Semin Liver Dis 2000; Volume 20(Number 03): 265-272
DOI: 10.1055/s-2000-9391
Copyright © 2000 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel.: +1(212) 584-4662

Hepatic Secretion of Conjugated Drugs and Endogenous Substances

DIETRICH. KEPPLER, JÖRG. KÖNIG
  • From the Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany
Further Information

Publication History

Publication Date:
31 December 2000 (online)

Table of Contents #

ABSTRACT

Conjugate export pumps of the multidrug resistance protein (MRP) family mediate the ATP-dependent secretion of anionic conjugates across the canalicular and the basolateral hepatocyte membrane into bile and sinusoidal blood, respectively. Xenobiotic and endogenous lipophilic substances may be conjugated with glutathione, glucuronate, sulfate, or other negatively charged groups and thus become substrates for export pumps of the MRP family. The apical isoform, MRP2 (gene symbol ABCC2), has been localized to the apical membrane of several polarized epithelia and particularly to the canalicular membrane of hepatocytes. Absence of functionally active MRP2 glycoprotein from this membrane domain prevents the secretion of many anionic conjugates into bile. Prototypic endogenous substrates of high affinity for recombinant human MRP2 include bisglucuronosyl bilirubin, monoglucuronosyl bilirubin, and the glutathione S-conjugate leukotriene C4. Several mutations in the human MRP2 gene have been identified that lead to the absence of MRP2 from the canalicular membrane and to the conjugated hyperbilirubinemia of Dubin-Johnson syndrome. MRP2-mediated conjugate export represents a decisive final step in the detoxification of drugs, toxins, and endogenous substances. The basolateral isoform, MRP3 (gene symbol ABCC3), is upregulated in MRP2 deficiency and in extrahepatic cholestasis. MRP3 mediates the ATP-dependent transport of anionic conjugates, particularly of glucuronides and sulfoconjugates, across the basolateral hepatocyte membrane into sinusoidal blood. The inverse regulation of MRP3 and MRP2 expression under many conditions is consistent with their distinct localization and with a compensatory role of MRP3 in the hepatic secretion of anionic conjugates during impaired transport into bile.

Hepatocytes actively convert exogenous and endogenous substances into anionic conjugates with glutathione, glucuronate, sulfate, or other negatively charged moieties. This conjugation of lipophilic substances precedes their transport into the extracellular space. Under physiologic conditions, these conjugates are secreted across the canalicular membrane into bile by an ATP-dependent conjugate export pump.[1] [2] [3] [4] [5] [6] The molecular identification and cloning of this canalicular conjugate export pump (MRP2)[7] [8] [9] was a consequence of the discovery that the multidrug resistance protein 1 (MRP1) transports similar substrates, including glutathione S-conjugates, glucuronides, and sulfoconjugates.[10] [11] [12] [13] Mutant rat strains deficient in the hepatobiliary secretion of conjugates and additional organic anions[14] [15] were shown to lack the multidrug resistance protein 2 (MRP2) in the canalicular membrane[8] [9] due to mutations in the rat MRP2 gene, leading to premature termination codons.[8] [16] Observations in these mutant rats prove the concept that anionic conjugates of many lipophilic substances cannot exit across the plasma membrane into bile in the absence of the ATP-dependent export pump MRP2. The lack of this protein prevents the secretion into bile of substances such as the glutathione S-conjugate leukotriene C4 [15] and N-acetyl leukotriene E4,[17] both high-affinity substrates for MRP2.[3] [18] Evidently, there is no alternative transport protein in the canalicular membrane for the secretion of these conjugates. It is important to note, however, that many conjugates formed in the hepatocytes can be transported into the sinusoidal space, particularly under pathophysiologic conditions associated with an impaired function of MRP2.[17] [19] The recent localization of another member of the MRP family, MRP3, to the basolateral membrane of hepatocytes[20] [21] suggests that basolateral isoforms of the MRP family can support the secretion of conjugates from hepatocytes into blood.

In this article we consider the role of MRP family members in the hepatocellular secretion of anionic substances formed in the conjugation phase (phase II) of detoxification of drugs and endogenous substances. Moreover, we review consequences of mutations in the MRP2 gene leading to the Dubin-Johnson syndrome in humans.

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CONJUGATE EXPORT PUMPS OF THE MRP FAMILY

Members of the MRP family have been identified in a variety of different organisms, including yeast, nematodes, plants, and mammals. The major function of this ATP-binding cassette (ABC) transporters is the export of anionic conjugates from cells. In humans, the MRP family currently comprises six characterized members, known as MRP1 (symbol ABCC1); MRP2 (ABCC2), also known as canalicular MRP or canalicular multispecific organic anion transporter; MRP3 (ABCC3); MRP4 (ABCC4); MRP5 (ABCC5); and MRP6 (ABCC6). The deduced amino acid numbers range from 1,325 amino acids for MRP4 to 1,545 amino acids for MRP2 (Table [1]). The length of the proteins and the membrane topology discriminates the MRPs from the MDR Pglycoproteins. In contrast to the typical six plus six transmembrane segment topology described for members of the MDR family,[22] [23] four of the six current members of the MRP family exhibit an additional aminoterminal membrane-spanning domain represented by an extension of approximately 200 amino acids.[24] In addition to computational methods, the topology of MRP1 and MRP2 has been studied by mutational analyses, limited proteolysis experiments,[25] and epitope insertion studies.[26] [27] [28] Both transporters are predicted to consist of a P-glycoprotein-like core structure with two ATP-binding domains and two transmembrane regions, in addition to a third transmembrane region located amino-proximal in front of the core sequence. A remarkable topologic feature of MRP1 and MRP2 represents their amino-terminus, which was predicted to be extracellular. This was first described for MRP2 on the basis of topology prediction programs[9] and subsequently experimentally established both for MRP1 by epitope insertion studies[29] and for MRP2 by direct immunofluorescence microscopy.[18]

MRP1 and MRP2 are the best characterized members of the MRP family, and both share a similar substrate specificity.[30] MRP1, the founding member of the family, was cloned from a drug-selected human lung cancer cell line.[31] The MRP1 gene spans approximately 200 kilo-base pairs (kbp) and is located on chromosome 16p13.12-13; the gene contains 31 exons.[32] The human MRP2 protein consists of 1,545 amino acids and is encoded by a gene located on chromosome 10q23-q24.[9] [18] [19] [33] [34] [35] [36] The MRP2 gene spans approximately 45 kbp and contains 32 exons ranging in size from 56 to 255 bp.[37] Each nucleotide binding domain of the MRP2 gene is encoded by three exons. The comparison of the genomic organization of MRP2 [37] [38] with the genomic organization of the MRP1 gene[32] displays remarkable similarities indicated by the size and number of exons and by 21 identical splice sites when viewed on the amino acid level.[37] Recently, the genomic organization of the MRP3 gene became accessible as a genomic cosmid clone (GenBank accession AC004590). The MRP3 gene contains 31 exons. Interestingly, MRP1, MRP2, and MRP3 have 21 identical splice sites when viewed on an amino acid alignment. Despite the fact that these three MRP isoforms share only a relatively low degree of amino acid identity, a close evolutionary relationship of these transporters is indicated by their similar genomic organization.

The identification of MRP3, MRP4, MRP5, and MRP6 was mainly based on the analysis of the expressed sequence tag database[39] followed by the cloning of partial cDNA sequences[40] and subsequently of the complete cDNA of the respective transporter. MRP1-5 are encoded by genes located on different chromosomes (Table [1]). Among the more recently identified MRP isoforms (MRP3-5), MRP3 is best characterized with respect to its tissue-specific expression, its transport function, and its localization.[20] [21] [41] [42] [43] Unlike the substrates for MRP1 and MRP2, glutathione S-conjugates are poor substrates for MRP3.[42] On the other hand, other glucuronosyl conjugates, including 17β-glucuronosyl estradiol, are substrates for MRP1, MRP2, and MRP3. In contrast to MRP1 and MRP2 from rat and human, rat MRP3 is able to transport sulfated and nonsulfated bile salts.[42] Moreover, MRP3 is also able to confer drug resistance against several epipodophyllotoxins and methotrexate.[21] The tissue distribution of MRP3 exhibits similarities with the tissue distribution of MRP2, and both proteins are expressed in liver and colon.[20] [21] [30]

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APICAL CONJUGATE EXPORT PUMP MRP2

MRP2 has been the second member of the MRP family to be cloned, localized, and functionally characterized.[7] [8] [9] [16] [18] [19] [30] [33] [34] [35] [36] [44] [45] [46] [47] [48] [49] Up to now, it is the only conjugate export pump detected in the apical (canalicular) membrane domain of hepatocytes. MRP2 has also been localized to the apical membrane of kidney proximal tubules,[47] [48] of intestinal epithelia, and various polarized cells in culture.[49] [50] [51] [52] It should be noted that other known members of the human MRP family have been localized to the basolateral domain of polarized cells.[30] The localization of MRP2 is dynamic as shown by the endocytic retrieval and exocytic insertion of the protein in rat hepatocytes.[53] [54] [55] [56] [57] Endocytic retrieval followed by downregulation of MRP2 gene expression is also observed in rat hepatocytes after endotoxin-induced cholestasis[56] [57] and after duct ligation.[55] The endotoxin-induced decrease of MRP2 in the canalicular membrane explains the well-known impairment of the excretion of MRP2 substrates into bile in endotoxemia. This is exemplified by the early and potent inhibition of cysteinyl leukotriene secretion across the canalicular membrane into rat bile after endotoxin administration.[58] [59]

The substrate specificity of MRP2 has been elucidated in a stepwise fashion, starting from hepatobiliary elimination studies in MRP2-deficient mutant rats,[6] [14] [15] [60] leading to measurements of ATP-dependent transport using inside-out-oriented canalicular membrane vesicles derived from normal and MRP2-deficient mutant rat liver,[2] [3] [4] [5] [6] [7] [9] and finally by transport measurements using recombinant MRP2 from human and other species.[18] [30] [36] [46] Based on the work with recombinant human MRP2 studied in membrane vesicles, the prototypic high-affinity substrates include monoglucuronosyl bilirubin, bisglucuronosyl bilirubin, and the endogenous glutathione S-conjugate leukotriene C4 (Table [2]). A large number of glutathione S-conjugates, glucuronides, and sulfoconjugates of drugs and other xenobiotics are also substrates for MRP2, although the affinity for most drug conjugates is much lower than for the prototypic endogenous substrates.[61] In addition, a number of nonconjugate amphiphilic anions, such as the lipophilic pentaanion Fluo3[49] [52] and the monoanionic para-aminohippurate,[62] are MRP2 substrates.

Because MRP2 shares only 48% identical amino acids with MRP1 (Table [1]), it cannot be anticipated that known inhibitors for MRP1[63] will also interfere with MRP2-mediated transport. Inhibitors acting on MRP2, although less potently than on MRP1,[11] include the quinoline derivative MK571[9] and cyclosporin A.[52] Selective inhibitors for MRP2 may be of interest to overcome MRP2-mediated drug resistance[18] [36] and to enhance the intestinal absorption of drugs that are MRP2 substrates and are otherwise transported back into the intestinal lumen.

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MEMBERS OF THE MRP FAMILY LOCALIZED TO THE BASOLATERAL MEMBRANE

The founding member of the MRP family, MRP1,[31] has been localized to the basolateral membrane of polarized pig kidney cells after transfection with human MRP1 cDNA.[64] In hepatocytes, however, the expression of MRP1 mRNA is extremely low[31] and the immunoreactive proteins detected at the basolateral hepatocyte membrane by use of antibodies, raised against human MRP1,[7] [19] could only be fully identified after the more recent cloning of the isoforms MRP3[20] [21] and MRP6.[30] [65] [66] [67] MRP3 and MRP6 have been localized to the basolateral membrane of human hepatocytes and other polarized epithelial cells.[20] [21] [30] [65] MRP6 is constitutively and highly expressed in hepatocytes and kidney,[66] but its substrate specificity has not yet been elucidated. MRP3 is low under normal conditions in hepatocytes and upregulated in cholestatic liver disease.[20] [21] [41] [42] [43] Under many conditions, MRP3 is regulated inversely when compared with MRP2.[68] It is of interest that MRP3 has also been localized to the basolateral membrane of cholangiocytes[21] and intestinal epithelia where it may contribute to the enterohepatic circulation of bile salts.[42] [43] [69]

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MRP2 DEFICIENCY IN DUBIN-JOHNSON SYNDROME

Some mutations in the MRP2 gene are associated with the absence of the MRP2 protein from the hepatocyte canalicular membrane.[34] [37] Several different mutations in the MRP2 gene have been discovered in humans[34] [37] [38] [70] and rats.[8] [16] The Dubin-Johnson syndrome in humans is an autosomal recessively inherited disorder characterized by conjugated hyperbilirubinemia and pigment deposition in the liver.[71] [72] [73] The deficient transport of monoglucuronosyl bilirubin and bisglucuronosyl bilirubin and other anionic conjugates from hepatocytes into bile is caused by the absence or the functional impairment of the MRP2 protein in the canalicular membrane.[19] [34] [37] [74] So far, however, mutations in the MRP2 gene leading to a functionally deficient protein inserted into the hepatocyte canalicular membrane have not been identified. Furthermore, we have neither detected truncated MRP2 protein in the hepatocytes from a Dubin-Johnson syndrome patient with a stop codon in exon 23 nor in a patient with a 6-nucleotide deletion in exon 30.[37] Current knowledge on the sites of mutations in the coding sequence and in splice sites of the MRP2 gene in Dubin-Johnson syndrome together with the exon-intron organization of the human MRP2 gene are depicted in Figure [1]. Determination of the exon-intron organization of the gene has been a prerequisite for the elucidation of mutations underlying Dubin-Johnson syndrome.[37] [38] The currently known mutations in the MRP2 gene are scattered preferentially over the 3′-proximal half of the mRNA including the exons encoding both nucleotide-binding domains (Fig. [1]).

Established mutations in patients with Dubin-Johnson syndrome include a nonsense mutation leading to a premature termination codon,[34] [37] a missense mutation affecting the first nucleotide-binding domain,[38] [70] a deletion mutation leading to the loss of two amino acids in the second nucleotide-binding domain,[37] splice junction mutations leading to exon deletions and premature termination codons,[38] [70] [75] and a mutation causing an isoleucin to phenylalanine exchange in position 1173.[76] Furthermore, mutations were identified in two well-characterized hyperbilirubinemic rat strains, which have been considered as animal models of the human Dubin-Johnson syndrome, the GY/TR- mutant rat[8] and the Eisai hyperbilirubinemic rat.[16] These mutations introduce premature termination codons at codon 401 and 855 in GY/TR- and Eisai hyperbilirubinemic rat mutant rats, respectively. In both mutant livers, however, no truncated proteins were detected,[9] and the MRP2 mRNA was below detectability as analyzed by Northern blotting.[8] [9] [16] The introduction of premature termination codons may lead to a decrease in the level of mRNA by a mechanism termed ``nonsense mediated decay.''[77] In the case of a stop codon 5′ of the last splice site, this is recognized during translation, and the mRNA is subjected to decay.[77] It is likely that the absence of the MRP2 protein from the hepatocyte canalicular membrane in certain cases of Dubin-Johnson syndrome[19] [37] [74] is also a consequence of the rapid degradation of the mutated mRNA. Other mutations in the MRP2 gene may lead to a reduced stability of the protein, may affect the interaction of the MRP2 protein with chaperone proteins, may influence trafficking and apical localization of the protein, or may lead to an apically localized but functionally deficient MRP2 protein. These alternatives should be considered and possibly investigated for each of the mutations in the MRP2 gene, unless mutations leading to a premature termination codon allow for a simpler interpretation of the consequences.

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INTERACTION OF THE APICAL AND BASOLATERAL CONJUGATE EXPORT PUMPS: MRP2 AND MRP3

The release on anionic conjugates from hepatocytes into sinusoidal blood under conditions of extrahepatic cholestasis or MRP2 deficiency can be mediated by the basolateral export pump MRP3 (Fig. [2]). This is indicated by the basolateral localization of MRP3 in human and rat hepatocytes[20] [21] and by the substrate preference of MRP3 for anionic conjugates formed inside hepatocytes both under normal conditions and during cholestasis or MRP2 deficiency.[42] [78] [79] Substrates for MRP3 include glucuronosyl bilirubin,[78] 17β-glucuronosyl estradiol,[42] [79] and sulfated bile salts such as sulfatolithocholyl taurine and sulfatochenodeoxycholyl taurine,[42] [79] whereas glutathione conjugates are relatively poor substrates for MRP3 when compared with MRP1 and MRP2.[79] The pronounced expression of MRP3 in the basolateral membrane of hepatocytes from patients with Dubin-Johnson syndrome[20] serves as a compensatory transport pathway for conjugates that cannot be secreted into bile and must therefore be released into blood followed by renal excretion. The conjugated hyperbilirubinemia in Dubin-Johnson syndrome suggests that the release of bilirubin glucuronides from hepatocytes via MRP3 is more rapid than renal excretion. The compensatory role of MRP3 in MRP2 deficiency and in extrahepatic cholestasis is also consistent with the upregulation of MRP3 in rat liver after bile duct ligation and in rats with hereditary MRP2 deficiency.[80] The noninvasive assessment of hepatobiliary and renal secretion of anionic conjugates in rats under such conditions has indicated a prolonged time period for intracellular storage and metabolism and an approximately threefold extension of hepatic excretion half-times followed by the complete renal elimination of substances that are eliminated under normal conditions via MRP2 into bile.[17]

The molecular mechanisms leading to the upregulation of MRP3 have not been sufficiently elucidated.[42] [68] [79] [80] [81] Characterization of the 5′-flanking region of human MRP3 demonstrates that MRP3 is under the control of a TATA-less promoter and that Sp1 binding sites may be involved in the regulation of its expression.[81] Evidence has not been obtained supporting a direct action of bilirubin as an inducer.[81] The basal promoter activity of human MRP3 is only 4 % of that measured for MRP2[68]; however, MRP3 promoter activity, mRNA, and protein are markedly increased after disruption of microtubules by nocadazol.[68] This treatment leads, on the other hand, to a downregulation of promoter activity, mRNA, and protein of MRP2, exemplifying one of several conditions under which MRP3 and MRP2 are inversely regulated in liver and hepatocyte-derived cells.[68]

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ABBREVIATIONS

ABC ATP-binding cassette

ABCC ATP-binding cassette transporter subfamily C

kbp kilo-base pair

MRP multidrug resistance protein

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Figure 1. Mutations in the MRP2 gene leading to Dubin-Johnson syndrome. The genomic organization of the human MRP2 gene is characterized by 32 exons and a size of the total gene of about 45 kbp.[37] The exon-intron boundaries (GenBank accession AJ132244), the number of exons, and both ATP-binding domains are indicated. Arrows indicate the sites of mutations currently identified. F1-F4 correspond to mutations identified in Fukuoka,[38] [70] S1 indicates a mutation identified in Saga,[75] H1 and H2 correspond to mutations studied in Heidelberg,[37] A1 is an identical mutation as H1 and was first described in Amsterdam,[34] and T1 designates the mutation present in a large group of Iranian Jews analyzed in Tel Aviv.[76]

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Figure 2. Localization and function of the conjugate export pumps MRP2 and MRP3 in normal and MRP2-deficient hepatocytes. (Top) The uptake of various substances across the basolateral membrane, followed by conjugation, and MRP2-mediated export across the apical (canalicular) membrane is shown. (Bottom) Situation in extrahepatic cholestasis or MRP2 deficiency with the compensatory function of MRP3 mediating export of conjugates across the basolateral membrane into sinusoidal blood.

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  • 51 Bock K W, Eckle T, Ouzzine M. Coordinate induction by antioxidants of UDP-glucuronosyltransferase UGT1A6 and the apical conjugate export pump MRP2 (multidrug resistance protein 2) in Caco-2 cells.  Biochem Pharmacol . 2000;  59 467-470
  • 52 Cantz T, Nies A T, Brom M. MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles of Hep G2 cells.  Am J Physiol Gastrointest Liver Physiol . 2000;  278 G522-G531
  • 53 Kubitz R, D'Urso D, Keppler D. Osmodependent dynamic localization of the multidrug resistance protein 2 in the rat hepatocyte canalicular membrane.  Gastroenterology . 1997;  113 1438-1442
  • 54 Roelofsen H, Soroka C J, Keppler D. Cyclic AMP stimulates sorting of the canalicular organic anion transporter (Mrp2/cMoat) to the apical domain in hepatocyte couplets.  J Cell Sci . 1998;  111 1137-1145
  • 55 Trauner M, Arrese M, Soroka C J. The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis.  Gastroenterology . 1997;  113 255-264
  • 56 Vos T A, Hooiveld G J, Koning H. Up-regulation of the multidrug resistance genes, Mrp1 and Mdr1b, and down-regulation of the organic anion transporter, Mrp2, and the bile salt transporter, Spgp, in endotoxemic rat liver.  Hepatology . 1998;  28 1637-1644
  • 57 Kubitz R, Wettstein M, Warskulat U. Regulation of the multidrug resistance protein 2 in the rat liver by lipopolysaccharide and dexamethasone.  Gastroenterology . 1999;  116 401-410
  • 58 Hagmann W, Denzlinger C, Keppler D. Role of peptide leukotrienes and their hepatobiliary elimination in endotoxin action.  Circ Shock . 1984;  14 223-235
  • 59 Keppler D, Hagmann W, Rapp S. The relation of leukotrienes to liver injury.  Hepatology . 1985;  5 883-891
  • 60 Takikawa H, Sano N, Narita T. Biliary excretion of bile acid conjugates in a hyperbilirubinemic mutant Sprague-Dawley rat.  Hepatology . 1991;  14 352-360
  • 61 Suzuki H, Sugiyama Y. Transporters for bile acids and organic anions. In: Amidon GL, Sadée W (eds). Membrane transporters as drug targets New York: Kluwer Academic/Plenum, 1999: 387-439
  • 62 Leier I, Hummel-EisenbeissJ, C ui. ATP-dependent para-aminohippurate transport by apical multidrug resistance protein MRP2.  Kidney Int . 2000;  57 1636-1642
  • 63 Norman B H. Inhibitors of MRP1-mediated multidrug resistance.  Drugs Future . 1998;  23 1001-1013
  • 64 Evers R, Zaman G J, van Deemter L. Basolateral localization and export activity of the human multidrug resistance-associated protein in polarized pig kidney cells.  J Clin Invest . 1996;  97 1211-1218
  • 65 Borst P, Evers R, Kool M. The multidrug resistance protein family.  Biochim Biophys Acta . 1999;  1461 347-357
  • 66 Kool M, van der Linden M, de Haas M. Expression of human MRP6, a homologue of the multidrug resistance protein gene MRP1, in tissues and cancer cells.  Cancer Res . 1999;  59 175-182
  • 67 Madon J, Hagenbuch B, Landmann L. Transport function and hepatocellular localization of mrp6 in rat liver.  Mol Pharmacol . 2000;  57 634-641
  • 68 Stöckel B, König, J, Nies A T. Characterization of the 5′-flanking region of the human multidrug resistance protein 2 (MRP2) gene and its regulation in comparison with the multidrug resistance protein 3 (MRP3) gene.  Eur J Biochem . 2000;  267 1347-1358
  • 69 Hirohashi T, Suzuki H, Chu X Y. Function and expression of multidrug resistance-associated protein family in human colon adenocarcinoma cells (Caco-2).  J Pharmacol Exp Ther . 2000;  292 265-270
  • 70 Wada M, Toh S, Taniguchi K. Mutations in the canalicular multispecific organic anion transporter (cMOAT) gene, a novel ABC transporter, in patients with hyperbilirubinemia II/Dubin-Johnson syndrome.  Hum Mol Genet . 1998;  7 203-207
  • 71 Dubin I N, Johnson F B. Chronic idiopathic jaundice with unidentified pigment in liver cells: A new clinicopathologic entity with report of 12 cases.  Medicine . 1954;  33 155-179
  • 72 Sprinz H, Nelson R S. Persistent nonhemolytic hyperbilirubinemia associated with lipochrome-like pigment in liver cells: Report of four cases.  Ann Intern Med . 1954;  41 952-962
  • 73 Roy Chowdhury J, Roy Chowdhury N, Wolkoff A W. Heme and bile pigment metabolism. In: Arias IM, Boyer JL, Fausto N, et al (eds). The liver: Biology and pathobiology. New York: Raven Press, 1994: 471-504
  • 74 Kartenbeck J, Leuschner U, Mayer R. Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome.  Hepatology . 1996;  23 1061-1066
  • 75 Kajihara S, Hisatomi A, Mizuta T. A splice mutation in the human canalicular multispecific organic anion transporter gene causes Dubin-Johnson syndrome.  Biochem Biophys Res Commun . 1998;  253 454-457
  • 76 Mor-Cohen R, Zivelin A, Rosenberg N. Identification of a common ile1173phe mutation in the canalicular multispecific organic anion transporter gene in patients with Dubin-Johnson syndrome of Iranian-Jewish origin.  Am J Human Genetics . 1999;  581 (581)
  • 77 Thermann R, Neu-Yilik G, Deters A. Binary specification of nonsense codons by splicing and cytoplasmic translation.  EMBO J . 1998;  17 3484-3494
  • 78 Keppler D, Kamisako T, Leier I. Localization, substrate specificity, and drug resistance conferred by conjugate export pumps of the MRP family.  Adv Enzyme Regul . 2000;  40 339-349
  • 79 Hirohashi T, Suzuki H, Sugiyama Y. Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).  J Biol Chem . 1999;  274 15181-15185
  • 80 Hirohashi T, Suzuki H, Ito K. Hepatic expression of multidrug resistance-associated protein-like proteins maintained in Eisai hyperbilirubinemic rats.  Mol Pharmacol . 1998;  53 1068-1075
  • 81 Takada T, Suzuki H, Sugiyama Y. Characterization of 5′-flanking region of human MRP3.  Biochem Biophys Res Commun . 2000;  270 728-732
#

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  • 64 Evers R, Zaman G J, van Deemter L. Basolateral localization and export activity of the human multidrug resistance-associated protein in polarized pig kidney cells.  J Clin Invest . 1996;  97 1211-1218
  • 65 Borst P, Evers R, Kool M. The multidrug resistance protein family.  Biochim Biophys Acta . 1999;  1461 347-357
  • 66 Kool M, van der Linden M, de Haas M. Expression of human MRP6, a homologue of the multidrug resistance protein gene MRP1, in tissues and cancer cells.  Cancer Res . 1999;  59 175-182
  • 67 Madon J, Hagenbuch B, Landmann L. Transport function and hepatocellular localization of mrp6 in rat liver.  Mol Pharmacol . 2000;  57 634-641
  • 68 Stöckel B, König, J, Nies A T. Characterization of the 5′-flanking region of the human multidrug resistance protein 2 (MRP2) gene and its regulation in comparison with the multidrug resistance protein 3 (MRP3) gene.  Eur J Biochem . 2000;  267 1347-1358
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  • 72 Sprinz H, Nelson R S. Persistent nonhemolytic hyperbilirubinemia associated with lipochrome-like pigment in liver cells: Report of four cases.  Ann Intern Med . 1954;  41 952-962
  • 73 Roy Chowdhury J, Roy Chowdhury N, Wolkoff A W. Heme and bile pigment metabolism. In: Arias IM, Boyer JL, Fausto N, et al (eds). The liver: Biology and pathobiology. New York: Raven Press, 1994: 471-504
  • 74 Kartenbeck J, Leuschner U, Mayer R. Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome.  Hepatology . 1996;  23 1061-1066
  • 75 Kajihara S, Hisatomi A, Mizuta T. A splice mutation in the human canalicular multispecific organic anion transporter gene causes Dubin-Johnson syndrome.  Biochem Biophys Res Commun . 1998;  253 454-457
  • 76 Mor-Cohen R, Zivelin A, Rosenberg N. Identification of a common ile1173phe mutation in the canalicular multispecific organic anion transporter gene in patients with Dubin-Johnson syndrome of Iranian-Jewish origin.  Am J Human Genetics . 1999;  581 (581)
  • 77 Thermann R, Neu-Yilik G, Deters A. Binary specification of nonsense codons by splicing and cytoplasmic translation.  EMBO J . 1998;  17 3484-3494
  • 78 Keppler D, Kamisako T, Leier I. Localization, substrate specificity, and drug resistance conferred by conjugate export pumps of the MRP family.  Adv Enzyme Regul . 2000;  40 339-349
  • 79 Hirohashi T, Suzuki H, Sugiyama Y. Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).  J Biol Chem . 1999;  274 15181-15185
  • 80 Hirohashi T, Suzuki H, Ito K. Hepatic expression of multidrug resistance-associated protein-like proteins maintained in Eisai hyperbilirubinemic rats.  Mol Pharmacol . 1998;  53 1068-1075
  • 81 Takada T, Suzuki H, Sugiyama Y. Characterization of 5′-flanking region of human MRP3.  Biochem Biophys Res Commun . 2000;  270 728-732
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Figure 1. Mutations in the MRP2 gene leading to Dubin-Johnson syndrome. The genomic organization of the human MRP2 gene is characterized by 32 exons and a size of the total gene of about 45 kbp.[37] The exon-intron boundaries (GenBank accession AJ132244), the number of exons, and both ATP-binding domains are indicated. Arrows indicate the sites of mutations currently identified. F1-F4 correspond to mutations identified in Fukuoka,[38] [70] S1 indicates a mutation identified in Saga,[75] H1 and H2 correspond to mutations studied in Heidelberg,[37] A1 is an identical mutation as H1 and was first described in Amsterdam,[34] and T1 designates the mutation present in a large group of Iranian Jews analyzed in Tel Aviv.[76]

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Figure 2. Localization and function of the conjugate export pumps MRP2 and MRP3 in normal and MRP2-deficient hepatocytes. (Top) The uptake of various substances across the basolateral membrane, followed by conjugation, and MRP2-mediated export across the apical (canalicular) membrane is shown. (Bottom) Situation in extrahepatic cholestasis or MRP2 deficiency with the compensatory function of MRP3 mediating export of conjugates across the basolateral membrane into sinusoidal blood.

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