The leucotriene C4 binding sites in multidrug resistance protein 1 (ABCC1) include the first membrane multiple spanning domain

The multiple drug resistance protein 1 (MRP1 or ABCC1) transports anticancer drugs and normal cell metabolites. Leucotriene C 4 (LTC 4 ) is one of the highest affinity substrates of MRP1. In this study, we have synthesizedand characterized a novel photoreactive azido analogue of LTC 4 (AALTC 4 ). The specificity of AALTC 4 binding to MRP1 was confirmed using an LTC 4 -specific monoclonal antibody. Moreover, binding with radioiodinated [ 1 2 5 I]AALTC 4 (or IAALTC 4 ) to MRP1 was dramatically competed with unmodified LTC 4 and to a lesser degree by glutathione (GSH). Oxidized glutathione (GSSG) slightly increased IAALTC 4 binding to MRP1, while MK571, verapamil, and vincristine inhibited IAALTC 4 binding to MRP1. Using AALTC 4 together with a panel of epitope-specific and LTC 4 -specific monoclonal antibodies, we identified LTC 4 binding sites in MRP1. Western blotting of large tryptic fragments of MRP1 with three well-characterized epitope-specific mAbs (MRPrl, QCRL1, and MRPm6) showed LTC 4 binding in both the N- and C-terminal halves of MRP1. Furthermore, a peptide corresponding to the N-terminal membrane-spanning domain of MRP1 (MSD0) was photoaffinity labeled by AALTC 4 , indicating that MSD0 contains an LTC 4 binding site. Higher resolution mapping of additional LTC 4 binding sites was obtained using eight MRP1 variants with each containing hemaglutanin A (HA) epitopes at different sites (at amino acid 4, 163, 271, 574, 653, 938, 1001, or 1222). MRP1 variants were photoaffinity labeled with IAALTC 4 and digested with trypsin to isolate specific regions of MRP1 that interact with LTC 4 . These results confirmed that sequences in MSD0 interact with IAALTC 4 . Other regions that were photoaffinity labeled by IAALTC 4 include TM 10-11, TM 16-17, and TM 12, shown previously to encode MRP1 drug binding site(s). Together, our results show a high-resolution map of LTC 4 binding domains in MRP1 and provide the first direct evidence for LTC 4 binding within MSD0.