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
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ABSTRACT
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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 FKBP51s 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 FKBP52s 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.
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INTRODUCTION
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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 GRs
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, FKBP51s
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 FKBP51s PPIase domain is less accessible. In the
present report, we have more closely examined FKBP51s preferential
association with steroid receptor complexes and have mapped out the
region of FKBP51 responsible for its preference.
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RESULTS
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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. 1
), 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- , 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.
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As seen in the upper panel of Fig. 1
, the recoveries of
Hsp70, Hip, and Hop with PR complexes all attain a maximum in the first
510 min and then drop to lower levels. Hsp90 recovery contrasts by
reaching a sustained plateau of maximal binding in 1015 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. 1
, lower
panel). Cyp40 and FKBP52 achieve maximal binding levels over 515
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 FKBP51s binding was not
quantitated relative to FKBP52 and Cyp40. To our knowledge, GR
complexes have not been directly tested for FKBP51. In Fig. 2
, 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. 2
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.
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Mutagenic Analysis of FKBPs
FKBP51 and FKBP52 are homologous proteins with highly conserved
domain structures, as illustrated by their sequence alignment (Fig. 3A
). 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 143147) 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 FKBP542s
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 32138), and a
putative NLS-binding motif in hFKBP52 (residues 143147) 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 397413). 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.
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As diagrammed in Fig. 3B
, 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. 57

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. 5 , 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.
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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 FKBP51s 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. 4A
). However, radiolabeled wt51 and
FD67DV associated equally with PR complexes during cell-free assembly
(Fig. 4B
). 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.
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Hsp90 Binding and PR Association by FKBP Truncation Mutants
For the data in Fig. 5
, 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. 5A
, gel autoradiograph
in the bottom panel) were added to RL, and the mixtures were
immunoprecipitated with the anti-Hsp90 antibody H9010. 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 H9010 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. 5
, B and C, and all
remaining figures were selected to illustrate relative binding levels
of various FKBP mutant forms. Similar to Fig. 5A
(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. 5B
) 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. 5A
, 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. 5C
)
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. 6A
). As in Fig. 5A
, 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 52T1s 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. 5B
). 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 FKBP51s 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. 7A
).
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. 7B
) 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. 7C
) contained only amino acids 404433 of FKBP51. Preferred PR
association was observed with this chimera.
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DISCUSSION
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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. 2
). The results of an in vitro PR assembly
time course analysis (Fig. 1
) 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. 47


as well as others, are summarized in Fig. 8A
. Two conclusions from this study are
that the TPR domain of FKBP51 is context-sensitive for Hsp90 binding
(Fig. 8B
, 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. 4
). 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 FKBP51s
preferred association with PR complexes, this is not the case. As shown
in Fig. 6
, replacing FKBP51s 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. 7
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 1520 amino acid region of FKBP51 immediately
downstream from the CaMBC (Fig. 7
). 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. 5A
). 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. 5B
). Perhaps not coincidentally, these
additional amino acids of FKBP51 colocalize with the sequence that
confers preferential association with PR complexes (Fig. 7C
).
A more striking difference in the FKBPs was observed with chimeras
involving the TPR domain (Fig. 6
). 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
FKBP51s 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
|
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Assembly Time Course for PR Complexes
In vitro expression plasmids for PR-associated
proteins (chicken Hsp90-
, 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-
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 3.510 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 FKBP52s 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 H9010 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 (H9010).
 |
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 R0148218.
Received for publication October 24, 1997.
Revision received December 9, 1997.
Accepted for publication December 11, 1997.
 |
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