FKBP51 lacks two of the glutamic acids and has a phenylalanine in place of FKBP542’s threonine. No other deletions/insertions are apparent in comparing the overall sequences.



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Figure 3. FKBP Sequences and Mutant Constructs

A, Alignment of deduced amino acid sequences for human FKBP51 and FKBP52. Protein sequences for the two FKBPs were aligned using Gap (Wisconsin Package Version 9.0, Genetics Computer Group, Madison, WI). Identical amino acids are included in shaded boxes. The N-terminal PPIase domain homologous to FKBP12 is indicated by a single underline (hFKBP51 residues 32–138), and a putative NLS-binding motif in hFKBP52 (residues 143–147) is indicated by a shaded bar. In the C-terminal half of each sequence, the three units in the TPR region are indicated by double underlines, and a consensus calmodulin-binding motif is indicated by a dashed underline (hFKBP51 residues 397–413). B, Diagram of FKBP mutants and chimeric swaps. The major domains and structural motifs shared by FKBP51 and FKBP52 (shaded boxes) are identified above the first diagram. One double-point mutant was generated (FD67DV) targeting the PPIase activity of FKBP51. Two sets of truncation mutants were prepared, one set each for FKBP51 and FKBP52, but only the FKBP52 mutants are named on the left. Chimeric proteins were generated by alternately swapping cDNA sequences coding for each of the protein regions shown. Only the chimeras mentioned in later figures are named.

 
As diagrammed in Fig. 3BGo, a number of FKBP mutants were prepared. The small box filled with slanted lines in FKBP51, corresponding to the putative NLS-binding sequence in FKBP52, indicates the apparent deletion of two amino acids. In the TPR domain, the first shaded box corresponds to the first TPR unit, and the larger box corresponds to the second and third TPR units. FD67DV is a double-point mutation generated in the PPIase domain of FKBP51 only. A set of truncation mutants for both FKBP51 and FKBP52 was prepared; only the FKBP52 mutants are named on the left, but the corresponding FKBP51 mutants are each shorter by two amino acids. Two sets of chimeric swap mutants were generated. All were analyzed, but only those chimeras used in Figs. 5–7GoGoGo are named. The first part of the name (52 or 51) indicates the host sequence (FKBP52 or FKBP51, respectively), the lettered portion designates the particular region swapped, and the final number (1 or 2) indicates the source for the swapped region (FKBP51 or FKBP52, respectively).



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Figure 5. Hsp90 Binding by FKBP Truncation Mutants

A, Plasmid-based cDNAs encoding wild-type FKPB51 (wt51), FKBP52 (wt52), or truncation mutants were expressed in vitro to generate radiolabeled protein products. Aliquots of the synthesis mixtures were separated by SDS-PAGE and the dried gel autoradiographed (bottom panel); bands were quantitated by laser scanning densitometry to determine the relative specific activity of each product. Radiolabeled products were separately added to RL supplemented with an ATP-regenerating system and containing an anti-Hsp90 immunoaffinity resin. FKBP52 mutants and wt52 were included at a 5-fold higher molar amount than FKBP51 products to match the molar ratio of endogenous FKBPs in RL. After a 30-min incubation at 30 C, resin pellets were washed, bound components were extracted with SDS sample buffer, and proteins were separated by SDS-PAGE. The Coomassie-stained gel profile of total proteins is shown (top panel) along with an autoradiograph of the dried gel (middle panel). The label above each lane indicates the particular radiolabeled FKBP form included in the reaction mix. Protein bands identified on the left are Hsp90, the major associated proteins Hop and Hsp70, and antibody heavy and light chains (HC and LC). For reference, wild-type immunophilins comigrate with HC. The band just below HC in the top panel and other minor bands on the image are RL proteins that bind nonspecifically to immunoaffinity resins. The migration positions for molecular weight markers are indicated on the right. B, A similar assay was performed using a progressive series of C-terminal truncation mutants. The Coomassie-stained gel profile is shown on top with a corresponding autoradiograph below. C, An additional assay was performed in which RL mixtures contained recombinant chicken PR-A immobilized on an anti-PR immunoaffinity resin. PR complexes were allowed to assemble at 30 C for 30 min, and resin pellets were treated as in panels A and B. Bands identified in the Coomassie-stained profile are PR, the receptor-associated proteins Hsp90 and Hsp70, and the anti-PR heavy chain components (HC).

 


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Figure 6. Hsp90 Binding by Chimeras Involving the TPR Domain of FKBPs

Similar to the assays shown in Fig. 5Go, radiolabeled FKBP forms were added to RL (wt52 and all chimeras at a 5-fold molar excess over wt51). Either PR complexes or Hsp90 complexes were isolated after 30-min incubations at 30 C and analyzed by SDS-PAGE with Coomassie staining and autoradiography. A, Wild- type FKBPs, an FKBP52 chimera containing the TPR domain from FKBP51 (52T1), and the converse FKBP51 chimera (51T2) were assessed for PR association and Hsp90 binding. B, Similar to panel A, except only the second and third TPR units were exchanged.

 


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Figure 7. Mapping of FKBP51 Region Required for Preferred Association with PR Complexes

PR complexes were assembled in vitro in the presence of radiolabeled FKBP forms. Nonmutated forms of FKBP51 (wt51) and FKPB52 (wt52) were included in PR assembly reactions at a 1:5 molar ratio to reflect relative endogenous levels of FKBPs in RL. Labeled chimeric proteins were included at the same molar concentration as wt52. Coomassie-stained gel patterns are shown in upper panels and autoradiographs of the dried gels in lower panels. A, PR association of chimeras in which the C-terminal 67 amino acids of FKBP52 are replaced by the sequence from FKBP51 (52C1) or in which the converse switch was made (51C2). B, PR association of a chimera in which the final 53-amino acid variable region of FKBP52 is replaced by the corresponding region of FKBP51 (52V1). C, PR association of an FKBP52 chimera (52Va1) in which 29 amino acids encompassing the end of the CaMBC and beginning of the variable region are replaced by the corresponding sequence from FKBP51.

 
PPIase Activity Not Required for FKBP51 Protein Interactions
It has been difficult to demonstrate the physiological importance of PPIase activity of immunophilins. FK506 and similar drugs that bind the PPIase active site in FKBPs and inhibit their enzymatic activity have been used experimentally, but it is not clear that FK506 can freely access FKBP51 while it is in a complex with Hsp90 (10). As an alternative means to disrupt FKBP51’s PPIase activity and to avoid potential artifacts associated with FK506, the double-point mutant FD67DV was prepared. Codons were altered to replace residues Phe-67 and Asp-68 in the PPIase domain with Asp and Val, respectively. These are highly conserved residues throughout the FKBP family, and the corresponding positions in FKBP12 are known to participate in FK506 binding (31) and are required for PPIase activity (32, 33).

Purified, recombinant wild-type FKBP51 (wt51) and mutant FD67DV were tested in a PPIase assay, and enzymatic activity of the mutant was reduced by more than 90% relative to wt51 (Fig. 4AGo). However, radiolabeled wt51 and FD67DV associated equally with PR complexes during cell-free assembly (Fig. 4BGo). Thus, PPIase activity does not appear to be important for FKBP51 assembly or preferred association with PR complexes.



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Figure 4. Role of PPIase Activity in FKBP51 Association with PR

Two conserved amino acids that are required for PPIase activity in FKBP12 were mutated in FKBP51 (FD67DV). A, Purified recombinant forms of wild-type FKBP51 (wt51) and mutant FD67DV were purified and compared in a colorimetric assay for PPIase activity. B, Radiolabeled wt51 and FD67DV were compared for their abilities to enter PR complexes. Plasmids encoding wt51 and FD67DV were expressed in vitro in the presence of [35S]methionine, and the level of incorporated radioactivity in each synthesis mixture was quantitated. Equimolar amounts of each radiolabeled product were included in cell-free PR assembly reactions. The Coomassie-stained gel pattern of PR complexes is shown in the upper panel. Bands representing PR and the PR-associated proteins Hsp90, Hsp70, and Hop are identified on the left. Also indicated are the heavy chain (HC) bands of antibody used to immunoadsorb PR. An autoradiograph of the dried gel (lower panel) reveals radiolabeled FKBP51 forms associated with PR complexes.

 
Hsp90 Binding and PR Association by FKBP Truncation Mutants
For the data in Fig. 5Go, cDNAs encoding truncated protein products were expressed in vitro to generate radiolabeled protein products, and these products were compared for their abilities to coprecipitate with Hsp90 or to associate with PR complexes. Truncations of the entire sequences upstream (51/270-C and 52/272-C) or downstream (51/N-389 or 52/N-391) from the TPR domain were examined first. Equimolar amounts of the radiolabeled wild-type or mutant proteins (Fig. 5AGo, gel autoradiograph in the bottom panel) were added to RL, and the mixtures were immunoprecipitated with the anti-Hsp90 antibody H90–10. The 51/270-C lane contains two radiolabeled forms consistent with initiation of cell-free translation from both Met270 and the internal Met285.

The protein profiles of Hsp90 immunoprecipitates from RL mixtures were visualized on a Coomassie-stained gel (top panel). Note that similar levels of Hsp90 and coprecipitating rabbit proteins were obtained for each sample. Bands representing Hsp90 and the major associated proteins Hop and Hsp70 are identified on the left along with the H90–10 heavy and light chains (HC and LC). The dried gel was autoradiographed to reveal any radiolabeled FKBP forms coprecipitating with Hsp90 (middle panel). Only the full-length FKBPs coprecipitated with Hsp90, suggesting that sequences both upstream and downstream of the TPR domain are necessary for Hsp90 binding. However, the conformational level at which the deleted sequences are needed for TPR binding to Hsp90 is not immediately apparent from these large truncations.

A similar assay was performed for association of FKBP forms with PR; as with Hsp90 complexes, only the full-length FKBPs were recovered in PR complexes (results not shown). The concordance of Hsp90 and PR coprecipitation patterns is not surprising here since the FKBPs, at least initially, indirectly associate with PR through their binding to Hsp90.

The gel and autoradiograph images shown in Fig. 5Go, B and C, and all remaining figures were selected to illustrate relative binding levels of various FKBP mutant forms. Similar to Fig. 5AGo (bottom panel), in each set of analyses the specific activity of radiolabeled mutant forms was determined, and molar amounts of radiolabeled mutant forms added to assembly mixtures were adjusted as indicated in the figure legends. Furthermore, all relative binding results illustrated are consistent with at least two additional replicate experiments.

FKBP mutants, progressively truncated from the C terminus toward the TPR domain, were tested for Hsp90 binding and PR association. FKBP52 binding to Hsp90 (Fig. 5BGo) was maintained when 43 amino acids were truncated from the C terminus (52/N-416) but not when an additional 25 amino acids, including the CaMBC, were removed (Fig. 5AGo, 52/N-391). FKBP51 differs from FKBP52, since Hsp90 binding requires sequences beyond the CaMBC. Mutant 51/N-414 failed to bind Hsp90, contrasting with the retention of binding by the corresponding FKBP52 mutant (52/N-416); however, mutant 51/N-431 retains Hsp90 binding.

Qualitatively, association of FKBP truncation mutants with PR (Fig. 5CGo) followed a similar pattern as that seen for Hsp90 binding. 52/N-416 was recovered in PR complexes, although at a lower level than wt52 or mutants 52/N-433 and 52/N-448 having lesser truncations. Recovery of FKBP51 mutants was either at levels equivalent to wt 51 (51/N-446 and 51/N-431) or, in agreement with loss of Hsp90 binding, was absent (51/N-414). The quantitative differences in PR-associated proteins were consistently observed in two additional assays.

FKBP Chimeras Involving the TPR Domain
The entire TPR domain, including the first TPR unit, a non-TPR intervening sequence, and the final two TPR units in tandem, was swapped between FKBP51 and FKBP52 (Fig. 6AGo). As in Fig. 5AGo, bottom panel, radiolabeled wild-type and chimeric constructs were generated and examined by SDS-PAGE and autoradiography for expression levels and specific activity (not shown). In all cases, expression levels were similar. FKBP51 containing the TPR domain from FKBP52 (51T2) displayed preferential association with PR complexes similar to wt51 (left-hand panel). Unexpectedly, however, the corresponding FKBP52 chimera containing the TPR from FKBP51 (52T1) failed to associate with PR, even though this chimera was present in the mixture at a level equivalent to wt52 or 51T2. Upon examination of Hsp90 binding by the chimeras (right-hand panel), it was found that 51T2 bound similar to wild-type FKBPs while 52T1 failed to bind Hsp90, thus accounting for 52T1’s absence in PR complexes.

To more closely identify sequences within the TPR domain of FKBP51 responsible for loss of Hsp90 binding in chimeric FKBP52, chimeras between the less highly conserved intervening sequences were tested, but no changes in Hsp90 binding or PR association were observed (results not shown). Similarly, the first TPR unit was swapped, but again no changes in protein interactions were observed. Finally, the second and third tandem TPR units were swapped, and the effects seen with complete TPR swaps were replicated (Fig. 5BGo). Thus, it appears that some feature localized to the second and third TPR units of FKBP51 requires an additional FKBP51 sequence for Hsp90 binding. FKBP52 TPR units apparently do not require this additional sequence since they function equally well in the FKBP52 or FKBP51 background (wt52 and 51Tbc2). FKBP51 chimeras were generated in which a comprehensive set of FKBP52 sequences upstream or downstream from the TPR were swapped into FKBP51. These were then tested for coprecipitation with Hsp90. However, none of these chimeras displayed the loss of Hsp90 binding that would be expected if a required sequence in FKBP51 had been replaced (results not shown). Additional studies will be needed to understand the context dependence of FKBP51’s TPR domain.

FKBP Chimeras Involving the C-Terminal Region
Chimeras swapping FKBP sequences from the end of the TPR domain down to the C terminus were tested for Hsp90 binding and PR association. No differences were found between chimeras and wild type in binding to Hsp90 (results not shown), but quantitative differences were noted in PR association (Fig. 7AGo). The C-terminal 70 amino acids of FKBP51 confer preferred PR association to FKBP52 (52C1); conversely, the corresponding region of FKBP52 removes preferred association from FKBP51 (51C2).

This region was subdivided to resolve sequences within the C terminus of FKBP51 responsible for preferred PR association. The exchanged region in 52V1 (Fig. 7BGo) lacks the CaMBC but contains the more variable C-terminal 40 amino acids. Preferred PR association followed this fragment. This region was further subdivided such that chimera 52Va1 (Fig. 7CGo) contained only amino acids 404–433 of FKBP51. Preferred PR association was observed with this chimera.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Three Hsp90-binding immunophilins, FKBP51, FKBP52, and Cyp40, have been identified in steroid receptor complexes. FKBP51, when compared with FKBP52 and Cyp40 in cell-free assembly assays, displays a striking preference for associating with PR and GR complexes, though not ER complexes (Fig. 2Go). The results of an in vitro PR assembly time course analysis (Fig. 1Go) suggests that the less abundant FKBP51-Hsp90 complex has a competitive advantage in PR associations over more abundant FKBP52- or Cyp40-Hsp90 complexes. To aid in mapping the sequences in FKBP51 responsible for its preferred association with PR complexes, a variety of FKBP mutants were generated and tested for Hsp90 binding and PR association. The results from all tests, those illustrated in Figs. 4–7GoGoGoGo as well as others, are summarized in Fig. 8AGo. Two conclusions from this study are that the TPR domain of FKBP51 is context-sensitive for Hsp90 binding (Fig. 8BGo, region 1) and that preferred PR binding maps to a variable sequence adjacent to the CaMBC of FKBP51 (region 2).



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Figure 8. Summary of Hsp90-Binding and PR Association Results

A, Binding of each FKBP51 and FKBP52 construct to Hsp90 is scored with a plus sign to denote detectable binding or a minus sign if no binding was observed. PR association is scored with an asterisk to denote preferred association, a plus sign for nonpreferred binding, or a minus sign if no interactions were observed. B, Illustration of binding properties mapped to the C-terminal region of FKBP51.

 
Preferred Association of FKBP51 with PR
In its preferred association with PR complexes, FKBP51 may convert Hsp90 into a form that is competitively favored in PR complexes. Alternatively, once FKBP51 indirectly enters PR complexes through its association with Hsp90, direct contacts may be established between FKBP51 and PR that help stabilize its association with the receptor complex. The results presented here do not resolve these possibilities, but they exclude the potential role of several FKBP51 features while highlighting a region of previously unrecognized significance. The PPIase activity of FKBP51 would appear to play little role in its preferential association with PR, since the PPIase-negative mutant FD67DV retains normal association with PR complexes (Fig. 4Go). The putative NLS-binding sequence was swapped between FKBP51 and FKBP52, but no difference was noted in the in vitro assembly of chimeras with PR (not shown). It is interesting, however, that FKBP51 in comparison with FKBP52 has two less glutamic acid residues and a threonine to phenylalanine substitution, changes that would reasonably be expected to alter any NLS-binding activity. Perhaps functional differences related to this sequence would be noted in the cellular context, or perhaps this sequence is not an NLS-binding site in either FKBP.

Since Hsp90 binding is a necessary characteristic for immunophilins to enter receptor complexes, the TPR/Hsp90-binding domain might be expected to alter interactions of the immunophilins with steroid receptors. In one sense this is true, as will be discussed below in relation to Hsp90 binding, but in the sense of conferring FKBP51’s preferred association with PR complexes, this is not the case. As shown in Fig. 6Go, replacing FKBP51’s TPR segments with those from FKBP52 does not alter preferred PR association.

Finally, differential interactions of FKBPs with calmodulin through their CaMBCs, which are similar but nonidentical, might influence PR associations. Again, the results in Fig. 7Go argue against a role for this sequence in preferred association. In fact, we have reported earlier (34) and have found through additional unpublished experiments that the assembly of PR complexes in vitro is no different when performed in an excess of Ca2+ or in the presence of the specific Ca2+ chelator EGTA. Some reports (35, 36) have suggested a role for calmodulin in GR function in vivo, but the exact mechanism for calmodulin action has not been resolved.

A sequence sufficient for conferring preferred association with PR complexes maps to the 15–20 amino acid region of FKBP51 immediately downstream from the CaMBC (Fig. 7Go). There is little similarity between FKBP51 and FKBP52 in this region, and it is not clear how this region influences immunophilin·receptor associations.

Hsp90 Binding by FKBPs
Previous studies on FKBP52 (37) and Cyp40 (38, 39) first identified the TPR domain of these immunophilins as required for Hsp90 binding. The present studies support the role of FKBP TPR domains in Hsp90 binding but extend the requirement to sequences outside the TPR. FKBP truncation mutants lacking sequences either up- or downstream from the TPR domain failed to bind Hsp90 (Fig. 5AGo). Upstream sequences required for Hsp90 binding were not further analyzed, but downstream sequences were. FKBP52 and FKBP51 differed in that Hsp90 binding was retained in an FKBP52 truncation ending with the CaMBC site while FKBP51 binding to Hsp90 was only observed when an additional 20 amino acids were present (Fig. 5BGo). Perhaps not coincidentally, these additional amino acids of FKBP51 colocalize with the sequence that confers preferential association with PR complexes (Fig. 7CGo).

A more striking difference in the FKBPs was observed with chimeras involving the TPR domain (Fig. 6Go). In FKBP52 chimeras containing the entire TPR domain or only the second and third repeats of FKBP51, no Hsp90 binding was observed. Interestingly, though, the converse FKBP51 chimeras retained Hsp90 binding. Whether the behavioral difference in FKBP51’s TPR resides strictly within its latter TPR units or also involves extraneous regions of FKBP51 has not been resolved, but the distinction in the corresponding TPR regions suggest structural differences in the two FKBPs that could relate to distinctive interactions with Hsp90. In a recent study comparing immunophilin binding by an assortment of Hsp90 mutants, several Hsp90 mutants, either deletions or a point mutation in the C-terminal half of Hsp90, could distinguish the two FKBPs (29). Thus, while the immunophilins bind competitively to Hsp90 (25, 40), their interactions with Hsp90 are nonidentical.

Implications for Distinct Immunophilin Functions
Despite the overall similarity of FKBP51 and FKBP52, we have observed in previous studies (10, 25, 26, 27, 29) and in the present study that the two immunophilins interact with Hsp90 and with Hsp90-targeted steroid receptors in distinctive manners. Collectively, these observations make a compelling case for FKBP51 and FKBP52 having functional distinctions. If so, then steroid receptor complexes containing FKBP51 as opposed to FKBP52 may function differently. Suggestive biological evidence for this comes from the altered ratio of FKBPs observed in glucocorticoid-resistant squirrel monkey lymphoblasts (24), and studies are currently underway to directly test the causal relationship between elevated FKBP51/depressed FKBP52 levels in these cells and reduced hormone binding affinity by GR.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Assembly Time Course for PR Complexes
In vitro expression plasmids for PR-associated proteins (chicken Hsp90-{alpha}, rat Hsc70, human Hop, Hip, FKBP51, FKBP52, and Cyp40) were prepared and used in a combined transcription/translation system (TnT Lysate, Promega, Madison, WI) to generate radiolabeled protein products as described in previous publications (25, 28, 29, 30). The radiolabeled products were combined in two ways with normal RL (1:1 lysate, Green Hectares, Oregon, WI) supplemented with an ATP-regenerating system. One mixture contained radiolabeled Hsp90, Hsp70, Hop, and Hip, and the other contained radiolabeled Hsp90, FKBP51, FKBP52, and Cyp40. These mixtures (each 2 ml total) were used for the cell-free assembly of PR complexes as described previously (2) with some modifications. Briefly, recombinant chicken PR-A (~10 µg) was immunoaffinity purified from Sf9 cell extracts using monoclonal antibody PR22 adsorbed to protein G-Sepharose (Pharmacia, Piscataway, NJ). The PR-resin pellet was divided equally, and RL assembly mixtures (prewarmed) were added to the pellets and incubated at 30 C. Aliquots (200 µl, equivalent to approximately 0.5 µg PR) were removed from the assembly mixes at 1, 2, 3, 4, 5, 7, 10, 15, 30, and 60 min after initiation of assembly. Each aliquot was immediately quenched in 1 ml ice-cold wash buffer (WB; 20 mM Tris, pH 7.4, 50 mM NaCl, 0.5% Tween 20) to inhibit further assembly reactions. Resin pellets were washed four times in cold WB and extracted with SDS sample buffer. Proteins were separated by SDS-PAGE and visualized by Coomassie blue-staining and autoradiography of the dried gel. Bands on the stained gel and x-ray film were quantitated by laser scanning densitometry (Molecular Dynamics, Sunnyvale, CA), and the absorbance values for radiolabeled bands were normalized to the absorbance of stained PR-A in each sample.

Two-Dimensional Gel Analysis of Steroid Receptor Complexes
Recombinant chicken PR-A and human ER-{alpha} were immunoaffinity purified from Sf9 insect cell extracts using monoclonal antibodies PR22 and Mab-17, respectively. GR was immunoaffinity purified from L-cell extracts using monoclonal antibody BuGR-2 (Affinity Bioreagents, Golden, CO). To assemble receptor complexes, approximately 0.5 µg of each receptor (adsorbed to a 10-µl pellet of protein G-Sepharose and 10 µg antibody) was incubated with 500 µl RL plus an ATP-regenerating system at 30 C for 45 min. Resin pellets were washed four times in WB and extracted in isoelectric focusing (IEF) sample solution. Proteins were separated in IEF dimension with ampholines in the pH 3.5–10 range (Pharmacia). The second dimension SDS-PAGE gels were silver stained to visualize protein spots.

General Approach to Production and in Vitro Expression of Mutant cDNAs
Plasmids containing cDNAs encoding human FKBP51 and FKBP52 (25) were used to produce more than 30 mutant and chimeric FKBP cDNAs for these studies. Details on the generation of a few mutant plasmids are given below. For other mutants, the sequences of oligonucleotide mutagenic primers, conditions used for each mutagenesis reaction, and procedures for subcloning of cDNA fragments will be provided on request. In general, site-directed mutagenesis (QuickChange kit, Stratagene, La Jolla, CA) was used to introduce: 1) stop codons for C-terminal truncation mutants, 2) altered codons for point mutants, and 3) suitable restriction enzyme sites to facilitate creation of chimeric FKBP cDNAs. Sequence changes in all mutated plasmids were confirmed by automated sequencing.

Each of the plasmids was expressed in vitro by combined transcription/translation (TnT lysate, Promega) in the presence of [35S]methionine (DuPont/NEN, Boston, MA; specific activity, 1200 Ci/mmol). The synthesis of radiolabeled protein products was monitored by separation of 2 µl of each synthesis mixture by SDS-PAGE followed by autoradiography of the dried gel. Bands on x-ray film were quantitated by laser scanning densitometry (Molecular Dynamics) to determine relative levels of incorporated radioactivity.

Preparation of PPIase-Deficient FKBP51 Mutant
FKBP51/pET30, a bacterial expression plasmid that codes for human FKBP51 with an N-terminal polyhistidine fusion (25), was mutated at two adjacent, highly conserved codons in the PPIase domain. Using site-directed mutagenesis, codon Phe-67 was changed to Asp, and Asp-68 was changed to Val; the alterations were verified by automated sequencing of the mutant cDNA. Mutant and wild-type plasmids were expressed in bacteria, recombinant proteins (FD67DV and wtFKBP51) were purified from cell extracts, and proteins were assayed for PPIase activity, as previously described (25).

Preparation of FKBP51/FKPBP52 Chimeras
The in vitro expression plasmid pSPUTK (Stratagene), containing a cDNA insert for either human FKBP51 or human FKBP52, was used for the preparation of chimeric cDNAs. Plasmids encoding chimeric proteins (51C2 and 52C1) in which the C-terminal 66 amino acids of each protein is swapped were generated by first using site-directed mutagenesis to introduce a PvuII site overlapping codon 391 of FKBP51 cDNA. Taking advantage of the unique PvuII site at the corresponding position (codon 393) in FKBP52’s cDNA and a pSPUTK NheI site upstream of the insertion site for FKBP cDNAs, plasmids were double-digested with NheI and PvuII. After separation of digestion products on agarose gels, the NheI/PvuII fragments were religated to the alternate plasmid-containing fragments.

Plasmids encoding 51T2 and 52T1, chimeric proteins in which the TPR domains have been swapped, were generated as follows. An AccI restriction enzyme site was introduced by site-directed mutagenesis at codon 279 of wtFKBP52; this site corresponds to an endogenous AccI site in wtFKBP51. Using the FKBP51 plasmid with a PvuII site introduced at codon 391, both plasmids were double-digested with AccI and PvuII; the resulting fragments were isolated from agarose gels and cross-ligated to produce cDNAs encoding the chimeric proteins.

A plasmid encoding 52V1, an FKBP52 with the C-terminal 53 amino acids of FKBP51, was produced by taking advantage of unique SmaI(Arg-406) and SapI (Glu-443) restriction sites in wtFKBP52 cDNA and a unique SapI site in the 3'-untranslated region of FKBP51 cDNA. A PCR product was generated using a high-fidelity polymerase (Deep Vent Exo+, New England Biolabs, Beverly, MA) wtFKBP51 cDNA as template, a forward primer (5'-TATACCCGGGACCGCAGGAT-ATACGC) introducing a SmaI site at codon 404, and a reverse primer (5'-ATATGCTCTTCTG-CTTCCAGAATCACATAGC) overlapping the SapI site. The FKBP51 PCR product and the FKBP52 plasmid were both digested with SmaI and SapI, and the appropriate gel bands were religated.

Plasmids encoding chimeric proteins 51Tbc2 and 52Tbc1, in which the second and third TPR units are swapped between FKBPs, were generated from the earlier chimeric constructs 51C2 and 52C1. The latter plasmids were digested with NheI, a pSPUTK site upstream from the insertion site, and MscI, a site within the coding region for the second TPR unit in both FKBPs. The digestion products were separated by gel electrophoresis, and the NheI/MscI fragments were religated to the alternate plasmid-containing fragments.

Interactions of FKBP Forms with PR and Hsp90
PR and Hsp90 complexes were assembled in vitro using RL supplemented with radiolabeled FKBP forms. In some cases, equimolar amounts of FKBP forms were included; in others, FKBP52 forms were included at a 5-fold molar excess over FKBP51 forms to reflect the ratio of endogenous FKBPs in RL. Relative molar quantities were estimated after quantification of incorporated radioactivity in each synthesis mixture with adjustments for the methionine content of each product.

Recombinant cPR-A immobilized on immunoaffinity resin was added to RL mixtures and incubated at 30 C for 30 min. Each sample contained 0.5 µg PR protein on a 10-µl resin pellet and 300 µl RL mixture containing an ATP-regenerating system and one of the radiolabeled FKBP forms. Resin pellets were washed four times in cold WB and extracted with SDS sample buffer. Proteins were separated by SDS-PAGE and visualized by Coomassie staining and autoradiography of the dried gel.

Hsp90 complexes were similarly analyzed. The anti-Hsp90 monoclonal antibody H90–10 was preadsorbed to protein A-Sepharose (Pharmacia), and resin was aliquoted into separate tubes (10 µg antibody on a 10-µl resin pellet per tube). Each sample was incubated with 100 µl RL mixture before gel separations and analysis.


    ACKNOWLEDGMENTS
 
Antibodies were generously provided by Richard Miksicek (Mab-17) and David Toft (H90–10).


    FOOTNOTES
 
Address requests for reprints to: David F. Smith, Department of Pharmacology, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-6260.

This work was supported by NIH Grant R01–48218.

Received for publication October 24, 1997. Revision received December 9, 1997. Accepted for publication December 11, 1997.


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 DISCUSSION
 MATERIALS AND METHODS
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