Analysis of substrates transport mechanism and structural modification of MRP1

我们先前报道了一种新型MRP1调节剂Agosterol-A(AG-A)的光类似物azidoAG-A，它能在谷胱甘肽(GSH)存在的情况下与MRP1的C末端结合，并与其他抗癌药物共享结合部位。我们更准确地分析了azidoAG-A的结合部位。胰酶消化后最小的光解片段的相对分子质量为16 kDa，来自TM15与TM16或TM17与NDB2之间的细胞质部分。我们重点研究了TM16-17附近的带电氨基酸残基精氨酸，由于我们发现TM16-17和1295的细胞质区域是药物结合所必需的。1202TH R的点突变不能被叠氮AG-A光标记，但具有LTC4转运活性。而1249R突变体不能被光标记并运输LTC4。因此，1249R对MRP1的转运活性是必不可少的。药物转运以及与碱性磷酸酶的偶联机制，特别是MRP1的核苷酸结合域(NBD)上的特征序列(Ss)的功能尚不清楚。我们研究了签名序列(G771D和G1433D)突变对叠氮-ATP光标记和钒酸捕获的影响。G771D突变完全抑制了Nbd2的捕获，并显著抑制了NBD1的捕获。但是，G1433D突变也显著地抑制了NBD1的捕获。由于这两个突变都降低了LTC4的运输活性，所以这两个突变都参与了碱性磷酸酶的水解，并且必须完整地参与MRP1的运输。

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.