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J. Biol. Chem., Vol. 276, Issue 50, 47715-47724, December 14, 2001

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The Conserved Sites for the FK506-binding Proteins in Ryanodine Receptors and Inositol 1,4,5-Trisphosphate Receptors Are Structurally and Functionally Different^*

Geert Bultynck^§, Daniela Rossi^¶, Geert Callewaert, Ludwig Missiaen, Vincenzo Sorrentino^¶, Jan B. Parys, and Humbert De Smedt^**

From the  Laboratorium voor Fysiologie, K.U.Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium, the ^¶ Section of Molecular Medicine, Department of Neuroscience, University of Siena, Siena I-531000, Italy, and  DIBIT, San Raffaele Scientific Institute, I-20132 Milan, Italy

Received for publication, July 13, 2001, and in revised form, September 21, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We compared the interaction of the FK506-binding protein (FKBP) with the type 3 ryanodine receptor (RyR3) and with the type 1 and type 3 inositol 1,4,5-trisphosphate receptor (IP₃R1 and IP₃R3), using a quantitative GST-FKBP12 and GST-FKBP12.6 affinity assay. We first characterized and mapped the interaction of the FKBPs with the RyR3. GST-FKBP12 as well as GST-FKBP12.6 were able to bind ~30% of the solubilized RyR3. The interaction was completely abolished by FK506, strengthened by the addition of Mg²⁺, and weakened in the absence of Ca²⁺ but was not affected by the addition of cyclic ADP-ribose. By using proteolytic mapping and site-directed mutagenesis, we pinpointed Val²³²², located in the central modulatory domain of the RyR3, as a critical residue for the interaction of RyR3 with FKBPs. Substitution of Val²³²² for leucine (as in IP₃R1) or isoleucine (as in RyR2) decreased the binding efficiency and shifted the selectivity to FKBP12.6; substitution of Val²³²² for aspartate completely abolished the FKBP interaction. Importantly, the occurrence of the valylprolyl residue as -helix breaker was an important determinant of FKBP binding. This secondary structure is conserved among the different RyR isoforms but not in the IP₃R isoforms. A chimeric RyR3/IP₃R1, containing the core of the FKBP12-binding site of IP₃R1 in the RyR3 context, retained this secondary structure and was able to interact with FKBPs. In contrast, IP₃Rs did not interact with the FKBP isoforms. This indicates that the primary sequence in combination with the local structural environment plays an important role in targeting the FKBPs to the intracellular Ca²⁺-release channels. Structural differences in the FKBP-binding site of RyRs and IP₃Rs may contribute to the occurrence of a stable interaction between RyR isoforms and FKBPs and to the absence of such interaction with IP₃Rs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intracellular Ca²⁺ stored in the sarco/endoplasmic reticulum of cells can be released into the cytosol by two different types of intracellular Ca²⁺-release channels, the ryanodine receptor (RyR)¹ and the inositol 1,4,5-trisphosphate receptor (IP₃R); both are represented by three different isoforms (1, 2). RyR1 and RyR2 are highly expressed in skeletal and cardiac muscle cells, respectively, and play a crucial role in Ca²⁺ mobilization during excitation-contraction coupling (3, 4). RyR3 is more ubiquitously expressed, but at much lower levels, and may exert a functional role in Jurkat T cells, the central nerve system, as well as in neonatal and adult skeletal muscle contraction (5-7). The different IP₃R isoforms have a very broad expression pattern and occur in almost all cell types. They play a crucial role in many cellular processes, ranging from cell proliferation and apoptosis to gene regulation and secretion (8).

The function of these intracellular Ca²⁺-release channels is regulated by different cellular factors. One of these regulators is the group of the FK506-binding proteins (FKBP) belonging to the immunophilin family (9). FKBP12 and its analogue FKBP12.6 are tightly associated with the skeletal RyR1 and the cardiac RyR2, respectively (10-13). The interaction between FKBPs and RyR1 is very well documented and is essential for stabilization, activation, and proper functioning of these channels and for the coordinated gating of neighboring channels (12-17). The FKBP12-binding domain of RyR1 was mapped to its central regulatory domain between aa 2401-2840 (18), whereby the valylprolyl residue 2461-2462 was shown to be critical for establishing this high affinity FKBP12 interaction (19). The interaction of RyR2 with FKBP12.6 is also well documented (12, 13, 20). Recent data have clarified the role of FKBP12.6 in the regulation of RyR2 and its physiological importance in cardiac hypertrophy (21-23). Recently, it has been demonstrated that FKBPs can also interact with RyR3 (18, 24, 25), but their interaction site has not yet been determined, and the characteristics of this interaction have not been well documented.

Although the interaction of FKBP12 with RyR isoforms is generally accepted, the proposed interaction of FKBP12 with the different isoforms of the IP₃R is controversial (26). It has been postulated that FKBP12 was associated with the IP₃R1 of rat cerebellum and functioned as a stabilizing protein as well as an adaptor protein for the Ca²⁺/calmodulin-dependent phosphatase calcineurin (27, 28). The core of the FKBP12-binding site in the IP₃R1 was identified by the yeast two-hybrid technique and was limited to an 11-amino acid stretch (aa 1396-1406), containing the critical leucylprolyl residue 1400-1401 (29). The interaction between IP₃R1 and FKBP12 as observed by a yeast two-hybrid assay is compatible with the fact that the primary sequence of the FKBP12-binding site on RyR and IP₃R isoforms is quite well conserved (29). However, other studies found neither functional nor biochemical evidence for the proposed interaction of FKBP12 with different isoforms of the IP₃R (18, 30-33). Recently, it has been reported that FK506 enhanced phosphorylation of the IP₃R1 by protein kinase C via inhibition of calcineurin hereby suppressing intracellular Ca²⁺ oscillations, although no direct interaction between IP₃R1 and FKBP12 was shown in this study (34).

The aim of the present study was to investigate the characteristics of the binding of FKBP12 and of FKBP12.6 to the RyR3 and to clarify the seemingly contradictory results obtained for RyRs and IP₃Rs, although both channel families contain a conserved sequence representing a putative FKBP-binding site. For this purpose we used HEK293 and COS-1 cells heterologously expressing either wild type RyR3, mutated forms of the RyR3, and a chimeric RyR3, containing the core of the proposed FKBP12-binding site of IP₃R1. In addition, we analyzed mouse IP₃R1 and human IP₃R3, which respectively contain leucylprolyl or valylprolyl residues in the core of their putative FKBP12-binding site. The predicted secondary structure of the FKBP-binding site in the different RyR and IP₃R isoforms was compared using several secondary structure prediction methods.

Our data indicate that the primary sequence of the FKBP12-binding site, which is similar in RyR and IP₃R isoforms, is by itself not sufficient to create a stable interaction between FKBP12 and the intracellular Ca²⁺-release channels. We have demonstrated that the local structural microenvironment plays a crucial role in targeting the FKBPs to their binding sites on RyRs and in establishing a high affinity interaction between both proteins. Furthermore, the differences in the predicted secondary structure between the FKBP12-binding site in the RyR isoforms and the IP₃R isoforms could explain that in contrast to RyRs, IP₃Rs have only a weak or transient interaction with FKBPs, which may not be functionally important for the mature protein.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Microsomal Preparations-- COS-1 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum, non-essential amino acids (Invitrogen 11140-035), Gluta-max (Invitrogen 35050-038), penicillin (100 units/ml), and streptomycin (100 µg/ml). RyR3-overexpressing HEK293 (35) and 16HBE14o cells (36) were cultured as described (35, 37). Cells were resuspended in homogenization medium, containing 10 mM Tris-HCl, pH 7.4, 1 mM EGTA, and protease inhibitors (0.83 mM benzamidine, 0.23 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 0.5 µg/ml aprotinin, 0.5 µg/ml pepstatin A), sonicated by a probe sonicator (MSE, Crawley, Surrey, UK) on ice for 3 times 20 s at an amplitude of 15 µm and centrifuged at 125,000 × g for 25 min. Microsomal pellets were resuspended in 20 mM Tris-HCl, pH 7.4, 300 mM sucrose, and protease inhibitors. Protein concentrations were determined by the Lowry assay and samples were stored at 80 °C.

Construction of Glutathione S-Transferase (GST) Fusion Proteins, Encoding FKBP12 and FKBP12.6-- PCR was used to amplify the cDNA encoding human FKBP12 and FKBP12.6. For FKBP12, we have used 5'-GCCGGATCCATGGGAGTGCAGGTGGAAACC-3' as forward primer and 5'-CCAAGAATTCTCATTCCAGTTTTAGAAGCTCC-3' as reverse primer, and for FKBP12.6, we have used 5'-GGGGGATCCATGGGCGTGGAGATCGAGACC-3' as forward and 5'-CCTTCGAATTCATCACTCTAAGTTGAGCAGCTCCACG-3' as reverse primer. Respective BamHI and EcoRI restriction sites are underlined. PCR products were digested with BamHI and EcoRI. These fragments were purified by PAGE and ligated into the BamHI-EcoRI sites of pGEX-2T and transformed into DH5 Escherichia coli for large scale production of DNA.

Expression of Wild Type, Mutated, and Chimeric RyR3-- The cDNA of the full-size mink RyR3 was cloned in the pcDNA3.1(+) vector (38). A fragment (bp 5715-10,528) of the full-size cDNA, containing two NheI restriction sites, was subcloned in the pBlueScript II SK(+) vector. This construct was used as template for the insertion of point mutations at Val²³²² of the RyR3. The sequence of the primers used for mutagenesis were as follows with indication of the mutated residues: V2322L, 5'-CTGCGCTCCCTGCTTCCCACAGAAGACCTGGTTGG-3' and 5'-CCAACCAGGTCTTCTGTGGGAAGCAGGGAGCGCAG-3'; V2322I, 5'-CTGCGCTCCCTGATTCCCACAGAAGACCTGGTTGG-3' and 5'-CCAACCAGGTCTTCTGTGGGAATCAGGGAGCGCAG-3'; V2322D, 5'-CTGCGCTCCCTGGATCCCACAGAAGACCTGGTTGG-3' and 5'-CCAACC- AGGTCTTCTGTGGGATCCAGGGAGCGCGCAG-3'.

The QuickChange^TM XL Site-directed Mutagenesis Kit was used according to the manufacturer's instructions. The insertion of the mutation was verified by sequence analysis. Construction of the chimeric RyR3 was done by overlap PCR of two PCR fragments, both amplified from the full-size RyR3 cDNA. Fragment A was amplified with 5'-GGTGCAGAGAAGTCTCAGATTGC-3' as forward primer (primer A-1F) and 5'-TGTCATCGAGCGGGAGCAAGGAGTTGCAGATTGACCTGATCCGGATGGC-3' as reverse primer, and fragment B was amplified with 5'-TCCTTGCTCCCGCTCGATGACATCGTTGGCATCATCAGCATCCCCTTG-3' as forward primer and 5'-GAAGAGCTTCTCCGTCAGGTGC-3' as reverse primer (primer B-2R). The inserted IP₃R1 sequence and its reverse complement at the 5'-end of the primers are underlined. The overlap PCR was done with primer A-1F and primer B-2R, using fragments A and B as templates. All fragments containing mutations were cloned in the NheI-cut pcDNA3.1 (+)/RyR3 (eukaryotic expression vector) and analyzed by sequence analysis. COS-1 cells were plated at 3 × 10⁶ cells/plate (150 cm²) and were transfected 1 day later, using 15 µg of DNA and 45 µl of Fugene^TM transfection reagent (Roche Molecular Biochemicals). Cells were harvested 3 days after transfection by scraping the cells in phosphate-buffered saline without Ca²⁺ and Mg²⁺, centrifuging at 400 × g for 5 min, and washing the cells twice in phosphate-buffered saline.

GST-FKBP12 and GST-FKBP12.6 Affinity Chromatography-- The GST-FKBP12 and GST-FKBP12.6 affinity chromatography assay was done exactly as described previously (18, 39). Quantification of the ratio between RyR3 and GST-FKBP12 or GST-FKBP12.6 immobilized on the column was performed after SDS-PAGE and staining with Sypro^TM Orange. For determining absolute amounts, values were compared with bovine serum albumin standards.

Mapping of FKBP12-binding Site on RyR3-- Microsomes of RyR3-overexpressing HEK293 cells (2 mg of protein) were partially trypsinized as described previously (18). In short, microsomal fractions were washed twice with 20 mM Tris-HCl, pH 7.4, and 150 mM NaCl and subsequently treated with trypsin (1:24 w/w) for 6 min at room temperature. Trypsinization was stopped by the addition of a 10-fold excess of soybean trypsin inhibitor and 0.23 mM phenylmethylsulfonyl fluoride. The binding of the trypsinized fragments to GST-FKBP12 and GST-FKBP12.6 was analyzed as described previously (18). The hydropathicity of the generated polypeptide fragments was predicted following the Kyte and Doolittle model (40).

SDS-PAGE, Western Blot Analysis, and Antibodies-- Full-size RyRs and IP₃Rs were analyzed by 3-12% SDS-PAGE followed by wet electrophoretic transfer to a polyvinylidene difluoride membrane (Immobilon-P, Millipore Corporation, Bedford, MA) performed for 16 h in transfer buffer (20 mM Tris, 192 mM glycine). After blocking the membranes with phosphate-buffered saline containing 1% (v/v) Tween 20 and 5% (w/v) non-fat dry milk powder, the membranes were subsequently incubated with the primary antibody and with alkaline phosphatase-conjugated secondary antibody. The immunoreactive bands were visualized after conversion of the Vistra^TM ECF substrate (Amersham Pharmacia Biotech) and quantified using ImageQuant 4.2 as described previously (41). Values are given as mean ± S.D., and statistical comparisons were performed by the Student's t test. The primary antibodies used in this study are m34C (Affinity BioReagents, Golden, CO) recognizing all RyR isoforms including RyR3 (24) and Rbt03 recognizing specifically IP₃R1 (42) and MMAtype3 (Transduction Laboratories, Lexington, KY) for IP₃R3 (43).

Secondary Structure Prediction of Primary Amino Acid Sequences of Peptides-- Prediction of the secondary structure of peptide sequences originating from RyR and IP₃R isoforms was performed with the PSIPRED version 2.0 method, available at insulin.brunel.ac.uk/psipred/. PSIPRED is a secondary structure prediction method, incorporating two separated feed-forward neural networks that perform an analysis on output obtained from PSI-BLAST (44, 45). The method is based on position-specific scoring matrices and contains three stages as follows: generation of a sequence profile, prediction of initial secondary structure, and finally the filtering of the predicted structure. Additional analysis was also performed by other secondary structure prediction methods, including the SAM-T99 method, available at www.cse.ucsc.edu/research/compbio/sam.html (46), and the SSpro2 method, available at promoter.ics.uci.edu/BRNN-PRED/ (47).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Interaction of RyR3 with FKBP12 and FKBP12.6-- We have developed a semi-quantitative affinity assay to investigate the FKBP-binding properties of the RyR3. After solubilization of microsomes from RyR3-overexpressing HEK293 cells, the supernatant was incubated with an excess of immobilized GST, GST-FKBP12, or GST-FKBP12.6 (~1 nmol or ~40 µg of protein per sample). Immunoblot analysis of the different fractions showed that the RyR3 was specifically retained with similar efficiency by GST-FKBP12 as well as by GST-FKBP12.6 (31 ± 6 and 33 ± 7%, respectively), but not by GST (Fig. 1A). The binding between RyR3 and both FKBP isoforms was completely abolished by 20 µM FK506, indicating a specific interaction (Table I). In our experimental conditions, GST-FKBP12 (40 µg, 1000 pmol) was in large excess (more than 500-fold) as compared with the amount of RyR3 protein. Hence, the RyR3 signal increased proportionally to the amount of solubilized fraction applied to the GST-FKBP12 column (Fig. 1B). The interaction between GST-FKBP12 and RyR3 represents specific (FK506-sensitive) and saturable (as shown in Fig. 1C) binding. The binding efficiency for the different constructs and different conditions can therefore be reliably used for estimation of the relative FKBP12 binding affinity.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Affinity purification of RyR3 using GST-FKBP12 or GST-FKBP12.6 immobilized on glutathione-Sepharose 4B. A, a microsomal fraction of RyR3-expressing HEK293 cells was used to characterize the interaction between RyR3 and GST-FKBP12 or GST-FKBP12.6. RyR3-containing microsomes (Micros) were solubilized (Sol) and incubated with GST, GST-FKBP12, or GST-FKBP12.6 immobilized on glutathione-Sepharose 4B. The retained proteins were eluted with 500 µl of Laemmli sample buffer, subjected to SDS-PAGE, and analyzed by immunoblotting with anti-RyR (m34C, 1:3000). Equivalent amounts of samples (5 µg) were used in the lanes with the microsomal fraction and the solubilized fraction and a 4-fold higher amount in the lanes with the eluate of GST, GST-FKBP12. and GST-FKBP12.6 affinity columns. B, different amounts of the solubilized fraction were incubated with the GST-FKBP12 matrix, containing ~40 µg of protein (or ~1000 pmol) per reaction tube. The samples were treated as described in A. The RyR3-immunoreactive bands were quantified by the ImageQuant 4.2a software. Each value is indicated as mean ± S.D. (n = 4) and is shown in arbitrary units, relative to the signal measured under standard conditions (400 µg of the solubilized fraction). The standard condition is indicated by a dashed line. C, identical amounts of the solubilized fraction (400 µg) were incubated with different amounts of the GST-FKBP12 matrix. Samples were treated as in A. The percent RyR3 bound, i.e. the relative amount of protein retained on the matrix compared with the solubilized amount, is plotted as a function of the amount of immobilized GST-FKBP12. Values are indicated as mean ± S.D. (n = 4).

Table I further indicates that the interaction between RyR3 and FKBP12 or FKBP12.6 was Ca²⁺-dependent, as 1 mM EGTA significantly decreased the binding efficiency (p < 0.05, n = 4). Moreover, the addition of 10 mM Mg²⁺, which inhibits RyR channel activity, significantly increased the binding efficiency by 1.5-fold (p < 0.01, n = 5). The addition of 10 µM cyclic ADP-ribose (cADPr), a putative RyR3 agonist, did not affect the binding efficiency, indicating that neither FKBP12 nor FKBP12.6 dissociated from RyR3 upon activation by cADPr.

Localization of the FKBP-binding Site on RyR3-- To identify the smallest domain on RyR3 that is sufficient for stable binding of FKBP12 and (or) FKBP12.6, we performed the GST-FKBP affinity assay on partially trypsinized microsomes from RyR3-overexpressing HEK293 cells. RyR3-containing microsomes were first trypsinized (Fig. 2A, Tryps) and solubilized (Fig. 2A, Sol). The fragments were very similar to those obtained for RyR1 (18, 48). After GST-FKBP12 or after GST-FKBP12.6 affinity chromatography, an ~40-kDa polypeptide, appearing as a double band, was retained (Fig. 2A, GSTFKBP12 and GST-FKBP12.6). Binding of the proteolytic fragments to the GST-FKBP column was much less efficient than for the full-size RyR3 (~2% versus ~30% of the solubilized amount). A higher amount of material was therefore loaded on the gel, whereby some nonspecific binding of the 40-kDa polypeptide fragment to GST was observed, which, however, never exceeded 0.4% of the solubilized fraction. Furthermore, the association of these RyR3 fragments to the FKBP matrix was specifically disrupted by the addition of 20 µM FK506 (data not shown). The epitope of m34C (aa 2619-2665) was localized close to the putative FKBP12-binding site based on the analogy with the FKBP-binding site proposed for RyR1, RyR2, and IP₃R1. Our immunoblots with m34C therefore indicate that the FKBP-binding site for RyR3 is within the same boundaries. The formation of this ~40-kDa proteolytic fragment of RyR3, encompassing the FKBP12- and FKBP12.6-binding site and the m34C epitope, therefore allows the identification of the FKBP-binding pocket on the native RyR3 protein. The generation of a doublet can be explained by the prediction of two successive hyperhydrophilic regions (HHR2 and HHR3) at the C-terminal part of the polypeptide fragments (Fig. 2, B and C). Both hyperhydrophilic regions contain multiple potential trypsin cleavage sites (Arg-X and Lys-X). The lower molecular mass fragment therefore probably arose from cleavages at HHR1 and HHR2, leading to a polypeptide from aa ~2265 to ~2665, whereas the higher molecular mass fragment probably arose from cleavages at HHR1 and HHR3, leading to a polypeptide from aa ~2265 to ~2700.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Localization of the putative FKBP-binding site on RyR3. A, after partial fragmentation of RyR3 by limited trypsin digestion, the microsomal fraction of RyR3-expressing HEK293 cells was solubilized and incubated with GST, GST-FKBP12, or GST-FKBP12.6 immobilized on glutathione-Sepharose 4B. Affinity-purified fragments were separated on SDS-PAGE and analyzed by immunoblotting with site-specific anti-RyR (m34C, 1:2500). The microsomal fraction (Micros), the trypsinized microsomes (Tryps), the supernatant after centrifugation of the trypsinized microsomes (Sn), the CHAPS-solubilized fraction of the trypsinized microsomes (Sol), the fraction bound to immobilized GST, GST-FKBP12, and GST-FKBP12.6 were loaded in successive lanes. Equivalent amounts of samples (10 µg) were used for the 1st 4 lanes, and 10-fold higher amounts were used in the last 3 lanes. The arrows on the left indicate the molecular masses of the protein standards. The upper arrow on the right indicates the full-size RyR3. The arrow labeled FKBP-BD indicates the two ~40-kDa peptide fragments, which are the smallest proteolytic fragments that still bound FKBP12 and FKBP12.6. B, linear presentation of the primary sequence of the relevant part of the central modulatory domain of the mink RyR3 (aa 2201-2750). The putative FKBP12-binding site (VP), the m34C epitope, and the three predicted hyperhydrophilic regions (HHR1, HHR2, and HHR3) are indicated. C, schematic presentation of the relevant part of the central modulatory domain of the mink RyR3 (aa 2201-2750) with indications of the putative FKBP12-binding site (V-P), the m34C epitope, and the three predicted hyperhydrophilic regions (HHR1, HHR2, and HHR3).

Comparison of the Structure of the FKBP-binding Site in RyR3 and IP₃R1-- Finally, we identified the molecular determinants for the binding of FKBP12 and FKBP12.6 to the RyR3 channel by site-directed mutations. Furthermore, we analyzed the difference in FKBP-binding properties of RyRs and IP₃Rs by the construction of a chimeric RyR3, containing the proposed FKBP12-binding site for IP₃R1 and by comparison with the full-size RyR3, IP₃R1, and IP₃R3. Val²³²² together with Pro²³²³ forms a valylprolyl residue largely conserved in both the RyR and IP₃R isoforms. For RyR1, these residues were shown to be at the core of the actual FKBP12-binding site (19). To elucidate the role of the valylprolyl residue in RyR3, we generated three mutants by substituting Val²³²² for leucine (V2322L mutant), isoleucine (V2322I mutant), or aspartate (V2322D mutant) (Fig. 3A). To investigate the differences between FKBP12-binding properties between the RyR and IP₃R, we also constructed a chimeric form of the RyR3, where 11 amino acids (aa 2318-2328) were substituted for the proposed core of the FKBP12-binding site of the mouse IP₃R1 (aa 1396-1406) (29) (Fig. 3A). In addition, we used 16HBE14o cells to analyze the human IP₃R3 isoform, which is predominantly expressed in these cells (37). The latter IP₃R isoform is the only one known to contain a valylprolyl residue (aa 1391-1392) in the presumed FKBP12-binding site (Fig. 5) (29). Mutated RyR3 as well as wild type RyR3 and wild type IP₃R1 were expressed in COS-1 cells. Microsomal fractions of the different transfected COS-1 cells were separated on SDS-PAGE and analyzed by immunoblotting with anti-RyR antibody (m34C) (Fig. 3B). No RyR3-immunoreactive band was present in the native COS cells or in the COS cells transfected with the pcDNA3.1(+) vector alone or transfected with IP₃R1. However, upon transfection with wild type RyR3 or altered RyR3 constructs, a RyR3-immunoreactive band was detected, indicating the heterologous expression of these proteins (Fig. 3B). The FKBP12- and FKBP12.6-binding properties of the different RyR3 forms, the heterologously expressed IP₃R1, and the endogenous human IP₃R3 were investigated by affinity chromatography (Fig. 4). Fig. 4A shows the interaction of RyR3 from RyR3-expressing COS-1 cells with FKBP12 and FKBP12.6. Substituting Val²³²² for Leu or for Ile significantly decreased the binding of the RyR3 for the FKBPs. Furthermore, these mutated RyR3 proteins became more selective for FKBP12.6 than for FKBP12. Replacing the Val²³²² for a negatively charged amino acid (Asp) completely disrupted the FKBP-binding properties of this RyR3 form. This was also observed for RyR1 (19) and is indicative of the hydrophobic nature of the FKBP-interaction site on RyR3. Furthermore, the 11-aa core of the FKBP12-binding site of IP₃R1 inserted in the full-size RyR3 was able to interact with both FKBP12 and FKBP12.6. This chimeric RyR3 construct bound FKBP12.6 more efficiently than FKBP12. As already shown previously (18), the wild type IP₃R1 did not interact with either GST-FKBP12 or GST-FKBP12.6 affinity column (Fig. 4F). Also the human IP₃R3, which contains a valylprolyl residue in the proposed FKBP12-binding core did not bind to GST-FKBP12 or GST-FKBP12.6.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Heterologous overexpression of wild type RyR3 and different altered RyR3 species in COS-1 cells. A, three mutant RyR3 proteins were constructed, each with a single amino acid substitution in the valine moiety of valylprolyl residues 2322-2323. The substituted amino acids are underlined. A chimeric RyR3/IP3R1 was generated, containing an 11-amino acid stretch (underlined), relatively well conserved in different RyR and IP3R isoforms and encompassing the proposed FKBP12-binding site for IP3R1. B, the microsomal fractions of COS-1 cells (40 µg), expressing native and altered RyR3 proteins or expressing IP3R1, were separated by SDS-PAGE and analyzed by immunoblotting with an anti-RyR antibody (m34C, 1:2000). The relevant portion of the gel is shown, and the position of RyR3 is indicated by the arrow. Untransfected COS-1 cells and COS-1 cells transfected with pcDNA3.1(+) were taken as negative control.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   GST-FKBP12 and GST-FKBP12.6 affinity chromatography applied for the wild type and mutated RyR3 from transfected COS-1 cells. Microsomes were solubilized and incubated with GST, GST-FKBP12, or GST-FKBP12.6 immobilized on glutathione-Sepharose 4B. The retained proteins were eluted with Laemmli sample buffer. After SDS-PAGE, the proteins were analyzed by immunoblotting. The proteins (A-F) were obtained from microsomes of COS-1 cells transfected with the following: A, RyR3; B, RyR3/V2322L; C, RyR3/V2322I; D, RyR3/V2322D; E, RyR3/IP3R1-(2318-2328) ; F, IP3R1; G, human IP3R3 obtained from 16HBE14o microsomes. Microsomal samples were 25 µg/lane, except for A and F where 40 µg/lane and G where 50 µg/lane were used. 4-Fold higher amounts of the solubilized fraction (Sol) were used compared with the microsomal fraction (Micros), except for A, F, and G were equivalent amounts were used. 4-Fold higher amounts were used in the GST, GST-FKBP12, and GST-FKBP12.6 lanes compared with the solubilized fraction (Sol). H shows the quantitative analysis of the binding efficiencies (% bound, i.e. the relative amount of protein retained on the matrix compared with the amount of solubilized protein) of the different RyR3 constructs, IP3R1 and IP3R3.

Because the expression levels of the RyR3 mutants were not identical, we calculated the relative amount of the RyR3 bound to GST, GST-FKBP12, or GST-FKBP12.6 compared with the total amount of solubilized RyR3, which was set as 100%. These values were obtained from the relative intensities of the immunoreactive bands and are represented in Fig. 4G. The results show that the RyR3/V2322L and RyR3/V2322I mutants had about an 8-fold lower binding efficiency for FKBP12 and about a 3-fold lower binding efficiency for FKBP12.6. The binding efficiency of the chimeric RyR3 was about 10-fold lower for FKBP12 and about 6-fold lower for FKBP12.6 than that of the wild type RyR3.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The immunophilins FKBP12 and FKBP12.6 are cis/trans-peptidylprolyl isomerases. They exert their enzymatic activity by interacting with and lowering the energy of the twisted amide transition state intermediate of a peptidylprolyl bond. This catalytic mechanism is an important feature in the restructuring of the peptide backbone during protein maturation and in the proper folding of the tertiary structure of proteins (49). However, these FKBPs can also form stable protein complexes with different cellular targets, particularly intracellular Ca²⁺-release channels (50). Compelling evidence was provided for an interaction of FKBP12 with the RyR1 (10, 11, 17, 19, 20) and for FKBP12.6 with the RyR2 (12, 13, 20, 21, 51, 52). The interaction with RyR3 is less well documented (18, 24), whereas the interaction with the IP₃R family is controversial (18, 26-29, 33). In this study, we have characterized the FKBP-binding properties of the RyR3 and compared these properties with those of IP₃R1 and of IP₃R3 at the molecular level.

Interaction of RyR3 with FKBP12 and FKBP12.6-- Due to the very low cellular expression levels of RyR3 and the fact that it is usually present together with much higher levels of the other RyR isoforms, it is very difficult to characterize the specific properties of the RyR3 from natural sources. We therefore used two different heterologous RyR3-expression systems, permanently RyR3-expressing HEK293 cells and transiently RyR3-transfected COS-1 cells. Our data show that FKBP12 as well as FKBP12.6 interacted with high affinity with the RyR3, allowing its purification from solubilized microsomes of RyR3-expressing HEK293 cells. About 30% of the solubilized RyR3 proteins were retained by the immobilized GST-FKBP12 and GST-FKBP12.6. The same observation was made for transiently RyR3-transfected COS-1 cells. The interaction between RyR3 and FKBP12 or FKBP12.6 was completely abolished by the presence of 20 µM FK506. The high affinity interaction of the RyR3 with FKBP12.6 has recently been used for purification of the RyR3 and its three-dimensional reconstruction (25). The binding efficiency of FKBP12 and FKBP12.6 to RyR3 was modulated by Ca²⁺ and Mg²⁺. Removal of Ca²⁺ significantly decreased the binding efficiency, whereas the addition of 10 mM Mg²⁺ clearly potentiated the association between the FKBPs and the RyR3. A similar effect of Mg²⁺ was observed for the binding of FKBP12 to the RyR1 (18). In contrast to what was observed for RyR2 (53), cADPr did not affect FKBP binding to the RyR3. cADPr-induced Ca²⁺ release from pancreatic islets microsomes is thought to be mediated by the binding of cADPr to FKBP12.6 and the subsequent dissociation of FKBP12.6 from the RyR2 (53). In contrast to the well documented role of cADPr in the activation of RyR2 (54-58), the action of cADPr on RyR3 is less clear as different results were reported, the majority of which found no response. Our GST-FKBP affinity assay showing no effect of cADPr is therefore in agreement with the latter observations (59). This potentially indicates an important difference in the regulation of the various RyR isoforms by cADPr.

Localization of the FKBP-binding Domain on RyR3-- FKBP12 as well as FKBP12.6 were shown to interact with an ~40-kDa proteolytic domain of the RyR3, located in the central modulatory domain of the RyR3 and recognized by the m34C antibody. This fragment is very similar to the 45-kDa fragment, obtained from proteolytic cleavage of the RyR1 (18), indicating that the boundaries of the FKBP-binding domain for RyR3 were similar to those for the FKBP12-binding domain of RyR1. However, the binding efficiency of the proteolytic fragment of RyR3 was more than 10-fold decreased in comparison with the full-size RyR3. A similar shift in binding efficiency was also observed for RyR1 and its proteolytic fragments (18). Furthermore, bacterial or mammalian recombinantly expressed proteins corresponding to the above described proteolytic fragments of RyR1 completely lost their ability to interact with FKBP12.² These differences in interaction between proteolytic fragments and recombinant domains on the one side and between proteolytic fragments and full-size proteins on the other side indicate that the higher order structure of the FKBP12-binding pocket is extremely important in the full-size RyR for a stable interaction with FKBP12. In addition, it cannot be excluded that other parts of the RyR structure outside the boundaries of this 40-kDa domain are involved in stabilizing the interaction with FKBP12.

To establish the molecular characteristics of the binding core, we performed site-directed mutations at Val²³²² in the full-size RyR3. Substitution of Val²³²² for the hydrophobic amino acids leucine or isoleucine decreased the binding efficiency and shifted the selectivity toward FKBP12.6, whereas substitution for the negatively charged amino acid aspartate abolished FKBP binding. These data therefore clearly indicate that similarly to Val²⁴⁶¹ of RyR1 (19) or Ile²⁴²⁷ of RyR2 (21), the Val²³²² residue of RyR3 is critical for FKBP interaction. The observation that the binding of FKBP12.6 was less affected by substitution of Val²³²² with leucine or isoleucine may reflect its larger catalytic pocket compared with FKBP12, because FKBP12.6 contains the smaller Phe⁵⁹ residue instead of the Trp⁵⁹ residue in FKBP12 (60).

Secondary Structure of the FKBP-binding Site-- Although RyR isoforms contain more than 50 valylprolyl, leucylprolyl, and isoleucylprolyl residues, only one critical valylprolyl residue (2461-2462 in RyR1 and 2322-2323 in RyR3) or one critical isoleucylprolyl residue (2427-2428 in RyR2) was the effective target for FKBP12 and (or) FKBP12.6, and only one FKBP12 interacted per subunit (10, 61). To explain this selectivity, we analyzed the secondary structure of these sequences by PSIPRED 2.0, which is a secondary structure prediction method based on position-specific scoring matrices (45). The accuracy of secondary structure prediction methods for a target sequence is related with the number of homologous sequences found in the Protein Data Base (62). In our case, sequences of 1800 amino acids of the different RyR and IP₃R isoforms containing the proposed FKBP12-binding site were sent to the server. The PSI-BLAST server was unable to find any other sequences similar to these sequences in the current sequence data banks that are installed on the PSIPRED server. This means that there is no evolutionary information available for PSIPRED to use. The expected average accuracy for a prediction based on a single sequence is about 68%. Fig. 5 shows that the valylprolyl residue 2461-2462 (RyR1) or 2322-2323 (RyR3) and isoleucylprolyl residue 2427-2428 (RyR2) had a unique secondary structure. These residues occur as a coil-coil residue flanked by an -helix at both sides, thereby acting as an -helix breaker. No other valylprolyl residues in the RyR3 sequence had a similar secondary structure. Similar results were obtained with secondary structure prediction methods, such as the SAM-T99 method and SSpro2 method, both also based on position-specific scoring systems. Unequivocal evidence and determination of the structural organization of the different domains in the RyR and IP₃R isoforms can only be obtained from the crystal structure of these proteins by x-ray diffraction, which will reveal the higher order structure of these gigantic proteins. Such analysis is however not yet possible. It is known that prolyl residues in an -helix may act as an -helix breaker, which is originating from side chain constraints and disrupting the H-bond network of the -helix. This will lead to a kink in the -helical structure. This structural aspect may therefore lock the valylprolyl residue in a twisted amide state, preventing the complete catalysis of the binding to a cis or trans configuration. The structural microenvironment may therefore play a crucial role in establishing the FKBP12/FKBP12.6 interaction (63). This -helix breaking structure is not only conserved among the different RyR isoforms but also during evolution in the different classes of vertebrates and in the Drosophila RyR (Fig. 6). In the Caenorhabditis elegans RyR, the critical prolyl residue is replaced by a serine residue, which altered the predicted secondary structure of this region to a long uninterrupted -helix. The importance of the conserved structural microenvironment of this site during evolution is reflected by experimental evidence, showing that FKBPs were able to interact with the RyR1 of skeletal muscle (16) and the RyR2 of cardiac muscle (64) from the different classes of vertebrate animals. Until now, it has not been investigated whether the RyR isoforms of Drosophila and of C. elegans are able to bind FKBPs.

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Presentation of the predicted secondary structure of the FKBP-binding site in the different RyR and IP3R isoforms and in the mutated RyR3 constructs. The secondary structure of the primary amino acid sequences of the different RyR and IP3R isoforms was predicted by version 2.0 of PSIPRED, using the maximal sequence length authorized by the server (i.e. 900 aa at both sites of the critical X-Pro residue). The 1st line shows the primary amino acid sequence. The 2nd line shows the predicted secondary structure elements as follows: H, helix; E, strand; and C, coil. The 3rd line shows the probability of the predicted structure with 0 = low and 9 = high. The critical X-Pro is indicated by an arrow, and the helical structures are indicated by a box. The substituted amino acids in the mutated RyR3 proteins are underlined.

View larger version (48K): [in this window] [in a new window]   Fig. 6.   Predicted secondary structure of the FKBP-binding site in the different RyR isoforms during evolution. The secondary structure of the primary amino acid sequences of the different RyR and IP3R isoforms was predicted by version 2.0 of PSIPRED. Method and description are identical to Fig. 5.

The FKBP-binding Site on IP₃R Isoforms-- IP₃R isoforms also contain in their primary sequence a putative FKBP12-binding site (29). However, its conservation during evolution is more divergent, because the corresponding FKBP-binding site in the Drosophila IP₃R contains a serine residue instead of a proline residue (65). At least for the murine IP₃R1, there is evidence from a yeast two-hybrid assay that the region surrounding the residues Leu¹⁴⁰⁰-Pro¹⁴⁰¹ directly interacts with FKBP12. By using GST-FKBP affinity chromatography, however, FKBP binding could not be detected for IP₃R1 and IP₃R3 from different cellular sources (18), which indicated that the interaction was much weaker than for the RyRs. Human IP₃R3, which similarly to RyR1 and RyR3 has a valylprolyl residue in the putative FKBP12-binding pocket, did not bind either FKBP12 or FKBP12.6. Therefore, it can be concluded that the absence of a high affinity FKBP-binding site in the IP₃R is not related to the absence or presence of a valyl residue in the putative binding pocket. From the secondary structure analysis of the IP₃R isoforms, it was evident that the valylprolyl residue in human IP₃R3 or the leucylprolyl residue in the other IP₃R sequences did not occur as an -helix breaker (Fig. 5). The importance of the higher order structure is emphasized by the FKBP-binding properties of a chimeric RyR3, which contains the 11-aa core of the FKBP12-binding site of the IP₃R1. This FKBP12-binding site from IP₃R1 in the RyR3 adopts the -helical breaking feature. Hence, these 11 amino acids were ineffective in the context of the IP₃R1, but when expressed within the RyR3, these amino acids were a target for the interaction with the FKBPs. The correct higher order structure of the valylprolyl, leucylprolyl, or isoleucylprolyl residue may therefore contribute to the specificity and high affinity interaction of the FKBP isoforms with the RyRs. In the case of the IP₃R, the valylprolyl or the leucylprolyl residue may be a substrate for FKBP12 and may undergo a complete isomerization to cis or trans state. FKBP12 will participate in the reaction by binding to and stabilizing the high energy intermediate transition state but will be released from the protein when the isomerization is complete. FKBP12 may act as an enzyme on the IP₃R substrate, catalyzing the cis/trans isomerization reaction (63).

In conclusion, our data showed that both FKBP12 and FKBP12.6 interacted with RyR3 but not with IP₃R1 or IP₃R3. The core of the interaction site was at the Val²³²²-Pro²³²³ residues in the modulatory domain of RyR3. Furthermore, this study showed that valylprolyl residues displayed the highest affinity for FKBP12 and FKBP12.6, whereas leucylprolyl (as in IP₃R1) and isoleucylprolyl (as in RyR2) preferentially interacted with FKBP12.6 instead of with FKBP12. For the IP₃R isoforms, however, the corresponding Leu¹⁴⁰⁰-Pro¹⁴⁰¹ in the murine IP₃R1 or even the Val¹³⁹¹-Pro¹³⁹² in the human IP₃R3 were not able to form a stable interaction with FKBP12 or FKBP12.6. Importantly, a chimeric RyR3/IP₃R1 construct, containing the proposed FKBP12-binding site of IP₃R1, could bind both FKBP isoforms. The difference in FKBP-binding properties of RyR and IP₃R isoforms could be attributed to a difference in the predicted secondary structure of the proposed interacting peptidylprolyl residues. The differences in secondary structure may therefore offer an explanation for the seemingly contradictory results between RyR and IP₃R isoforms concerning their FKBP-binding properties.

	ACKNOWLEDGEMENTS

We thank L. Bauwens, M. Crabbé, A. Florizoone, L. Heremans, Y. Parijs, J. Renders, M. Schuermans, and I. Willems for skillful technical assistance. We thank Dr. D. T. Jones (University College London, London, UK) for additional information on the PSIPRED secondary structure prediction method. We acknowledge Dr. A. Friedrich and Dr. K. Murato (Fujisawa GmbH, Munich, Germany) for providing us with FK506 and Dr. D. C. Gruenert (University of Vermont, Colchester, VT) for providing us with the 16HBE14o cells.

	FOOTNOTES

^* This work was supported in part by Grant 3.0207.99 from the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (to H. D. S. and J. B. P.), by Grant P4/23 from the Program on Interuniversity Poles of Attraction (to J. B. P., G. C., H. D. S., and L. M.), by Grant 99/08 from the Concerted Actions of the K. U. Leuven (to L. M., H. D. S., G. C., and J. B. P.), and by grants from Telethon and Murst (to V. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Recipient of a predoctoral fellowship from the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie.

^** To whom correspondence should be addressed: Laboratory for Physiology, K. U. Leuven, Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-345725; Fax: 32-16-345991; E-mail: humbert.desmedt@med.kuleuven.ac.be.

Published, JBC Papers in Press, October 11, 2001, DOI 10.1074/jbc.M106573200

² G. Bultynck, G. Callewaert, L. Missiaen, J. B. Parys, and H. De Smedt, unpublished results.

	ABBREVIATIONS

The abbreviations used are: RyR, ryanodine receptor; aa, amino acids; cADPr, cyclic ADP-ribose; FKBP, FK506-binding protein; GST, glutathione S-transferase; HHR, hyperhydrophilic region; IP₃R, inositol 1,4,5-trisphosphate receptor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

	REFERENCES

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