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Heather L. Tardif,1
Martin Hessling,2
Johannes Buchner,2 and
David F. Smith1*
Mayo Clinic Arizona, Scottsdale, Arizona 85259,1 Institut fur Organische Chemie und Biochemie, Technische Universitat Munchen, 85747 Garching, Germany2
Received 4 June 2007/ Returned for modification 9 July 2007/ Accepted 1 October 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The molecular mechanism by which either FKBP51 or FKBP52 alters receptor function has not been clearly defined. Both proteins consist of three functional domains (reviewed in reference 28): a C-terminal tetratricopeptide repeat (TPR) domain that contains the Hsp90 binding site, an N-terminal PPIase domain characteristic of FKBP family members, and an interceding FKBP-related domain that lacks PPIase activity. Three-dimensional crystallographic structures (27, 35) suggest very similar conformations within domains, although domain-domain orientations may differ somewhat between the two proteins. FKBP52 and FKBP51 share approximately 70% amino acid sequence similarity—greater within individual functional domains—and have similar enzymatic properties (21).
In a series of experiments utilizing a Saccharomyces cerevisiae model to study FKBP function and steroid signaling (25), FKBP51 did not on its own inhibit GR function in the absence of FKBP52; however, FKBP51 reduced the enhancement of GR function mediated by FKBP52. Additional studies in mammalian cells are consistent with an ability of FKBP51 to antagonize the actions of FKBP52 (5, 31, 34). FKBP52-dependent enhancement of receptor function requires binding of FKBP52 to Hsp90 and is targeted to the receptor ligand binding domain (25), which is the site for direct binding of Hsp90 in receptor heterocomplexes. FKBP51 and FKBP52 compete for a common binding site on Hsp90, and FKBP binding to Hsp90 can occur independently of the receptor (20). However, FKBP51 is recovered preferentially in some receptor complexes (1), and its association is sensitive to hormone binding (6, 29), suggesting that FKBPs somehow detect the nature of the receptor that is associated with Hsp90.
Observations that FKBP52, but not FKBP51, binds to the microtubular motor protein dynein have been made (6, 26, 34), and a model has been proposed in which FKBP52 assists in directing GR to the nucleus subsequent to hormone binding (reviewed in reference 22). Based on the highly dynamic interaction of FKBP cochaperones with receptor complexes at physiological temperatures (20) and a recent observation that FKBP52 directly binds tubulin and inhibits microtubule assembly (2), there are several issues that remain to be resolved in the receptor transport model. Moreover, the transport model would appear to be less relevant to AR or PR, whose subcellular distributions differ from GR but whose enhancement by FKBP52 is similar (4, 31). Finally, genetic approaches in the yeast model clearly dissociate dynein dependence from FKBP52-mediated enhancement of receptor function (25). Nonetheless, FKBP52 interaction with microtubule components might be physiologically relevant to mammalian sperm flagellar function, although in an AR-independent manner (13).
Does the PPIase activity of FKBP52 play a role in receptor enhancement, perhaps by altering receptor conformation and function through isomerization of receptor prolines? Despite the fact that FKBP51 and FKBP52 have similar enzymatic activities toward model peptide substrates (21), which would seem at odds with their distinct effects on receptor activity, three lines of experimental evidence support a role for FKBP52 PPIase (25). First, deletion of the PPIase domain eliminates FKBP52's ability to enhance receptor function. Next, the drug FK506, which binds the PPIase active site and inhibits enzymatic activity, inhibits FKBP52-dependent potentiation of receptor function in yeast. Third, a double point mutation in the PPIase domain that is known to reduce PPIase activity also inhibits FKBP52-dependent potentiation. That the PPIase domain is important seems clear. On the other hand, two considerations potentially limit the certitude of current evidence supporting the need for enzymatic activity. First, FK506 is a relatively large molecule and a significant portion of the drug is excluded from the PPIase pocket (33); this excluded portion could sterically hinder interactions involving the PPIase domain independent of enzymatic activity. Second, an independent mutagenic study (12) of a related FKBP family member showed that several point mutations of enzymatically critical residues within the PPIase pocket failed to disrupt binding of the FKBP to its partner, whereas a double mutation at the corresponding residues targeted in FKBP52 did disrupt binding. Therefore, further analysis of the FKBP52 PPIase role in receptor enhancement is warranted.
To complement approaches employing directed mutagenesis of FKBP52 to disrupt function, we randomly mutagenized FKBP51 in hopes of generating gain-of-function mutants that would convert FKBP51 from an inhibitor to an activator of receptor. The characterization of such mutants could provide alternative evidence of the mechanism by which FKBP52 acts and help to understand the relevant sequence differences that distinguish the opposing actions of FKBP52 and FKBP51.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ss-26X [reporter], pG/N795 [GR expression], and FKBP51, FKBP52 and FKBP51/52 chimera expression plasmids) have been previously described (25). Human AR and the mutant AR-P723S were cloned, respectively, into p424GPD and p424TEF (19). Mutations were introduced by site-directed mutagenesis (QuikChange II XL; Stratagene, San Diego, CA) into the wild-type human FKBP51 or FKBP52 gene cloned into p423GPD. Potentiation by these mutant FKBPs was measured in strain W303a transformed with the pUCss-26X reporter plasmid and a GR or AR expression plasmid. Dihydrotestosterone (DHT) (15 nM) was used in the AR hormone induction assays, and deoxycorticosterone (DOC) (10 nM) was used in the GR assays.
Selection for FKBP51 potentiation mutants and analysis.
The mutant FKBP51 library was made using error-prone PCR (GeneMorphII; Stratagene, CA) using the manufacturer's recommended conditions for high-frequency mutagenesis (5 ng target DNA per reaction). The template used was p425GPD-hFKBP51, and the primer binding sites were approximately 100 bases outside of the gene borders. The PCR product (400 ng, purified by agarose gel electrophoresis) and p424GPD vector (200 ng, linearized with EcoRI and SalI) were cotransformed into the selection strain YNK435 (MATa ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 pdr5::GT3Z his3::GT3H; gift of Natasha Kralli, Scripps Institute) containing the plasmid p425TEF-AR-P723S. FKBP51 potentiation mutants were selected on plates containing synthetic complete medium lacking tryptophan, leucine, and histidine (SC-WLH) supplemented with 10 nM DHT and 5 mM 3-amino-1,2,4-triazole. The concentrations of each were adjusted to maximize the growth differential between YNK435 strains expressing either FKBP51 or FKBP52. The colonies appearing after about 1 week of incubation at 30°C were purified on SC-WL plates. The mutant phenotype (potentiation of hormone signaling) was confirmed by assaying hormone-dependent expression of the ß-galactosidase reporter in the selection strain. Isolates that showed increases in both hormone-dependent growth and ß-galactosidase expression were confirmed by transferring the mutated FKBP51 gene into a clean genetic background. This was done by extracting the mutated FKBP51 gene using PCR amplification (LA Taq polymerase; Takara Shiga, Japan) from yeast lysates (Whole Cell Yeast PCR kit; MP Biochemicals, Irvine, CA). The same promoter/terminator primer pair used to construct the original mutant FKBP51 library was used in this PCR. The PCR product, purified by agarose gel electrophoresis, was cotransformed with the p424GPD vector (linearized with EcoRI and SalI) into strain W303a expressing AR-P723S and containing a plasmid-encoded hormone-responsive ß-galactosidase gene (pUCss-26X). Transformants were assayed for potentiation of hormone signaling as described above. Those mutated FKBP51 genes that retained the potentiation properties in this clean genetic background were then sequenced, and interesting mutations were put in individually and in combination into the FKBP51 gene by site-directed mutagenesis.
Hormone-induced gene expression in mouse cells. Immortalized mouse embryonic fibroblasts (MEF) derived from FKBP52 knockout mice (31) were cultured in 5% CO2 in minimum essential medium (Eagle) with Earle's balanced salts supplemented with 10% charcoal-stripped fetal bovine serum. Cells were cultured in 12-well plates until they were 80% confluent, and then they were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The transfection mix contained the following (per well): 25 ng of a constitutive ß-galactosidase-expressing plasmid (for transfection normalization); 200 ng of the pCI-neo plasmid (Promega, Madison, WI) expressing human AR; 300 ng of pCI-neo expressing FKBP51, -52, or variants; and 500 ng of a plasmid expressing firefly luciferase reporter driven by the androgen-dependent probasin promoter. Twenty-four hours after transfection, the growth medium was replaced with medium containing 30 pM DHT. Approximately 16 h later, the cells were lysed in 200 µl M-PER mammalian protein extraction reagent (Pierce, Rockford, IL) as recommended by the manufacturer. Luciferase expression was assayed by adding 100 µl of cell lysate to 100 µl of luciferase assay reagent (Promega, Madison, WI). ß-Galactosidase expression was assayed by adding 10 µl of lysate to 100 µl of Gal Screen reagent (Tropix, Bedford, MA). The 96-well plates were incubated at room temperature until maximum luminescence developed (about 5 min for the luciferase assay and 2 h for the ß-galactosidase assay), and then the luminescence was measured. The normalized level of reporter expression (luciferase RLU/ß-galactosidase RLU [RLU is relative light units]) reported is the average (± standard deviation [SD]) of at least three separate samples.
Hormone binding measurements in mouse cells. The 52KO MEF cell line was grown and transfected as described above in the hormone induction assay. Cells were seeded in six-well plates and cotransfected with the following constitutive expression plasmids: AR plasmid or empty vector (1 µg DNA/well), an FKBP plasmid expressing either wild-type or mutant FKBP (0.5 µg/well), and ß-galactosidase plasmid (25 ng/well) for transfection control. Three replicate wells at each hormone concentration contained AR plasmid, and one well at each hormone concentration contained empty vector to determine nonspecific hormone binding. After 2 days, medium was removed and replaced with fresh medium containing [3H]DHT (110 Ci/mmol; NEN, Waltham, MA) at final concentrations from 0 to 10 nM DHT. After 90 min, medium was removed, cells were washed twice in phosphate-buffered saline, and cells were lysed with a commercial lysis reagent (M-PER; Pierce, Rockford, IL). ß-Galactosidase activity was measured in an aliquot of each cell lysate, and radioactivity in the remaining lysate was measured by liquid scintillation counting. Radioactive counts were normalized to the corresponding ß-galactosidase activity in each sample. Specific hormone binding at each concentration of DHT was determined by subtracting radioactivity in the nonspecific sample (empty vector) from each of the three replicates containing AR and averaging the resulting values. The maximum specific binding density and dissociation constant (KD) were calculated using Prism software (GraphPad Software, San Diego, CA).
Western immunoblots. To confirm equivalent expression of each FKBP form, Western immunoblots were performed on extracts from cells used in the induction assays. Yeast extracts were prepared either with glass beads as previously described (25) or with Y-PER yeast protein extraction reagent (Pierce, Rockford, IL) as recommended by the manufacturer. Mammalian cell lysates were prepared with M-PER as described above. The following mouse monoclonal antibodies were used: anti-FKBP51 FF1 (epitope in FK1 domain), Hi51b (epitope in TPR domain), and anti-FKBP52 Hi52d (epitope in FK1 domain). To confirm sample loading, antibody against L3 ribosomal protein (yeast lysates) and antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (mammalian cell extracts) were used.
Protease-coupled PPIase assay. Proteins were expressed in the Escherichia coli strain BL21. Protein purification and PPIase assays were performed as described earlier (21). PPIase assays were performed using a protease-coupled assay. In brief, various concentrations of the purified PPIase were combined with the synthetic peptide substrate N-succinyl-Ala-Ile-Pro-Phe-p-nitroanilide (50 µM final concentration) in 40 mM HEPES, pH 7.5, supplemented with 1 mg/ml chymotrypsin. The fraction of peptide containing a cis-proline peptide bond is cleaved by protease in a slow reaction that allows determination of the rate of proline isomerization and catalytic efficiency.
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RESULTS |
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To further examine the role of FKBP52 PPIase in potentiation, we have now tested the effects of additional point mutations in conserved residues that are generally important for enzymatic activity (Fig. 1). Unlike residues Phe-67 and Asp-68, which are positioned around the mouth of the PPIase pocket, amino acids Tyr-57, Trp-90, and Phe-130 are located within the hydrophobic PPIase pocket (Fig. 1A). As seen with corresponding mutations in other FKBP family members (reviewed in reference 10), we confirmed that individual mutation of these amino acids greatly impairs PPIase activity (Fig. 1B). To determine the effects of mutation on receptor potentiation, we first used a yeast model (Fig. 1C). Yeast strains were stably transformed with three plasmids: one expressing the steroid hormone receptor, a second containing a ß-galactosidase reporter gene transcribed from a hormone-inducible promoter, and a third expressing wild-type or mutant FKBP. As we previously reported (25), the FD67DV double mutation abolishes FKBP52-dependent potentiation of GR (Fig. 1C, left panel) and partially reduces AR potentiation (Fig. 1C, right panel). In contrast, the Y57A, F67Y, W90L, and F130Y mutations have no effect on FKBP52 potentiation of either GR or AR signaling. None of the mutations significantly altered the steady-state levels of expressed proteins as measured by Western blot analysis of yeast extracts.
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Genetic selection for FKBP51 gain-of-potentiation mutations. A possible explanation for the functional differences between the FKBPs is that FKBP51 lacks one or more residues present in FKBP52 that are critical for productive interaction with a binding partner. Here we have used yeast genetics to identify such residues by selecting for gain-of-function mutations in FKBP51 that enable it to potentiate hormone signaling in a manner similar to that of FKBP52.
In the yeast strain used for this selection (YNK435; see Materials and Methods for details), the HIS3 gene, which is required for histidine biosynthesis, is transcribed from a weak promoter having an adjacent upstream hormone response element (Fig. 2). To inhibit growth due to leaky expression of the HIS3 construct in the absence of hormone, the His3p inhibitor 3-amino-1,2,4-triazole was added to the growth medium. In strain derivatives stably expressing AR, growth in medium lacking histidine was dependent upon the ligand dihydrotestosterone, and growth dramatically increased when FKBP52 was coexpressed. To further enhance this conditional, hormone-dependent growth, our selection strain expresses AR with the P723S point mutation, originally identified in an individual with complete androgen insensitivity syndrome (7), which is hyperdependent on FKBP52 for hormone-induced activity (4). In medium supplemented with DHT, little growth was observed in yeast strains expressing FKBP51, while strains expressing FKBP52 displayed robust growth (Fig. 2A).
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We isolated numerous independently derived mutants with the expectation that key mutations would repeatedly arise. A total of 25 gain-of-function mutants were isolated from eight independent libraries; of these, 11 had mutations in one of three residues located within a 6-amino-acid stretch in the FK1 domain: A116V/T, L119P, and S124P (Fig. 3). With the exception of one A116T mutation, each was isolated multiple times from independent libraries. The three residues at positions 116, 119, and 124 are all located in a loop that extends over the PPIase pocket (Fig. 4B and C). The amino acid sequences of FKBP51 and FKBP52 diverge only at positions 119 and 124 in this loop (Fig. 4D, arrows). Interestingly, the selected FKBP51 mutations at position 119 or 124 all introduced proline residues, as found in FKBP52 and other FKBP family members.
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Previous reports have shown that the presence of FKBP51 or FKBP52 in steroid receptor complexes can alter receptor hormone binding affinity (5, 8, 25, 31). An FKBP52-mediated increase in receptor hormone binding affinity can explain, at least in part, potentiation of hormone-dependent gene transactivation. In Fig. 5C, we present hormone binding curves generated from intact 52KO MEF cells coexpressing AR and one of the wild-type or mutant FKBP forms. AR hormone binding affinity was increased approximately fivefold in cells expressing exogenous FKBP52 (KD = 1.7 ± 0.2 nM) compared to cells expressing FKBP51 (KD = 9.3 ± 0.8 nM). FKBP51-L119P increased AR hormone binding affinity (KD = 3.7 ± 0.9 nM) compared to wild-type FKBP51, and FKBP52-P119L modestly decreased affinity (KD = 2.2 ± 0.4 nM) compared to wild-type FKBP52; both changes correlate well with the effects of mutant proteins on AR-dependent gene activation (Fig. 5B). The calculated maximum specific binding density values in all cases were statistically equivalent, which is consistent with equivalent AR protein levels in various MEF extracts (Fig. 5B).
Both FKBPs have alanine in position 116. In yeast and MEF cells, the A116V mutation in wild-type FKBP51 only modestly increased AR signaling (Fig. 6). This level of potentiation is insufficient to be readily detected in our genetic screen. On the other hand, A116V in combination with L119P displays receptor potentiation fully equivalent to FKBP52. The A116V mutation was selected when either the template used for random mutagenesis already contained P119 (Fig. 3, mutants F1, G1, H1, and G2), or the A116V and L119P mutations were simultaneously introduced (mutant B2). A116V mutation of FKBP52 does not significantly increase its ability to potentiate AR, suggesting that FKBP52 is already optimized for this function.
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As noted above (Fig. 7A, right panel), sequences outside the FK1 domain appear to contribute to potentiation in 52KO MEF cells. Additional chimeras were generated to map these extraneous features to either the FK2 or TPR domain (Fig. 7B). The chimera containing the FK1 and FK2 domains of FKBP52 and the TPR of FKBP51 (52/52/51, bar 4) has activity approximately equal to that of intact FKBP52, suggesting that the FK domain region of FKBP52 is sufficient for full potentiation and that TPR domain differences are not functionally relevant to potentiation. On the other hand, the chimera combining the FK1 and TPR domains of FKBP52 with the FK2 domain of FKBP51 (52/51/52, bar 3) has somewhat greater activity than the chimera containing only the FK1 domain of FKBP52 (52/51/51, bar 2), so unique interactions between FK2 and TPR domains, as suggested by FKBP52 and FKBP51 crystal structures, might influence FK1-mediated potentiation. Activity differences in chimeric proteins cannot be attributed to different levels of protein expression (Fig. 7B, lower right panel).
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DISCUSSION |
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FKBP51, which despite its many similarities to FKBP52 does not potentiate receptor activity, was randomly mutated, and a yeast genetic screen was devised to identify FKBP51 mutants that gain potentiation activity (Fig. 2). The major class of gain-of-function mutations identified by the genetic screen (summarized in Fig. 3) localized to a surface loop of FK1 that overhangs the PPIase active site (Fig. 4). Strikingly, interconversion of a single amino acid within this loop—Pro119 in FKBP52 and Leu119 in FKBP51—can largely confer potentiation activity to FKBP51 or ablate potentiation by FKBP52 in either the yeast model or in mouse cells (Fig. 5 and 6). A second loop mutation at nearby position 124 (S124P) was identified in the yeast screen as conferring some potentiation activity to FKBP51, but this activity was evident only vis-à-vis the AR-P723S point mutant, not with wild-type AR or GR (Fig. 5). Further implicating the loop as important for potentiation activity, two point mutations of amino acid 116 (A116V or A116T) were isolated that elevated potentiation by the gain-of-function FKBP51 L119P mutant to the level normally observed with wild-type FKBP52 (Fig. 6). Since FKBP51 and FKBP52 both naturally contain alanine in loop position 116, we tested whether an FKBP52-A116V mutation would have potentiation activity exceeding that of wild-type FKBP52 (Fig. 6); as only a small increase in potentiation was observed, we conclude that FKBP52 potentiation activity is already optimal.
The FKBP51 gain-of-function mutant results highlight the importance of the PPIase loop, in particular amino acid position 119, in FKBP-dependent potentiation of AR and GR activities. FKBP51-L119P activity can be optimized by combining with a novel mutation at position 116 or combining the mutant FK1-L119P domain with the FK2 and TPR domains of FKBP52 (Fig. 7), demonstrating that FKBP52-unique sequences outside the FK1 domain—probably in FK2—normally contribute to potentiation capacity.
PPIase loop interactions. To gain some insight into the nature of potential interactions by FKBP52 FK1, we refer to observations of protein-protein interactions involving single-domain FKBP PPIases. Several FKBP-protein interactions have been shown to be independent of PPIase enzymatic activity yet still involve residues in or near the PPIase active pocket. FKBP12 and FKBP13 form FK506-dependent complexes with calcineurin (18), which inhibits calcineurin activity and forms the basis for FK506-mediated immunosuppression. Calcineurin binding involves an exposed region of FK506 and FKBP residues outside the PPIase pocket. Furthermore, FKBP12 and FKBP12.6 interactions with ryanodine receptor (36) and FKBP12 binding to the transforming growth factor ß (TGF-ß) receptor (14) do not require PPIase activity, yet in each of these cases FK506 inhibits protein interaction.
The cocrystallographic structure of FKBP12 in complex with the TGF-ß receptor (Fig. 8A) illustrates features that could be analogous to FKBP52 interactions with the steroid receptor. FKBP12 (yellow chain) binds the C-terminal domain of the TGF-ß receptor (blue chain) through FKBP loop amino acids (gray region) as well as FKBP residues surrounding the PPIase pocket. Note that FKBP12 aspartic acid 37 (red), which corresponds to D68 in the FK1 domain of FKBP52, lies along the PPIase pocket and participates in interactions with the TGF-ß receptor. By analogy, alteration of this amino acid and the adjacent phenylalanine in the FD67DV mutant of FKBP52, aside from impairing enzymatic activity, might disrupt FK1 interactions with the receptor and explain why we observe loss of GR potentiation (25) (Fig. 1C). In the loop region, mutation of FKBP12 amino acids 89 and 90 (equivalent to positions 120 and 121 in FKBP52, as shown in Fig. 4D) was shown to disrupt binding of FKBP12 to the TGF-ß receptor. Tryptophan and phenylalanine side chains from the TGF-ß receptor (Fig. 8A, blue and purple) are seen to interact with FKBP12-P88 (orange), which corresponds to FKBP52-P119. The phenylalanine side chain (purple) actually projects into a hydrophobic notch formed along the top of the loop.
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Mechanism for FKBP52-mediated potentiation of steroid receptor activity. We propose that the FKBP52 FK1 domain, via the notch formed by P119 in the overhanging loop, forms a specific contact with the receptor ligand binding domain (LBD) in the context of Hsp90 heterocomplexes and that this contact potentiates receptor response to hormone. Several lines of evidence factor into this model. First, although FKBP52 binds Hsp90 directly and this binding is required for potentiation, we do not believe the relevant FK1 contact for potentiation is with Hsp90 itself. The reason is that FKBP52 selectively potentiates the activities of AR, GR, and PR without altering the activity of estrogen receptor (25, 31) or mineralocorticoid receptor (unpublished observations). An FKBP52-induced change in Hsp90 function would seem likely to affect all Hsp90-bound steroid receptors in a similar manner. Furthermore, we have previously shown with chimeric steroid receptors (25) that FKBP52-dependent potentiation localizes to the receptor LBD. The purpose, we presume, of Hsp90 binding by FKBP52 is to properly localize and orient the FK1 domain for productive interaction with the receptor LBD. We have been unable to detect direct interaction between FKBP52 and steroid receptor in the absence of Hsp90, but others have reported such an interaction (26). FK1 interaction with the LBD is likely weak except as mediated by Hsp90.
Related to the roles of Hsp90 binding and FK1 interactions, we considered whether FKBP12, which shares a proline equivalent to P119 in the overhanging loop and other structural similarities to FKBP52-FK1, might functionally substitute for FK1. FKBP12, like FKBP52 that lacks Hsp90 binding, does not potentiate receptor activity, perhaps because FKBP12 is not recruited to the receptor complex through an association with Hsp90. To test this possibility, we generated a chimeric protein in which FKBP12 was situated in frame with FK2 and TPR domains of FKBP52, but the chimera failed to potentiate receptor activity in yeast assays (results not shown). FKBP12 shares only 50% amino acid identity with FKBP52-FK1, so there likely are distinct amino acids in FKBP52-FK1 that participate in productive interactions with the receptor.
How does FK1 interaction with the LBD enhance receptor sensitivity to hormone? FKBP52 is known to increase the hormone binding affinity of GR (5, 25) and PR (31), and in Fig. 5C we demonstrate a similar FKBP52-mediated increase in AR hormone binding affinity. There is a fivefold increase in AR affinity for DHT in the presence of FKBP52 compared to FKBP51. Furthermore, the gain-of-function mutant FKBP51-L119P stimulates a two- to threefold increase in DHT binding affinity compared to wild-type FKBP51; this increase in hormone binding affinity corresponds well with FKBP51-L119P-mediated potentiation of reporter gene expression in AR transactivation assays (Fig. 5B). Since PPIase is not critical for receptor potentiation (Fig. 1), proline isomerization of the receptor LBD is unlikely to account for an increase in hormone binding affinity. As an alternative, perhaps FK1 interaction stabilizes a dynamic LBD conformational state that otherwise is transiently favorable for hormone binding. Unfortunately, little is precisely known about the LBD conformational state(s) of steroid receptors prior to hormone binding. Another possibility we have considered entails alternative orientations by which the LBD might interact with Hsp90. Currently, there is no direct evidence that Hsp90 binds to any particular client in more than one manner, but such a possibility would not be surprising if one considers the vast array of structurally distinct client proteins—steroid receptors, serine/threonine kinases, tyrosine kinases, etc.—that are bound by Hsp90. Given the diversity of structures recognized by Hsp90, we propose that any single client, e.g., the androgen receptor, might be bound by Hsp90 in either of two, or perhaps more, orientations. If Hsp90-associated FKBP52 forms a secondary contact with the receptor LBD, this contact may select an orientation that is more favorable for hormone binding.
Implications. If the FK1 domain of FKBP52 directly contacts the receptor LBD, we anticipate that the LBD site of interaction would be unique and specific to receptors responsive to FKBP52. We are currently pursuing directed genetic approaches with the AR LBD to help with identification of this putative interaction site. One would predict that receptor mutations in or near this FK1 interaction site could occlude FK1 interaction and potentiation, thus generating a receptor that is constitutively nonresponsive to FKBP52. Alternatively, receptor mutation could favor an LBD conformation or orientation vis-à-vis Hsp90 that mimics FKBP52-induced potentiation and thus produces a receptor that constitutively responds to hormone in a potentiated manner. Our identification of an FKBP51 gain-of-function point mutant underscores the opportunity for spontaneous FKBP mutation to influence hormonal responsiveness of tissues. For example, a single base change generating an L119P mutation of FKBP51, whose expression is highly induced by androgens, progestins, or glucocorticoids (reviewed in reference 28), could produce positive feedback that renders tissues hyperresponsive to hormone. Likewise, an inactivating mutation of FKBP52 could constrain tissue responsiveness to a limiting amount of hormone.
In summary, here we have shown that a single amino acid residue at position 119 within the PPIase loop plays a significant role in determining the functional consequence of FKBPs in steroid receptor complexes. Since the PPIase loop is often involved in protein interactions by FKBP family members, proline 119 in FKBP52 appears to specify a contact site that is functionally critical for steroid receptor interaction and potentiation. FKBP51 fails to potentiate receptor activity largely due to the presence of leucine at position 119. The identification of this critical residue is an important step toward understanding the mechanism of FKBP52-mediated potentiation of steroid signaling.
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ACKNOWLEDGMENTS |
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We thank Natasha Kralli for yeast strains and advice.
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FOOTNOTES |
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Published ahead of print on 15 October 2007.
Present address: Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX 79902.
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