Specific interaction of the cyclophilin-cyclosporin complex with the B subunit of calcineurin.
Calcineurin (CaN), a Ca2+/calmodulin-dependent serine/threonine phosphatase, has been shown to be inhibited by the complex of the immunosuppressant cyclosporin A (CsA) and its receptor, cyclophilin (CyP), but not by either alone. In the current study, the topological relationship between cyclophilin and the subunits of CaN has been explored using chemical cross-linking agents. In the presence of cyclosporin, 125I-CyP, shown to bind to CsA, is extensively cross-linked to the B subunit of CaN but not the catalytic A subunit. However, the A subunit is required for binding of the CyP.CsA complex, since cross-linking to recombinant B subunit alone does not occur. The kinetics of association indicate a saturable reaction with a Kd of less than 70 nM. Cross-linking to the B subunit occurs with cross-linkers that span from 0 to 16 A and employ different cross-linking chemistry, indicating direct contact between the B subunit and CyP. Similar cross-linking to the B subunit has been observed with the complex of 125I-labeled FK506 binding protein (FKBP) and FK506 but not with the FKBP-rapamycin complex. CyP.CsA cross-linking to CaN is Ca2+/calmodulin-dependent with intact CaN, but Ca2+/calmodulin-independent after digestion to remove the calmodulin binding and autoinhibitory domain.
Li
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Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Handschumacher
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RE
D000591:Amino Acid Isomerases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D003432:Cross-Linking Reagents; D016572:Cyclosporine / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D046911:Macromolecular Substances; D019696:Peptidylprolyl Isomerase; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011090:Polyenes / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D020123:Sirolimus; D016559:Tacrolimus / Q000378:metabolism; D022021:Tacrolimus Binding Proteins
D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D007166:Immunosuppressive Agents; D046911:Macromolecular Substances; D011090:Polyenes; D011994:Recombinant Proteins; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D000591:Amino Acid Isomerases; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase; D020123:Sirolimus; D016559:Tacrolimus