Rhodamine 123 binds to multiple sites in the multidrug resistance protein (MRP1)

The mechanisms of MRP1-drug binding and transport are not clear. In this st udy, we have characterized the interaction between MRP1 and rhodamine 123 ( Rh123) using the photoreactive-iodinated analogue, [I-125]iodoaryl azido-rh odamine 123 (or IAARh123). Photoaffinity labeling of plasma membranes from HeLa cells transfected with MRP1 cDNA (HeLa-MRP1) with IAARh123 shows the p hotolabeling of a 190 kDa polypeptide not labeled in HeLa cells transfected with the vector alone. Immunoprecipitation of a 190 kDa photolabeled prote in with MRP1-sepcific monoclonal antibodies (QCRL-1, MRPr1, and MRPm6) conf irmed the identity of this protein as MRP1. Analysis of MRP1-IAARh123 inter actions showed that photolabeling of membranes from HeLa-MRP1 with increasi ng concentrations of IAARh123 was saturable, and was inhibited with excess of IAARh123. Furthermore, the photoaffinity labeling of MRP1 with IAARh123 was greatly reduced in the presence of excess Leukotreine C-4 or MK571, but to a lesser extent with excess doxorubicin, colchicine or chloroquine. Cel l growth assays showed 5-fold and 14-fold increase in the IC50 of HeLa-MRP1 to Rh123 and the Etoposide VP16 relative to HeLa cells, respectively. Anal ysis of Rh123 fluorescence in HeLa and HeLa-MRP1 cells with or without ATP suggests that cross-resistance to Rh123 is in part due to reduced drug accu mulation in the cytosol of HeLa-MRP1 cells. Mild digestion of purified IAAR h123-photolabeled MRP1 with trypsin showed two large polypeptides (similar to 111 and similar to 85 kDa) resulting from cleavage in the linker domain (L1) connecting the multiple-spanning domains MSD0 and MSD1 to MSD2. Exhaus tive proteolysis of purified IAARh123-labeled 85 and I11 kDa polypeptides r evealed one (6 kDa) and two (similar to6 plus 4 kDa) photolabeled peptides, respectively. Resolution of total tryptic digest of IAARh123-labeled MRP1 by HPLC showed three radiolabeled peaks consistent with the three Staphyloc occus aureus V8 cleaved peptides from the Cleveland maps. Together, the res ults of this study show direct binding of IAARh123 to three sites that loca lize to the N- and C-domains of MRP1. Moreover, IAARh123 provides a sensiti ve and specific probe to study MRP1-drug interactions.