Molecular cloning and functional characterization of cynomolgus monkey multidrug resistance-associated protein 2 (MRP2)

Abstract

The monkey is an important experimental model in the pharmacological evaluation of new drugs. We isolated monkey multidrug resistance-associated protein 2 (MRP2) cDNA to examine expression profiles among various tissues and measured ATPase activity to assess substrate specificity. The amino acid sequence encoded by monkey MRP2 cDNA was very similar (96% identity) to the reported human MRP2 cDNA (GenBank accession no. NM_000392). The tissue distribution of MRP2 in monkeys was partially different from that in humans. We found relatively high expression of MRP2 in the monkey kidney and small intestine using Northern blotting. Substrate specificity was compared between human and monkey MRP2. The affinity of 17β-estradiol 17-(β-d-glucuronide), methotrexate, vinblastine, and probenecid to monkey MRP2 was higher than that to human MRP2. Functional and expression differences between human and monkey MRP2 should be incorporated into the evaluation of candidate drugs.

Introduction

The ATP-binding cassette (ABC) transporter family comprises many proteins, from eukaryotes to prokaryotes. They have important roles as membrane transporters, ion channels, and ion channel modulators (Higgins, 1992, Klein et al., 1999). Forty-eight human ABC transporter genes have been identified by molecular cloning and/or sequence data generated from the Human Genome Project. Based on the sequence similarity in the cassette region (also known as the nucleotide-binding domain), human ABC transporters are classified into seven gene subfamilies (A–G) (Dean et al., 2001; http://humanabc.4t.com/humanabc.htm).

The ABCC gene subfamily comprises multidrug resistance-associated proteins (MRPs) (Cole et al., 1992, Borst et al., 1999), sulfonylurea receptors (SUR1 and SUR2) (Bryan and Aguilar-Bryan, 1997), and cystic fibrosis transmembrane conductance regulator (Rommens et al., 1989). The human MRP subfamily comprises at least eight members (MRP1–MRP8) and exhibits a wide range of biological functions (Klein et al., 1999, Dean et al., 2001, Borst et al., 1999, Kool et al., 1999, Hopper et al., 2001, Yabuuchi et al., 2001).

MRP2 (ABCC2 according to the new nomenclature for human ABC transporter genes) is predominantly expressed in the hepatocyte canalicular membrane and is thought to be the most important efflux transporter in the biliary excretion process. MRP2 substrates include glutathione, glutathione-, sulfate-, and glucuronide-conjugates such as sulfated and glucuronide bile salts, and nonconjugated organic anions (Konig et al., 1999, Suzuki and Sugiyama, 1998). Mutations in MRP2 have been identified in patients with Dubin–Johnson syndrome (Paulusma and Elferink, 1997, Wada et al., 1998, Toh et al., 1999, Hashimoto et al., 2002). The MRP2 cDNA sequence has been reported in human (Taniguchi et al., 1996, Paulusma and Elferink, 1997), rat (Buchler et al., 1996, Paulusma et al., 1996, Ito et al., 1997), mouse (Fritz et al., 2000), rabbit (van Kuijck et al., 1996), rhesus monkey, dog (Conrad et al., 2001), and Macaca fascicularis (Ninomiya et al., 2006). Several reports have suggested a large interspecies difference in the biliary excretion clearance of organic anions. The ATP-dependent transport of glutathione conjugated and nonconjugated organic anions into bile canalicular membrane vesicles (CMVs) derived from human liver was less than that into CMVs from rat liver, although the ATP-dependent transport of glucuronide conjugates was similar between human and rat (Ishizuka et al., 1997, Niinuma et al., 1999). There is a large interspecies difference in the biliary excretion clearance of temocaprilat, an MRP2 substrate, in vivo as well as in the ATP-dependent transport activity of 2,4-dinitrophenyl-S-glutathione (DNP-SG) into CMVs prepared in vitro (from rat, mouse, guinea pig, rabbit, and dog liver) (Ishizuka et al., 1999). Despite these efforts, it is difficult to ascertain if the large differences are attributable solely to MRP2 because the canalicular membrane expresses multiple members of ABC transporter families. In addition, the expression levels of MRP2 in the canalicular membrane vary between species. The large interspecies difference in the biliary excretion clearance of organic anions between rat and dog depends on protein expression levels of MRP2 in the liver (Ninomiya et al., 2005). Isolating and comprehensively analyzing each transporter to distinguish the contribution of each member is essential.

The monkey is the primary choice for an animal model because its physiology is similar to that of humans. Interspecies differences must be considered when evaluating candidate drugs because the pharmacokinetics in animal models sometimes differ from those in humans. We isolated complete cDNA of MRP2 from cynomolgus monkey to examine tissue expression and kinetic parameters, and compared these features with those of its human ortholog.