Perspectives in Pharmacology Sponsored by the Drug Metabolism Division Regulation of Expression of the Multidrug Resistance- Associated Protein 2 (MRP2) and Its Role in Drug Disposition

The multidrug resistance protein 2 (MRP2; ABCC2) is an ATPbinding cassette transporter accepting a diverse range of substrates, including glutathione, glucuronide, and sulfate conjugates of many endoand xenobiotics. MRP2 generally performs excretory or protective roles, and it is expressed on the apical domain of hepatocytes, enterocytes of the proximal small intestine, and proximal renal tubular cells, as well as in the brain and the placenta. MRP2 is regulated at several levels, including membrane retrieval and reinsertion, translation, and transcription. In addition to transport of conjugates, MRP2 transports cancer chemotherapeutics, uricosurics, antibiotics, leukotrienes, glutathione, toxins, and heavy metals. Several mutagenesis studies have described critical residues for substrate binding and various naturally occurring mutations that eliminate MRP2 expression or function. MRP2 is important clinically as it modulates the pharmacokinetics of many drugs, and its expression and activity are also altered by certain drugs and disease states. The identification of the multidrug resistance protein 2 (MRP2; ABCC2) as the transporter that mediates the biliary excretion of numerous drugs and their metabolites has represented a major step forward in understanding the factors that regulate hepatic drug elimination and contribute to hepatic toxicity. MRP2 (ABCC2) is the second member identified in the now nine-member family of MRP membrane transporters (Dean et al., 2001). MRPs represent one branch of the ATP-binding cassette superfamily of transmembrane proteins that use the energy of ATP hydrolysis to translocate their substrates across biological membranes (Borst et al., 1999) (Fig 1A). The founding member, MRP1, was identified as the basis for resistance to a diverse spectrum of cancer chemotherapeutic agents and xenobiotics in tumor cells that did not express MDR1 P-glycoprotein (Hipfner et al., 1999). MRP1 was shown to transport organic anions of endogenous and exogenous origin that were conjugated to glutathione, glucuronide, and sulfate, including leukotriene C4, 2,4-dinitrophenyl-S-glutathione (DNP-SG), and estradiol-17 ( -Dglucuronide) (E217G). These same conjugates were identified as substrates of ATP-dependent transport in liver canalicular membranes; however, MRP1 expression in liver was much too low to account for the high-hepatic transport activity. Spontaneous mutant strains of hyperbilirubinemic rats deficient in biliary excretion of bilirubin glucuronides and glucuronide and glutathione conjugates of xenobiotics, the Groningen yellow/transport deficient Wistar rat (GY/TR ) and the Eisai hyperbilirubinemic Sprague-Dawley rat (EHBR) were critical to the cloning of the liver homolog of MRP1, termed Mrp2 (Buchler et al., 1996; Paulusma et al., 1996; Ito et al., 1997). Mrp2 is absent in TR and EHBR rats We gratefully acknowledge Public Health Service Grant GM55343 for support of the work from this laboratory cited here and the Reproductive Sciences Training Program (NIH T32 HD07436) for supporting P.M.G. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.035014. ABBREVIATIONS: MRP, multidrug resistance-associated protein (lower case refers to nonhuman); DNP-SG, 2,4-dinitrophenyl-S-glutathione; E217G, estradiol-17 ( -D-glucuronide); GY/TR , Groningen yellow/transport deficient Wistar rat; EHBR, Eisai hyperbilirubinemic Sprague-Dawley rat; TM, transmembrane domain; MSD, membrane-spanning domains; NBD, nucleotide-binding domain; DJS, Dubin-Johnson syndrome; MDR, multidrug resistance transporter; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; Bsep, bile salt export pump; PCN, pregnenolone 16 -carbonitrile; ER-8, everted repeat with an 8-base pair spacer; FXR, farnesoid X receptor; CAR, constitutive androstane receptor; PXR, pregnane X receptor; RXR, retinoid X receptor; RAR, retinoic acid receptor; SN-38, 7-ethyl-10-hydroxycamptothecin; BQ-123, cyclo(L-Leu-DTrp-D-Asp-L-Pro-D-Val); MK571, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid; NEM-SG, N-ethylmaleimide-glutathione; OATP, organic anion transporting polypeptide. 0022-3565/02/3022-407–415$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 302, No. 2 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 35014/999743 JPET 302:407–415, 2002 Printed in U.S.A. 407 at A PE T Jornals on July 8, 2017 jpet.asjournals.org D ow nladed from Fig. 1. A, phylogenetic tree showing the human MRP family, redrawn from Borst et al. (1999). SUR, sulfonylurea receptor; CFTR, cystic fibrosis transmembrane regulator. B, proposed membrane topology of Mrp2, adapted and redrawn from Ito et al., (2001d). C, amino acid sequence alignment for human MRP2 (Q92887), MRP1 (P33527), and MRP3 (O15438), and rat Mrp2 (Q63120), and Mrp3 (O88563), with respective NCBI protein accession numbers. Sequences were aligned using multiple sequence alignments on BCM Search Launcher (http://dot.imgen.bcm.tmc.edu:9331/multi-align/multialign.html) and formatted using BoxShade 3.21 (http://www.ch.embnet.org/ software/BOX_form.html). Approximate locations of putative TMs were obtained from the NCBI protein entries indicated above and may differ from those assumed in the references. Dots indicate mutations in MRP2 or Mrp2 discussed in the text. 408 Gerk and Vore at A PE T Jornals on July 8, 2017 jpet.asjournals.org D ow nladed from due to distinct mutations in the mrp2 gene, both of which create premature termination codons. Cloning of mrp2 has made possible an understanding of its structure function relationships, localization and regulation of expression, and characterization of the defect in patients with the DubinJohnson Syndrome (see below). Structure and Function of MRP2 The membrane topology predicted for MRP2 is like that of MRP1 and contains 17 transmembrane (TM) helices, which form three membrane-spanning domains (MSD1, -2, and -3) connected by poorly conserved linker regions (L0 and L1) and highly conserved nucleotide-binding domains (NBD1 and NBD2) (Fig. 1B) (Borst et al., 1999; Konig et al., 1999a). Both the predicted odd number of transmembrane domains and immunofluorescence studies indicate the extracellular localization of the amino terminus (Konig et al., 1999a). Studies in patients with Dubin-Johnson syndrome have revealed valuable information about MRP2 genomic organization and the structure and function of MRP2 protein. The classic Dubin-Johnson syndrome (DJS) consists of elevated total and direct bilirubin, increased urinary coproporphyrin I fraction ( 80%), and deposition of a dark pigment in the liver (Toh et al., 1999). Patients with DJS may also have a decreased biliary clearance of bromosulfophthalein and some degree of jaundice (Toh et al., 1999). DJS is linked to mutations in the MRP2 gene; these are summarized in Table 1 (Paulusma et al., 1997; Wada et al., 1998; Toh et al., 1999; Ito et al., 2001e; Mor-Cohen et al., 2001). Homozygous mutations lead to classic DJS, whereas heterozygous mutants have moderately elevated urinary coproporphyrin 1 fraction ( 40%) with normal total and direct bilirubin (Toh et al., 1999). Many of these mutations are localized to NBD1 or NBD2. Unlike other mutations, R1150H mutants of the MRP2 protein mature and are properly localized, but transport activity is impaired (Mor-Cohen et al., 2001). Future studies are needed to identify any polymorphisms and their effect on MRP2 function. Characterization of the substrate recognition/transport sites of MRP2 has been based on the importance of amino acid residues located in the MSD of other ATP-binding cassette transporters, MDR1, and cystic fibrosis transmembrane regulator, and the closely-related family members MRP1 and MRP3. MRP3 was cloned as a homolog of MRP1 and 2 and is highly expressed on the basolateral membrane of rat and human liver under cholestatic and hyperbilirubinemic conditions (Hirohashi et al., 1998; Kiuchi et al., 1998; Konig et al., 1999b). Hydropathy plot analysis has shown that the structures of MRP1 to 3 are very similar. Initial studies showed that the amino terminal MSD1 of MRP1 is nonessential for substrate transport (Bakos et al., 1998), although the linker region connecting MSD1 and MSD2 is essential for MRP1 transport of leukotriene C4. Likewise, no mutations in MSD1 of MRP2 have been identified in patients with DJS, consistent with this region lacking a critical function for MRP2 in humans (Table 1). Efforts have therefore focused on transmembrane segments TM6 to TM17 of MSD2 and MSD3 (Fig. 1, B and C). To characterize binding sites for typical anionic MRP2 substrates, studies altering charged (especially cationic) amino acids were performed. Ryu et al. (2000) used sitedirected mutagenesis to examine the participation of basic residues in TM6 to TM17 of MRP2 in the transport of a fluorescent substrate, glutathione-methylfluorescein. Thirteen basic residues (His, Arg, Lys) in these regions were substituted with alanine; four mutants (K324A in TM6, K483A in TM9, R1210A in TM16 and R1257A in TM17) were all delivered appropriately to the cell surface when expressed in COS-7 cells yet showed decreased efflux of the substrate (Ryu et al., 2000). Similar studies substituting 10 charged amino acids in rat Mrp2 have exploited the differences in substrate specificity between Mrp2, which transports glucuronide, sulfate, and glutathione conjugates with great efficiency versus Mrp3, which efficiently transports glucuronide and sulfate conjugates but not glutathione conjugates (Ito et al., 2001c). Site-directed mutagenesis of Lys to Met (K325M) and Arg to Leu (R586L) of rat Mrp2 markedly reduced transport of DNP-SG and leukotriene C4, without affecting transport capacity of model glucuronide and sulfate conjugates yet increased the affinity for transport of E217G (Ito et al., 2001c). Unlike other MRPs, Mrp3 also transports taurocholate, a bile acid. Site-directed mutagenesis studies substi-