Photolabeling of human and murine multidrug resistance protein 1 with the high affinity inhibitor [125I]LY475776 and azidophenacyl-[35S]glutathione.
Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent transporter of structurally diverse organic anion conjugates. The protein also actively transports a number of non-conjugated chemotherapeutic drugs and certain anionic conjugates by a presently poorly understood GSH-dependent mechanism. LY475776is a newly developed (125)I-labeled azido tricyclic isoxazole that binds toMRP1 with high affinity and specificity in a GSH-dependent manner. The compound has also been shown to photolabel a site in the COOH-proximal region of MRP1's third membrane spanning domain (MSD). It is presently not known where GSH interacts with the protein. Here, we demonstrate that the photactivateable GSH derivative azidophenacyl-GSH can substitute functionally for GSH in supporting the photolabeling of MRP1 by LY475776 and the transport of another GSH-dependent substrate, estrone 3-sulfate. In contrast to LY475776, azidophenacyl-[(35)S] photolabels both halves of the protein. Photolabeling of the COOH-proximal site can be markedly stimulated by low concentrations of estrone 3-sulfate, suggestive of cooperativity between the binding of these two compounds. We show that photolabeling of the COOH-proximal site by LY475776 and the labeling of both NH(2)- and COOH- proximal sites by azidophenacyl-GSH requires the cytoplasmic linker ( CL3) region connecting the first and second MSDs of the protein, but not the first MSD itself. Although required for binding, CL3 is not photolabeled by azidophenacyl-GSH. Finally, we identify non-conserved amino acids in the third MSD that contribute to the high affinity with which LY475776 binds to MRP1.[1]References
- Photolabeling of human and murine multidrug resistance protein 1 with the high affinity inhibitor [125I]LY475776 and azidophenacyl-[35S]glutathione. Qian, Y.M., Grant, C.E., Westlake, C.J., Zhang, D.W., Lander, P.A., Shepard, R.L., Dantzig, A.H., Cole, S.P., Deeley, R.G. J. Biol. Chem. (2002) [Pubmed]
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