Analysis of substrates transport mechanism and structural modification of MRP1

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We previously reported azidoAG-A, a photoanalog of a novel MRP1 modulating agent agosterol-A(AG-A), can bind to C-terminal part of MRP1 in the presence of glutathione (GSH) and share the binding sites with other anti-cancer drugs.We here analyzed binding sites of azidoAG-A more precisely.The molecular weight of the smallest photolabling fragment after trypsin digestion was l6kDa, which was derived from the cytoplasmic portion between TM15 and TM16 or TM17 and NDB2.We focused on charged amino acid residues arginine around TM16-17, since we found TM16-17 and cytoplasmic region up to1295 was essential for drug binding.The point mutant of 1202th R could not be photolabeled with azidoAG-A, but had LTC4 transport activity.However, 1249R mutant could not be photolabeled and transport LTC4.Thus 1249R is essential for transport activity of MRP1.The mechanisms of drug transportation and coupling with AlP hydrolysis, in particular the function of the signature sequences(ss)in the nucleotide binding domains(NBDs) of MRP1 are unknown.The effects of mutation of the signature sequences(G771D and G1433D)on the azido-ATP photolabeling and vanadate trapping were examined.The G771D mutation completely inhibited trapping at NBD2 and considerably inhibited trapping at NBD1.However, the G1433D mutation also considerably inhibited trap-ping at NBD1.Since either mutation decreased the transport activity of LTC4, both ss of MRP1 are involved in AlP hydrolysis and must be intact for the transport by MRP1.