DOMINANT MUTATIONS CONFER RESISTANCE TO THE IMMUNOSUPPRESSANT, RAPAMYCIN, IN VARIANTS OF A T-CELL LYMPHOMA

Rapamycin (RAP) disrupts signaling events implicated in cytokine-depen dent proliferation of lymphocytes and other cells. This action is know n to involve the formation of molecular complexes between the drug and intracellular binding proteins, termed FKBPs. However, the biochemica l target(s) for the effector RAP-FKBP complexes remain uncharacterized . As an approach to explore the mechanism of action of RAP, we have is olated three independent sets of somatic mutants of the YAC-1 murine T cell line with markedly reduced sensitivity to the drug's inhibitory effects on proliferation and on IL-l-induced IFN-gamma production. The se mutants were still fully sensitive to FR-506, an Immunosuppressant structurally related to RAP whose mode of action also involves an inte raction with FKBPs. Furthermore, the 12-kDa FKBP, FKBP12, was detectab le in immunoblots from cytosolic extracts and eluates from RAP-affinit y matrix in the mutants as in wild-type cells, suggesting that the res istance to RAP in the mutants is not due to a lack of FKBP12 expressio n. Cell fusion experiments were conducted to further define the nature of the alterations imparting RAP resistance in these mutants. Clones deficient in either thymidine kinase or hypoxanthine-guanine phosphori bosyltransferase, suitable as fusion partners for aminopterin-based se lection of hybrids were generated from the wild-type or mutant lines. In most instances, the hybrids derived from the fusion between RAP-sen sitive clones and RAP-resistant clones exhibited a RAP-resistant pheno type. Similar results were obtained with hybrids between RAP-resistant YAC-1 clones and the RAP-sensitive EL-4 cell line. Therefore, the mut ations that confer resistance to RAP in the present system are dominan t. Altogether, our observations are consistent with a model where phar macologically relevant targets for the RAP-FKBP complex, rather than F KBP, might be altered in the mutants such that the inactivation of the se targets by the effector complex is prevented. (C) 1995 Academic Pre ss, Inc.