Analysis of Human Multidrug Resistance Protein 1 (ABCC1) by Matrix-Assisted Laser Desorption Ionization/Time of Flight Mass Spectrometry: Toward Identification of Leukotriene C4Binding Sites

Abstract

Multidrug resistance in tumor cells may be caused by reduced drug accumulation resulting from expression of one or more proteins belonging to the ATP-binding cassette (ABC) transporter superfamily. In addition to their drug efflux properties, certain ABC proteins such as multidrug resistance protein 1 (MRP1) (ABCC1) mediate the ATP-dependent transport of a broad array of organic anions. The intrinsically photoreactive glutathione-conjugated cysteinyl leukotriene C4 (LTC4) is a high-affinity physiological substrate of MRP1 and is widely regarded as a model compound for evaluating the substrate binding and transport properties of wild-type and mutant forms of the transporter. In the present study, we have optimized high-level expression of recombinant human MRP1 in Pichia pastoris and developed a two-step purification scheme that results in purification of the transporter to >90% homogeneity. Peptide mapping by matrix-assisted laser desorption ionization/time of flight mass spectrometry of the peptides generated by in-gel protease digestions of purified underglycosylated MRP1 identified 96.7% of the MRP1 sequence with >98% coverage of its 17 transmembrane helices. Subsequent comparisons with mass spectra of MRP1 photolabeled with LTC4 identified six candidate LTC4-modified peptide fragments that are consistent with the conclusion that the intracellular juxtamembrane positions of transmembrane helices 6, 7, 10, 17, and a COOH-proximal portion of the cytoplasmic loop that links the first and second membrane spanning domains are part of the LTC4 binding site of the transporter. Our studies confirm the usefulness of mass spectrometry for analysis of mammalian polytopic membrane proteins and for identification of substrate binding sites of human MRP1.