Demonstration of ternary immunophilin-calcineurin complexes with the immunosuppressants cyclosporin and macrolide FK506.
The specificity of cyclosporin A (CsA) binding to the major intracellular receptor proteins, cyclophilin A and B, as well as the interaction of CsA with the phosphatase calcineurin were investigated. Binding of photoaffinity-labeled CsA (PL-CS), a photoaffinity probe of CsA, to recombinant human cyclophilin A and B is saturable and specific. Non-specific PL-CS binding to calcineurin is observed in the absence of cyclophilin and calmodulin. In the presence of cyclophilin, cyclosporin-calcineurin binding becomes specific. Ternary complexes containing an equimolar ratio of cyclophilin A or B, PL-CS and calcineurin are resolved using the chemical-crosslinking technique. The formation of these complexes is specific, calcium- but not calmodulin-dependent, and is only inhibitable by cyclosporins, which bind cyclophilin. The drug-immunophilin complex binds to the calcineurin A subunit. The proteolytic 43 kDa product of calcineurin A retains binding properties, suggesting that the C-terminal domains are not necessary for complex formation. A trimeric complex of FKBP-calcineurin is also formed with FK506, but not with rapamycin. As expected, these complexes are only competed with by homologous derivatives. Chemical crosslinking of photolabeled Jurkat T-cells strongly suggests that drug-calcineurin complexes are of biological relevance.