Characterization of the Human Multidrug Resistance Protein Isoform Mrp3 Localized to the Basolateral Hepatocyte Membrane

Abstract

The membrane proteins mediating the adenosine triphosphate (ATP)-dependent transport of anionic conjugates and amphiphilic anions, such as the bilirubin glucuronosides, 1 have been identified as members of the multidrug resistance protein (MRP) family. 2-11 Export pumps for anionic conjugates in yeast, 12 plants, 13 and Caenorhabditis elegans 14 were subsequently recognized too as members of the MRP family of ATP-dependent membrane transporters (for reviews Keppler and Kö nig 15 and Ishikawa et al. 16 ). In humans, six members of the MRP family, designated MRP1 through 6, have been identified. 17,18 At present, two of these, human MRP1 19 and MRP2,7,20,21 have been functionally characterized as conjugate export pumps. 2,3,10,11,22 The occurrence of distinct MRP isoforms in hepatocytes has been initially shown by amplification and sequencing of novel complementary DNA (cDNA) fragments, by immunoblotting, and by immunofluorescence microscopy. 5 The canalicular MRP isoform deficient in Dubin-Johnson syndrome was identified as MRP2, 21,23,24 however, the molecular identity of the MRP isoform(s) localized to the lateral or basolateral hepatocyte membrane 5,23,25 remained uncertain. RNase protection assays indicated that the liver expresses MRP2, MRP3, MRP5, and MRP6, but hardly MRP1. 18,26 Identification of MRP isoforms in liver by immunoblotting or immunofluorescence microscopy has been difficult because of the cross-reactivity of some antibodies with different MRP isoforms. 5,7,23 MRP3 has been recognized as a prominent MRP family member expressed in rat liver. 26,27 Human MRP3 has been cloned recently by Kiuchi et al. 28 and by our group (GenBank/EMBL accession Y17151 and this article), however, its localization and function in the hepatocyte remained unknown. It has been our hypothesis that a MRP isoform localized to the lateral or basolateral membrane may serve to excrete conjugates, which are formed inside the hepatocyte, into the sinusoidal blood under conditions of extrahepatic cholestasis or hereditary deficiency of the apical MRP isoform, MRP2. 5,25 Such a compensatory excretion across the basolateral membrane may be fulfilled by basolateral organic anion transporters functioning bidirectionally, in addition to MRP isoforms localized to the basolateral membrane domain. Cloning of MRP3 in the present study has enabled us to raise antibodies specifically directed against this MRP isoform and to localize MRP3 to the basolateral membrane domain of human hepatocytes. MATERIALS AND METHODSMaterials. Aprotinin, leupeptin, pepstatin, fetal calf serum, agar, and the protein standard mixture (M r 26,600 to 180,000) for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were from Sigma (Deisenhofen, Germany). Agarose was from Roth (Karlsruhe, Germany) and lysozyme and ampicillin were from Boehringer Mannheim (Mannheim, Germany). Marathon-Ready cDNA and Advantage cDNA Polymerase Mix were from Clontech (Heidelberg, Germany). RNase inhibitor RNAguard, StrataScript Moloney murine leukemia virus reverse trans...

Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma

“…The double-band pattern for MRP3, with a 190 and a 170 kDa band, was also seen in normal human liver and may be attributable to at least 2 different splice variants. 19 Using the QCRL1 antibody for MRP1, no bands were detected in the 4 tumor samples analyzed, indicating low, if any, MRP1 expression.…”

“…14 The FDS antibody was raised in rabbits against the 24 C-terminal amino acids (FDSPANLIAARGIFYG-MARDAGLA) and the ALL antibody against the 20 -amino acid peptide (ALLIEDTLSNHTDLTDNDPV) corresponding to amino acids 876 -895 of the human MRP3 sequence. 19 The monoclonal antibody (MAb) QCRL1 against MRP1 22 and the MAb M 2 III-6 against MRP2 23 were from Alexis (San Diego, CA).…”

“…Total RNA (5 g) was reversetranscribed using 5 g oligo(dT) 18 primer as described. 19 Synthesized sscDNAs were purified using Microcon (Bedford, MA) 100 filter units.…”

“…3a-d,f). In addition, the polyclonal ALL antibody, which is also directed against MRP3, 19 was used in some cases (Fig. 3e).…”

“…18 Another hepatic MRP isoform, MRP3, was cloned from human liver and localized to the basolateral membrane of hepatocytes. 19,20 MRP3 also confers resistance to chemotherapeutic agents such as etoposide and methotrexate. 20 In this study, we demonstrate by semi-quantitative RT-PCR that MRP2 and MRP3 mRNA expression in HCC was at least 10-fold higher than MRP1 mRNA expression.…”