MAPPING OF THE IMMUNOPHILIN-IMMUNOSUPPRESSANT SITE OF INTERACTION ON CALCINEURIN

The interaction of the immunosuppressive complexes cyclosporin A-cyclo philin A and FK506 binding protein-FK506 with the Ca2+- and calmodulin -dependent protein phosphatase calcineurin has been investigated by me ans of photoaffinity labeling and chemical cross-linking. Photolabelin g of purified bovine brain calcineurin with the affinity label [O-[4-[ 4-(1-diazo-2,2,2-trifluoroethyl)benzoyl] aminobutanoyl]-D-serine(8)]cy closporin in the presence of cyclophilin A results, in addition to the labeling of cyclophilin itself, in the transfer of some of the chemic al probe to both the catalytic subunit A and the regulatory subunit B of calcineurin. Chemical cross-linking studies with disuccinimidyl sub erate in the presence of either cyclophilin A, B, or C in complex with cyclosporin A or FK506 binding protein-FK506 result on the other hand in the apparently exclusive and strictly immunosuppressant-dependent formation of covalent immunophilin-calcineurin B subunit products. Cro ss-linking of immunophilins to calcineurin B subunit requires the pres ence of subunit A. In the present study, using a set of recombinant ma ltose-binding protein fusion products representing different stretches of the catalytic subunit A, we were able to map the minimal calcineur in A sequence necessary for immunophilin-ligand-calcineurin B interact ion to occur.