Binding of active cyclosporins to cyclophilin A and B, complex formation with calcineurin A.
The binding properties of several active and inactive cyclosporins to the major intracellular receptor proteins, cyclophilin A and B, as well as the interaction with the phosphatase calcineurin were investigated by ELISA and by means of a photoaffinity labeled probe (PL-CS). Binding to recombinant human cyclophilin A and B was rapid and saturable, and correlated with the in vitro immunosuppressive activity of cyclosporin derivatives. In the presence of cyclophilin A or B and calcium cyclosporin binds specifically to purified bovine calcineurin. PL-CS labeled only the calcineurin A subunit, but not the B subunit or calmodulin. Calcineurin A binding was competed by active (CsA, CsG or CsM), but not inactive (CsH, CsF) derivatives or the structurally unrelated macrolide immunosuppressant FK506. Ternary complexes containing equimolar ratios of cyclophilin A or B, PL-CS and calcineurin were resolved by chemical-crosslinking. The formation of these complexes was apparently specific, calcium-, but not calmodulin-dependent, and only inhibited by active cyclosporins. In vivo labelling of Jurkat T-cells revealed, that cyclophilin A and calcineurin A are the main labeled proteins, which form complexes in the presence of active cyclosporin. Thus, we demonstrate directly, that active cyclosporins have two recognition sites, which allow the in vivo recognition of cyclophilins and calcineurin A.
Institute of Toxicology, University of Zurich, Switzerland.
B
B
Ryffel
G
G
Woerly
M
M
Murray
H P
HP
Eugster
B
B
Car
D000345:Affinity Labels; D000591:Amino Acid Isomerases / Q000378:metabolism*; D001389:Azirines / Q000378:metabolism*; D019703:Calcineurin; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D021983:Cyclophilins; D003524:Cyclosporins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D019696:Peptidylprolyl Isomerase; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*
D000345:Affinity Labels; D001389:Azirines; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D003524:Cyclosporins; D011994:Recombinant Proteins; C082624:SDZ 212-122; C081786:cyclophilin B; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D000591:Amino Acid Isomerases; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase