Cyclic ADP-ribose binds to FK506-binding protein 12.6 to release Ca2+ from islet microsomes.

Cyclic ADP-ribose (cADPR) is a second messenger for Ca2+ mobilization via the ryanodine receptor (RyR) from islet microsomes for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). In the present study, FK506, an immunosuppressant that prolongs allograft survival, as well as cADPR were found to induce the release of Ca2+ from islet microsomes. After islet microsomes were treated with FK506, the Ca2+ release by cADPR from microsomes was reduced. cADPR as well as FK506 bound to FK506-binding protein 12.6 (FKBP12.6), which we also found occurs naturally in islet microsomes. When islet microsomes were treated with cADPR, FKBP12.6 dissociated from the microsomes and moved to the supernatant, releasing Ca2+ from the intracellular stores. The microsomes that were then devoid of FKBP12.6 did not show Ca2+ release by cADPR. These results strongly suggest that cADPR may be the ligand for FKBP12.6 in islet RyR and that the binding of cADPR to FKBP12.6 frees the RyR from FKBP12.6, causing it to release Ca2+.

RyRs have been purified from both skeletal and cardiac muscle (17,18), and FK506-binding protein 12 (FKBP12) and FK506binding protein 12.6 (FKBP12.6) were copurified with type 1 RyR from striated muscle and with type 2 RyR from cardiac muscle, respectively (19,20). FKBP12 and FKBP12.6 were shown to bind selectively to type 1 and type 2 RyR, respectively (21). It was reported that the type 1 RyR was activated by dissociation of FKBP12 from the RyR by the addition of FK506 to release Ca 2ϩ (22).
In the present study, we show that cADPR binds to FKBP12.6, that FKBP12.6 is present in islet microsomes, and that when cADPR was added to islet microsomes, FKBP12.6 was dissociated from the microsomes to release Ca 2ϩ .
Calcium Release Assay-Microsomes were prepared as described previously (4,6). In brief, 2,000 islets from Wistar male rats (240 -280 g) were homogenized with a Pellet mixer (Treff, Degersheim, Switzerland) in 0.2 ml of acetate intracellular medium composed of 250 mM potassium acetate, 250 mM N-methylglucamine, 1 mM MgCl 2 , and 20 mM Hepes (pH 7.2) supplemented with 0.5 mM ATP, 4 mM phosphocreatine, creatine phosphokinase (2 units/ml), 2.5 mM benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride. After the homogenates had been centrifuged for 45 s at 13,000 ϫ g, the microsomes were prepared by Percoll density gradient centrifugation at 20,000 ϫ g for 40 min at 10°C. Release of Ca 2ϩ was monitored in 0.6 ml of intracellular medium composed of 250 mM potassium gluconate, 250 mM N-methylglucamine, 1 mM MgCl 2 , and 20 mM Hepes (pH 7.2) supplemented with 1 mM ATP, 4 mM phosphocreatine, creatine phosphokinase (2 units/ml), 2.5 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 7 g/ml bovine brain calmodulin (6), and 3 M Fluo 3 with the addition of 30 l of the islet microsome fraction (10 g of protein) (4, 6). FK506 or cADPR (4,6) was added into the incubation, and Fluo 3 fluorescence was measured at 490 nm excitation and 535 nm emission with a JASCO CAF-110 intracellular ion analyzer (Tokyo, Japan) at 37°C (6). Total accumulated Ca 2ϩ in islet microsomes was estimated by the increase of Fluo 3 fluorescence caused by the addition of 200 nM ionomycin (Sigma) to the Ca 2ϩ release medium containing the microsomes, and the ambient free Ca 2ϩ concentration ([Ca 2ϩ ]) was calculated using the following equation, as described previously (6): where K d ϭ 400 nM. In response to 100 nM cADPR, islet microsomes exhibited 18 Ϯ 2 nmol Ca 2ϩ release/mg protein, which corresponded to 31 Ϯ 4% of the total accumulated Ca 2ϩ .
cDNA Cloning-1 l of rat islet cDNA library (2 ϫ 10 6 plaqueforming unit) (23,24) was used as a template for PCR (23)(24)(25). The sequences of sense and antisense primers were 5Ј-GGAATTCCGCGTC-CTTTTCCTCCTCCT-3Ј and 5Ј-GGAATTCCTTGAGGTTTATGGCAT-ATAGTT-3Ј for the isolation of rat FKBP12 cDNA, which corresponded to nucleotide sequences 48 -68 and 636 -658 of mouse FKBP12 mRNA * This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture, Japan and the Kanae Foundation of Research for New Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) D86641 and D86642.
‡ Present address: Dept. of Biochemistry, Kanazawa University School of Medicine, Kanazawa 920, Japan.
Immunoblot Analysis-Rat islet microsomes (100 g of protein) were chromatographed on 20% SDS-polyacrylamide gel electrophoresis (30) and transferred to Immobilon P. The membrane was incubated with a monoclonal antibody against human FKBP12 or with an anti-FKBP12.6 antiserum, which also reacts with rat FKBP12 (see Fig. 4A). The monoclonal antibody was diluted at 0.2 g/ml, and the antiserum was diluted 2000 times with 5% milk powder. After rinsing, the membrane was incubated with a sheep anti-mouse antibody labeled with horseradish peroxidase or with a goat anti-rabbit antibody labeled with horseradish peroxidase and developed using the ECL reagents as described (6,25).

RESULTS AND DISCUSSION
We have previously shown that islet microsomes release Ca 2ϩ in response to cADPR (4). In the present study, we found that FK506, one of the most widely used immunosuppressive agents, induced the release of Ca 2ϩ from islet microsomes. The dose-response curve of FK506 on the Ca 2ϩ release from islet microsomes was concentration-dependent, with half-maximal release occurring at 2 M, and maximal Ca 2ϩ release occurring at 10 M (Fig. 1A). In addition, as shown in Fig. 1B, after the islet microsomes were treated with FK506, the Ca 2ϩ release by cADPR from the microsomes was reduced depending on the concentration of FK506; the maximal reduction was seen at 5-25 M FK506. Because cADPR and FK506 appear to induce the release of Ca 2ϩ by a common mediator, we next tried to determine if this occurs by a targeting of the same ligand. The cellular target for FK506 is thought to be FKBP12 and FKBP12.6. Therefore, we isolated FKBP12 and FKBP12.6 cDNAs from a rat islet cDNA library. As shown in Fig. 2A, rat FKBP12 is composed of 108 amino acids and highly conserved with human (31,32), mouse (26), bovine (33), and rabbit (19) FKBP12. Rat FKBP12.6 is also a 108-amino acid protein and completely conserved with human (27,28) and bovine (33) FKBP12.6. RT-PCR analyses revealed that FKBP12 and FKBP12.6 mRNAs were ubiquitously expressed in rat tissues including pancreatic islets and streptozotocin/nicotinamide-induced insulinomas (Fig. 2B); FKBP12 mRNA was detected in RINm5F cells, but FKBP12.6 mRNA was not. RINm5F cells, rat insulinoma-derived immortal cells, which do not release Ca 2ϩ in response to cADPR (34), synthesize and secrete very little insulin and show negligible sensitivity to glucose (35). Streptozotocin/nicotinamide-induced insulinomas contain as much insulin mRNA as normal islets (36 -38) and retain the sensitivity to glucose (39). Therefore, it is FKBP12.6 rather than FKBP12 that plays a role in the CD38-cADPR signaling (5) in insulin secretion by glucose in islets, because CD38, which catalyzes the synthesis and degradation of cADPR (23,40,41), a second messenger for Ca 2ϩ mobilization in glucoseinduced insulin secretion in islets (4,5), is expressed in islets and streptozotocin/nicotinamide-induced insulinomas but not in RINm5F cells (23,24). We then isolated microsomes from rat islets and carried out immunoblot analyses. As shown in Fig. 2  (C and D), although islet microsomes did not contain FKBP12 (Fig. 2C, lane 4), the microsomes contained FKBP12.6 (Fig. 2D,  lanes 5 and 6), suggesting that FKBP12.6 is the target for FK506 and/or cADPR to release Ca 2ϩ from islet microsomes.
Next, we examined the binding of FKBP12.6 to cADPR. The recombinant rat FKBP12.6 bound to FK506 at a K d value of 32 nM. As shown in Fig. 3A, cADPR was found to bind to FKBP12.6 at a K d value of 35 nM. The cADPR binding was inhibited by FK506 and neither structurally nor functionally related analogues of cADPR inhibited the cADPR binding to FKBP12.6 (Fig. 3B). These results indicate that FKBP12.6 acts as a cADPR-binding protein and strongly suggest that cADPR is the actual ligand for FKBP12.6 because FK506 does not normally exist in mammalian cells. It was reported that FKBP12/12.6 bound to RyR tightly and that by the addition of FK506, FKBP12/12.6 were dissociated from RyR to form FK506-FKBP12/12.6 complexes (19, 22, 33).  6. B, Ca 2ϩ release by cADPR and FK506 from islet microsomes after the cADPR pretreatment. Islet microsomes (10 g of protein) were treated at 37°C for 3 min with the indicated concentrations of cADPR in the Ca 2ϩ release medium and centrifuged at 100,000 ϫ g for 60 min at 4°C. The resultant pellet was resuspended in 0.6 ml of the Ca 2ϩ release medium, and Ca 2ϩ release was induced by the addition of 100 nM cADPR (q) or 10 M FK506 (Ç) and measured as described under "Experimental Procedures." In addition, the open probability of the type 1 RyR Ca 2ϩ channel was greatly increased when FKBP12 was released from the RyR by the addition of FK506 (22). As shown in Fig. 4, after treatment of islet microsomes with cADPR, FKBP12.6 was not detected in the microsomes but was recovered in the supernatant (Fig. 4A), and Ca 2ϩ release from the microsome treated by cADPR or FK506 was reduced. FK506 as well as cADPR then had almost no effect in releasing Ca 2ϩ from the 1 M cADPRpretreated microsomes (Fig. 4B). Our recent experiment indicated that type 2 RyR is expressed in rat islets. 2 From these results, it is strongly suggested that when cADPR binds to FKBP12.6 in islet microsome RyR and causes the dissociation of FKBP12.6 from the RyR to form FKBP12.6-cADPR complex, the channel activity of the RyR is thereby increased to release Ca 2ϩ from the endoplasmic reticulum. As described previously (6), the RyR can also be activated by Ca 2ϩ /calmodulin-dependent protein kinase II. The interaction between the dissociation of FKBP12.6 from RyR and the phosphorylation of RyR by Ca 2ϩ /calmodulin-dependent protein kinase II remains to be elucidated.