Multidrug Resistance Protein

Abstract

Multidrug resistance protein (MRP) is a broad specificity, primary active transporter of organic anion conjugates that confers a multidrug resistance phenotype when transfected into drug-sensitive cells. The protein was the first example of a subgroup of the ATP-binding cassette superfamily whose members have three membrane-spanning domains (MSDs) and two nucleotide binding domains. The role(s) of the third MSD of MRP and its related transporters is not known. To begin to address this question, we examined the ability of various MRP fragments, expressed individually and in combination, to transport the MRP substrate, leukotriene C 4 (LTC 4 ). We found that elimination of the entire NH 2 -terminal MSD or just the first putative transmembrane helix, or substitution of the MSD with the comparable region of the functionally and structurally related transporter, the canalicular multispecific organic anion transporter (cMOAT/ MRP2), had little effect on protein accumulation in the membrane. However, all three modifications decreased LTC 4 transport activity by at least 90%. Transport activity could be reconstituted by co-expression of the NH 2 -terminal MSD with a fragment corresponding to the remainder of the MRP molecule, but this required both the region encoding the transmembrane helices of the NH 2 -terminal MSD and the cytoplasmic region linking it to the next MSD. In contrast, a major part of the cytoplasmic region linking the NH 2 -proximal nucleotide binding domain of the protein to the COOH-proximal MSD was not required for active transport of LTC 4 . Multidrug resistance protein (MRP)1 is a member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters (1, 2). When transfected into drug-sensitive recipient cells, MRP increases resistance to natural product chemotherapeutic agents including epipodophyllotoxins, Vinca alkaloids, and certain anthracyclines (3-6). The resistance of MRPtransfected cells is associated with an energy-dependent decrease in drug accumulation and an increase in drug efflux (4, 6). Thus MRP confers a form of multidrug resistance that shares several characteristics with that caused by overexpression of P-glycoprotein (P-gp).MRP can also act as a primary active transporter of a wide range of organic, anionic conjugates, some of which are potential physiological substrates (reviewed in Loe et al. (7)). These include a structurally diverse array of glutathione, glucuronide, and sulfate conjugates, with the cysteinyl leukotriene, LTC 4 , being one of the highest affinity MRP substrates characterized to date (8 -11). However, it has not been possible to detect binding or measure primary active transport of unmodified xenobiotics (8, 9, 12). We have been able to demonstrate MRP-mediated, ATP-dependent transport of vincristine, and more recently, aflatoxin B 1 , but only in the presence of physiological concentrations of GSH (8, 13).P-gp (encoded by the MDR1 gene) and MRP share only approximately 18% overall amino acid identity, but despite the lack of primary structure co...

Structural and Functional Consequences of Mutating Cysteine Residues in the Amino Terminus of Human Multidrug Resistance-associated Protein 1

“…It has been reported previously that co-expression of the MSD1 (MRP1 281N ) and the carboxyl-terminal core domain molecule of MRP1 can generate functional molecules, whereas the core domain alone is not functional (19,20). The results in Fig.…”

“…These observations suggest that the folding of MSD1 is different in the absence and presence of the core domain. It has been shown previously that functional MRP1 was produced when the MSD1 and the core domain were co-expressed (19). We thus conclude that the folding of the MSD1 in the presence of the core domain is likely correct and that the folding of MSD1 in the absence of the core domain is incorrect, which results in the formation of intermolecular disulfide bonds.…”

“…In a previous study, Gao et al (19) found that deletion of as few as 66 amino acids of the amino terminus of human MRP1 reduced 90% of LTC 4 transport activity of MRP1. This observation is consistent with the results of the Cys 7 mutation in this study.…”

The MRP family of drug efflux pumps

“…Notwithstanding evidence indicating that N-terminal deletion of the entire MSD0 does not alter several properties of the pump, it is anticipated that this domain has functions that have yet to be uncovered. Studies showing that MRP1 activity can be affected by point mutations in the extracellular portion of the N-terminus and in MSD0, and by N-terminal deletions that extend to and include the first transmembrane domain hint at this possibility, although it remains to be determined whether these perturbations influence actual drug binding and transport as opposed to preventing the assumption of correct topology in the plasma membrane (Gao et al, 1998;Yang et al, 2002;Leslie et al, 2003b).…”

Mutation of a Single Conserved Tryptophan in Multidrug Resistance Protein 1 (MRP1/ABCC1) Results in Loss of Drug Resistance and Selective Loss of Organic Anion Transport

“…Previous studies have shown that portions of the first 280 amino acids of MRP1 encompassing the N-terminal MSD (MSD1) and the cytoplasmic loop connecting it to the second MSD (MSD2) are important for its expression in mammalian cell membranes as well as its ability to transport at least some of its substrates (32,33). In the course of a mutational analysis of this region of MRP1, we derived a mutant in which Pro 196 was replaced with Ala by site-directed mutagenesis (34).…”