Analysis of Human Multidrug Resistance Protein 1 (ABCC1) by Matrix-Assisted Laser Desorption Ionization/Time of Flight Mass Spectrometry: Toward Identification of Leukotriene C4 Binding 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 C 4 (LTC 4 ) 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 Ͼ90% 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 Ͼ98% coverage of its 17 transmembrane helices. Subsequent comparisons with mass spectra of MRP1 photolabeled with LTC 4 identified six candidate LTC 4 -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 LTC 4 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.

The Human Breast Cancer Resistance Protein (BCRP/ABCG2) Shows Conformational Changes with Mitoxantrone

“…Expression of BCRP in Pichia pastoris was carried out as previously described (Mao et al, 2004; Wu et al, 2005). Further details for expression and purification of BCRP are described in the Supplemental Data.…”

Multidrug Resistance Protein 1 (MRP1, ABCC1), a “Multitasking” ATP-binding Cassette (ABC) Transporter

“…A notable example is Lys 332 in the first TM of MSD1 (Fig. 1B) (43,50). Mutation of TM6 Lys 332 to an opposite, neutral, or even same-charge amino acid completely abrogates MRP1 binding of the high affinity physiological substrate LTC 4 , but has virtually no effect on the binding or transport of any OA substrate lacking a GSH moiety.…”

Portrait of multifaceted transporter, the multidrug resistance-associated protein 1 (MRP1/ABCC1)

“…The GSHdependent azidoagosterol A labeling of the protein was detected only in TMD2 [130], whereas the azido analogs of GSH and unconjugated drugs, such as rhodamine123, or quinoline, labeled both halves of MRP1, reacting with TMHs 10-11 in TMD1 and TMHs 16-17 in TMD2 [29,65,126]. The iodoarylazido analog of LTC 4 labeled the above two regions as well as TMH 12 [66], while the labeling with unmodified LTC 4 showed that the TMHs 6, 7, 10, and 17 are parts of the LTC 4 binding site [176].…”