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.
Woerly
G
G
Institute of Toxicology, University of Zurich, Switzerland.
Weber
E
E
Ryffel
B
B
D000345:Affinity Labels; D000591:Amino Acid Isomerases / Q000737:chemistry / Q000378:metabolism; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D003432:Cross-Linking Reagents; D003511:Cyclohexanols / Q000737:chemistry / Q000378:metabolism*; D016572:Cyclosporine / Q000737:chemistry / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D019696:Peptidylprolyl Isomerase; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D011714:Pyrans / Q000737:chemistry / Q000378:metabolism*; D016559:Tacrolimus / Q000737:chemistry / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins
D000345:Affinity Labels; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D003511:Cyclohexanols; D006360:Heat-Shock Proteins; D011714:Pyrans; C066205:immunophilin ligand 506BD; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D000591:Amino Acid Isomerases; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase; D016559:Tacrolimus