Inducible gene expression and protein translocation using nontoxic ligands identified by a mammalian three-hybrid screen

The natural product rapamycin has been used to provide temporal and quantitative control of gene expression in animals through its ability to interact with two proteins simultaneously. A shortcoming of this approach is that rapamycin is an inhibitor of cell proliferation, the result of binding to FKBP12-rapamycin-associated protein (FRAP). To overcome this limitation, nontoxic derivatives of rapamycin hearing bulky substituents at its C16- position were synthesized, each in a single step. The isosteric isopropoxy and methallyl substituents with the nonnatural C16-configuration abolish both binding to FRAP and inhibition oft cell proliferation. Binding proteins for these derivatives were identified from libraries of cDNAs encoding mutants of the FKBP12-rapamycin-binding (FRB) domain of FRAP by using a mammalian three- hybrid transcription assay. Targeting of the mutations was guided by the structure of the FKBP12-rapamycin-FRB ternary complex. Three compensatory mutations in the FRB domain, all along one face of an α-helix in a rapamycin-binding pocket, were identified that together restore binding of the rapamycin derivatives. Using this mutant FRB domain, one of the nontoxic rapamycin derivatives induced targeted gene expression in Jurkat T ceils with an EC₅₀ below 10 nM. Another derivative was used to recruit a cytosolic protein to the plasma membrane, mimicking a process involved in many signaling pathways.