Identification and characterization of the murine FK506 binding protein (FKBP) 12.6 gene.

FK506 binding proteins (FKBP) are members of a family of proteins called immunophilins. This highly conserved gene family has been identified in prokaryotes, lower eukaryotes, plants, and mammals. The immunophilins are members of the peptidylpropyl cistrans isomerases (PPIases) enzyme family. PPIases catalyze the isomerization of peptide bonds on the amino side of proline residues from the naturally occurring trans configuration to a cis configuration. This isomerization has been observed in approximately 15% of structurally characterized proteins (for review see Kay 1996). The immunophilins are most notable for being the pharmacologic target of immunosuppressive agents including cyclosporin A, FK 506 (tacrolimus), and rapamycin. These drugs bind directly to the immunophilins; however, the observed immunosuppression is not mediated by alteration in PPIase activity. To date, at least six human FKBP subfamily members have been identified and named on the basis of calculated molecular weight (Kay 1996). Although these FKBP proteins are expressed in many tissues, the endogenous cellular function of these proteins remains unclear. A subfamily of the FKBP proteins, FKBP 12, are tightly associated with ryanodine receptor (RyR-1), a component of calcium release channels in the sarcoplasmic reticulum of skeletal muscle (Jayaraman et al. 1992). FKBP 12 may function to enhance formation of the multimeric subunits in calcium channels required for skeletal muscle excitation-contraction coupling (Marks 1996). It has also been suggested that FKBP 12 may function as an inhibitor of transforming growth factor b (TGFb) family Type I receptor signal transduction in specific cell types (Wang et al. 1996; Chen et al. 1997). Previously, it was demonstrated by mutational analysis that FKBP 12 specifically binds calcineurin, a Ser/Thr phosphatase (Yang et al. 1993). This suggests that FKBP 12 may act as a docking protein between the TGFb Type I receptor and cellspecific inhibitors, for example, calcineurin. FKBP 12 is released from the Type I receptor upon ligand interaction of the TGFb Type II receptor and phosphorylation of Ser/Thr residues on the Type I receptor by the activated Type II receptor. However, the specific role of FKBP 12 in TGFb signaling remains uncertain with the observation that primary fibroblasts and thymocytes from FKBP 12-deficient mice demonstrated normal TGFb-mediated transcription and growth responses (Bassing et al. 1998). Wiederrecht and colleagues have purified (Sewell et al. 1994) and cloned (Lam et al. 1995) a novel human FKBP 12 member, hFKBP 12.6. Human FKBP 12.6 is homologous to FKBP 12, binds FK 506 with similar affinity, and inhibits calcineurindependent signaling. Interestingly, FKBP 12.6 is associated with a cardiac specific isoform of the ryanodine receptor, RyR-2 (Lam et al. 1995). Here, we report the identification of a full-length murine cDNA clone for mFKBP 12.6 with characterization of genomic structure and tissue-specific RNA expression pattern. BLASTN analysis was used to identify the murine FKBP 12.6. The published sequence of the hFKBP 12.6 (Lam et al. 1995) was used to search a non-human expressed sequence tag (EST) database. Initially, this search identified a murine EST that was similar to the human FKBP 12 gene. Further analysis determined that the murine EST sequence was 91% identical to the reported hFKBP 12.6 cDNA sequence. An IMAGE clone (#390134) that contained the entire insert was subcloned into pBluescript SK (Stratagene, LaJolla, Calif.) and sequenced. This sequence analysis verified that the IMAGE clone contained sequence corresponding to the entire coding region for the hFKBP 12.6 gene (Fig. 1A). The predicted amino acid sequence of mFKBP 12.6 demonstrated 98.2% and 97.3% similarity to the published rat and human FKBP 12.6 proteins, respectively. The predicted amino acid sequence of mFKBP 12.6 is approximately 80.0% similar to the published mouse, rat, and human FKBP 12 proteins (Fig. 1B). On the basis of nucleotide and amino acid sequence alignments, we concluded that this clone was the murine homolog of hFKBP 12.6. In order to characterize the genomic organization of the mFKBP 12.6 gene, a series of PCR primers were designed to amplify across potential intron:exon boundaries, based on estimates of genomic organization and sequence similarities to the published human FKBP 12 gene (DiLella and Craig 1991). Mouse genomic DBA/1 DNA was used in long template PCR reactions across potential introns. A PCR fragment was identified and subcloned that spanned the sequence between the predicted exons 3 and 4. Based on the high homology between FKBP 12 and 12.6, it was thought that intronic sequence would result in a more specific probe for murine FKBP 12.6 genomic clones. This probe was subsequently used to identify two murine DBA/1 genomic lambda phage clones containing the entire coding region of the mFKBP 12.6 gene. Using exon-specific primers and one of the lambda phage clones as substrate, we used long template PCR to estimate the size of introns (Fig. 2A). Subsequently, these same primers were used to sequence the intron/exon boundaries (Fig. 2B). The exon boundaries were remarkably well conserved between the mFKBP 12.6 and hFKBP 12 genes, with only one exception. The mFKBP 12.6 gene has a single large exon 4 with coding sequence from the conserved methionine residue #67 and the last 42 amino acids of the mFKBP 12.6 carboxy terminal through the polyadenylation signal. This differs from the human FKBP 12 gene, which has a small 165-bp exon 4 from Met #67 through the coding sequence, followed by a 1.8-kb intron and a large exon 5 with 38 untranslated sequence and the polyadenylation signal (DiLella and Craig 1991). The expression pattern of the mFKBP 12.6 gene was studied relative to mFKBP 12. A murine multiple tissue Northern blot (Clontech, Palo Alto, Calif.) was hybridized with the entire cDNA sequence cloned in this report as probe for mFKBP 12.6 gene expression. After autoradiography, this same filter was stripped of radiolabel and re-hybridized with cDNA probe generated by PCR amplification of exon 5 of the mFKBP 12 gene. Exon 5 is a specific probe for mFKBP 12 because it contains a large stretch of Correspondence to: J.D. McNeish Mammalian Genome 9, 1069–1071 (1998).