Photoaffinity labeling of the multidrug resistance protein 2 (ABCC2/cMOAT) with a photoreactive analog of LTC4

Several studies have shown that the multidrug resistant protein MRP2 mediates the transport of chemotherapeutic drugs and normal cell metabolites, including Leukotriene C (LTC(4)); however direct binding of the LTC(4) to MRP2 has not been demonstrated. In this study, a photoreactive analog of LTC(4) (IAALTC(4)) was used to demonstrate its direct binding to MRP2. Our results show specific photoaffinity labeling of MRP2 with IAALTC(4) in plasma membranes from MDCKII(MRP2) cells. The photoaffinity labeling signal of MRP2 with IAALTC(4) was much lower than that of MRP1, consistent with previous studies whereby the measured K(m) values of MRP1 and MRP2 for LTC(4) were 1 μM and 0.1 μM LTC(4), respectively. Competition of IAALTC(4) photoaffinity labeling to MRP2 with MK571, a well characterized inhibitor of MRP2 function, showed ~75% reduction in binding in the presence of 50 μM excess MK571. Interestingly, unmodified LTC(4) enhanced the photoaffinity labeling of IAALTC(4) to MRP2, whereas excess GSH and Quercetin had no significant effect. Mild tryptic digestion of photoaffinity labeled MRP2 revealed several photoaffinity labeled peptides that localized the IAALTC(4) binding to a 15 kDa amino acid sequence that contains transmembrane 16 and 17. Together these results provide the first demonstration of direct LTC(4) binding to MRP2.