MECHANISMS OF IMMUNOSUPPRESSION BY FK506

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A novel immunosuppressive agent, FK506 and its structural analog rapamycin inhibit different signal transduction pathways yet both agents bind to the same family of intracellular receptors termed FK506-binding proteins, or FKBPs. FKBP12 (previously termed FKBP) is the predominant cellular receptor in T cells. The FK506-FKBP12 complex, like the CsA-CyP complex, has been shown to bind to and inhibit, in vitro, the activity of calcineurin (Cn), a Ca+2-calmodulin dependent serine/threonine phosphatase. We have confirmed that FK506 and CsA, but not rapamycin, inhibit calcineurin activity in vivo, and now propose to examine the role of calcineurin in T cell activation. In addition, we have cloned and characterized novel FK506 and rapamycin receptors. We propose to: 1. Analyze the role of calcineurin in T cell activation. Calcineurin protein levels will be correlated with biologic activity and with ability to inhibit with drug. 2. Characterize the FKBP multigene family. We have identified functionally active cell lines deficient in their expression of FKBP12 and of FKBP13. These cell lines will be transfected with wild type and mutated FKBP12 and FKBP13, and their function will be studied. The ability of FK506 and rapamycin to inhibit function will also be studied. Whether FKBP12 is the relevant receptor for T cell inhibition will be addressed, and a limited number of informative mutations will be made to confirm the predicted drug binding pocket. The subcellular localization of each of the cloned FKBPs will be studied, as will the biological role of the receptors. 3. Design experiments to isolate the rapamycin-FKBP target protein using both FKBP12 and FKBP25, assay for the presence of protein-protein interactions between the FKBPs and other cellular proteins, and isolate putative target proteins within the cell. We will attempt to immunoprecipitate endogenous proteins or peptides bound to the immunophilins. The role of rapamycin in regulating the upstream activators of p70 S6 kinases will be studied. A greater biological understanding of these proteins and of the mechanism of inhibition by FK506 and by rapamycin may allow rational design of agents with immunosuppressive activity but limited toxicity, or of agents that modulate specific signalling pathways. This approach may ultimately lead to the design of agonists or inhibitors for use in allograft transplantation, and possibly in autoimmune disorders.