GSH-dependent Photolabeling of Multidrug Resistance Protein MRP1 (ABCC1) by [125I]LY475776

Photolabeling of Human and Murine Multidrug Resistance Protein 1 with the High Affinity Inhibitor [125I]LY475776 and Azidophenacyl-[35S]Glutathione

“…1B). In contrast to the results obtained with LTC 4 , when the two halves of the protein were co-expressed, labeling occurred predominantly in the COOH-proximal half of the protein in the region extending from amino acid 932 to 1531, consistent with previous trypsinolysis studies using H69AR membranes (36). With the coexpressed half-molecules, we also detected very weak labeling of the NH 2 -proximal portion (Fig.…”

“…1, A and C, respectively) (36). No labeling of a comparably sized protein was detected when membranes from cells infected with a control vector encoding ␤-glucuronidase (␤-gus) were used, confirming that the protein was indeed MRP1 (Fig.…”

“…Concurrent with these studies we have also examined the binding of azidophenacyl-GSH, which we show can effectively support transport of a GSH-dependent substrate such as estrone sulfate as well as the photolabeling of MRP1 by LY475776. We have recently shown by limited trypsinolysis of full-length MRP1 that the major site photolabeled by LY475776 resides in the COOH-proximal segment of MSD3 (36). Using baculovirus co-expressed fragments of MRP1, we demonstrate that both halves of the protein are required for photolabeling of the site in MSD3, although photolabeling can occur in the absence of MSD1 (amino acids 1-203).…”

“…LY475776 is an iodinated azido tricyclic isoxazole that displays essentially complete dependence on GSH for binding to MRP1, and which has a higher affinity for the protein than LTC 4 2 (36,37). Concurrent with these studies we have also examined the binding of azidophenacyl-GSH, which we show can effectively support transport of a GSH-dependent substrate such as estrone sulfate as well as the photolabeling of MRP1 by LY475776.…”

The MRP family of drug efflux pumps

“…Experiments employing the quinoline-based drug IACI and the rhodamine analog IAARh123 identified TM10-11 in MSD1 and TM16-17 in MSD2 as drug binding determinants (Daoud et al, 2000a(Daoud et al, , b, 2001. Sites that overlap these two regions are also crosslinked by the high-affinity substrate LTC4, and LY474776, a glutathione-dependent inhibitor of the pump (Mao et al, 2002;Qian et al, 2002). LTC4 binding sites were mapped to a region encompassing MSD1-NBD1 and to TM14-17, and LY474776 binding was mapped to TM16-17.…”