Báo cáo khoa học: Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2) potx

Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2) Anne T. Nies 1 ,Jo¨rg Ko¨ nig 1 , Yunhai Cui 1 , Manuela Brom 1 , Herbert Spring 2 and Dietrich Keppler 1 1 Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany; 2 Division of Cell Biology, Deutsches Krebsforschungszentrum, Heidelberg, Germany The human multidrug resistance p rotein 2 (MRP2, symbol ABCC2) is a polytopic membrane glycoprotein of 1545 amino acids which exports anionic conjugates across the apical membrane of polarized cells. A chimeric protein composed of C-proximal MRP2 and N-proximal MRP1 localized to the a pical membrane of polarized Madin–Darby canine kidney cells (MDCKII) indicating involvement of the carboxy-proximal part of human MRP2 in apical sorting. When compared to other MRP family members, MRP2 has a seven-amino-acid extension at its C-terminus with the last three amino acids (TKF) comprising a PDZ-interacting motif. In order to analyze whether this extension is required for apical sorting of MRP2, we g enerated MRP2 constructs mutated a nd stepwise truncated at their C-termini. T hese constructs were fused via their N-termini to green fluorescent protein ( GFP) and were transiently transfected into polar- ized, liver-derived hu man HepG2 cells. Q uantitative analysis showed th at full-length GFP– MRP2 was localized to the apical membrane in 73% of transfected, polarized cells, whereas it remained o n i ntracellular membranes in 27% of cells. Removal of the C-terminal TKF peptide a nd stepwise deletion of up to 11 amino acids did not change this pre- dominant apical d istribution. However, apical localization was largely impaired when GFP–MRP2 was C-terminally truncated by 15 or more amino acids. Thus, neither the PDZ-interacting TKF motif nor the full seven-amino-acid extension were necessary for apical sorting of MRP2. Instead, our d ata indicate that a d eletion of at least 15 C-terminal amino acids impairs the localiz ation o f MRP2 to the a pical membrane of polarized cells. Keywords: epithelial polarity; green fluorescent protein; multidrug r esistance protein 2; protein trafficking. Members of the multidrug resistance p rotein (MRP) f amily are i ntegral membrane glycoproteins which m ediate the ATP-dependent export of amphiphilic anions across the plasma membrane [1]. MRP1, t he first cloned member o f the MRP family [2], is present in the plasma membrane of several cell types [3–5]. After t ransfection of MRP1 cDNA in polarized cells, MRP1 is localized to the basolateral membrane [6]. Several M RP family members are known to be endogenously expressed in polarized cells. Whereas MRP3 [7,8] and MRP6 [9,10] are localized to the basolateral membrane of rat and human hepatocytes, MRP2 is the only isoform identified so far that is localized exclusively to the apical membrane of polarized cells, s uch as hepatocytes and renal proximal tubule cells [1,11,12]. MRP2 was initially cloned f rom rat liver [11,13,14], and subsequently from human liver [11,15,16] and human tumor cells [17]. Trans- port studies using inside-out oriented membrane vesicles from liver [18,19] or from cells stably transfected with human MRP2 cDNA [16,20,21] demonstrated the transport of conjugated and unconjugated lipophilic anions by MRP2. The absence of MRP2 from the canalicular membrane of human hepatocytes is the molecular basis of the Dubin–Johnson syndrome [ 15,22–24], which is associ- ated with conjugated hyperbilirubinemia. Epithelial cell polarity is a result of the d omain-specific sorting of p roteins. Neither a pical nor basolateral trafficking seems to f ollow a ÔdefaultÕ pathway, rather, specific signals or interactions are required f or inclusion of proteins into apically or basolaterally destined transport v esicles within the trans Golgi network (TGN; reviewed in [25]). Basolat- eral sorting signals are m ost often tyrosine- o r dileucine- based motifs i n the cytoplasmic domains of proteins [26], however, other basolateral sorting signals have been also identified [27,28]. Several mechanisms have been described for a pical sorting. T hese include apical localization signals in the extracellular, transmembrane, or cytoplasmic domains [29]. For several apical proteins, clustering into cholesterol- and sphingolipid-rich, detergent-insoluble microdomains has been demonstrated to be important for the formation of apical vesicles from the TGN [30]. In addition to active sorting into specific transport vesicles within the TGN, selective stabilization of proteins in their respective membrane domains has been suggested [31]. One mechanism by which this may b e achieved is t he binding of membrane proteins via their C-termini to PDZ domain-containing proteins. The latter recognize a consen- sus s equence ( T/S-X-V/I) at the C-termini of membrane proteins [32]. Interaction of these PDZ-interacting motifs with PDZ domain-containing proteins has been shown to be required for the m embrane domain-specific sorting of some basolateral as well as of some apical membrane Correspondence to A. Nies, Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Fax: + 49 6221 422402, Tel.: + 49 6221 422403, E-m ail: a.nies@dkfz.de Abbreviations: GFP, green fluorescent protein; MRP2, multidrug resistance protein 2 (hum an genome nomenclature symbol ¼ ABCC2); PDZ, PSD-95/DlgA/ZO-1-like. (Received 23 January 2002, accepted 6 February 2002) Eur. J. Biochem. 269, 1866–1876 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02832.x proteins [33]. PDZ domain-containing proteins either bind directly or via adaptor proteins to the c ytoskeleton [33]. Present knowledge on the mechanisms by which MRP isoforms are targeted to t heir respective membrane domain in polarized cells is limited. We r ecently showed that a six- nucleotide d eletion w ithin t he human MRP2 gene causes Dubin–Johnson syndrome [24,34]. This mutation, leading to the loss of two amino a cids from the s econd nucleotide- binding domain [24], results in defective M RP2 m aturation and retention ofMRP2 in the ER, so that sorting o f M RP2 t o the a pical membrane i s i mpaired [34]. T he aim of the present study was t o identify structural d eterminants required f or apical sorting of human MRP2. B ecause MRP2 h as a seven- amino-acid extension at its C-terminus, which is not found in the basolaterally localized isoforms MRP1, MRP3, and MRP6 [7], it was hypothesized that this C-terminal extension contains a signal for apical localization of MRP2. In addition, the C-terminal three amino acids of MRP2 were identified as a motif interacting with a PDZ domain- containing protein [35]. A recent study described that deletion of this PDZ-interacting motif leads to localization of MRP2 predominantly in the basolateral membrane o f polarized Madin–Darby canine kidney (MDCK) cells [36]. This result may, however, be misleading because MRP2 was tagged at the C-terminus with GFP a nd interaction with P DZ domain-containing proteins may be disrupted by the addi- tion of amino acids to the C-terminal PDZ-interacting m otif [37,38]. In addition, hu man proteins m ay localize differently in canine cells. In the present work, we therefore used human MRP2 tagged with GFP at t he N-terminus, t hus leaving the C-terminus free for possible binding of interacting proteins. With this experimentalsetup, we show that, in contrast to our expectations, the C-terminal 11 amino acids of MRP2, including the PDZ-interacting motif, were not necessary for apical sorting of MRP2 in polarized human HepG2 cells. However, truncation b y more t han 15 amino acids resulted in impaired delivery of M RP2 to the apical membrane . MATERIALS AND METHODS Materials and antibodies Fetal bovine serum and agarose were from Sigma (St Louis, MO, USA). Pfu DNA polymerase, restriction enzymes, ligase, and m odifying e nzymes were from Stratagene (La Jolla, CA, USA) or Promega (Madison, WI, U SA). Lysozyme and ampicillin were from Roche Molecular Biochemicals (Indianapolis, IN, USA). R hodamine-conju- gated concanavalin A was from Vector Laboratories (Burlingame, CA, USA). All other chemicals were of analytical grade and obtained either from M erck (Da rm- stadt, Germany) or Sigma. The polyclonal rabbit antibody directed against the C-terminus of human MRP2, EAG5, has b een described previously [11,12]. The mouse mAb to dipeptidylpepti- dase IV (CD26; anti-DPPIV Ig; clone 202.36) was from Ancell (Bayport, MN, USA), and t he mouse m onoclonal antibody to protein disulfide isomerase (PDI; clone RL90) was purchased from Affinity Bioreagents (Golden, CO, USA). The mouse monoclonal a nti-villin Ig was from Transduction Lab oratories (Lexington, KY, USA). Rat anti-(ZO-1) Ig w as from Chemicon (Temecula, CA, USA). Goat anti-(rabbit IgG) Ig coupled to Alexa Fluor546 or Alexa Fluor488 were from Molecular P robes (Eugene, O R, USA). Donkey anti-(rat IgG) Ig coupled to TexasRed and Cy3-conjugated goat anti-(mouse IgG) Ig were from Jackson Immunoresearch (West Grove, PA, USA). Generation of a cDNA encoding a MRP1/2 chimeric protein The cDNA encoding the c himeric MRP1/2 p rotein (Fig. 1) was constructed by g enerating a Xba I restriction site in the cDNA sequence of human MRP1 in a PCR-based approach. I n detail, a MRP1 cDNA f ragm ent was amplified using the MRP1 cDNA, inserted into the vector pcDNA3.1(+), as template and the T7 v ector primer as forward primer. The reverse primer ochimrp1.r ev was used to generate the Xba I restriction site in the MRP1 c DNA. It has the sequence 5¢-AGAGGGGATCATCTAGAAG GTA-3¢ (position 2386 –2365) a nd has three base-pair substitutions when compared with the MRP1 wild-type sequence: 2370G fi A, 2371A fi G, a nd 2373 G fi T. These substitutions were necessary to generate the XbaI restriction site. A  2500 bp fragment was PCR amplified using the following cycles: 5 min 94 °C, 5 cycles with 45 s at 94 °C denaturation, 45 s 5 5 °C annealing and 120 s 72 °C elongation, 30 cycles with 45 s 94 °C denaturation, 45 s at 65 °C a nnealing, and 120 s at 72 °C e longation, followed by 10 min at 72 °C. The fragment was subcloned into the vector pCR2.TOPO (Invitrogen, C arlsbad, CA, U SA) resulting in the plasmid pmrp1/XbaI.topo. Human MRP2 cDNA ( GenBank/EMBL accession number X96395) was cloned into pcDNA3.1(+) as described previously ([16], pMRP2). For generating a full-length cDNA encoding the MRP1/2 chimera, pMRP2 was restricte d with No t I/XbaI and the MRP1 cDNA fragment f rom t he pmrp1/XbaI.topo plasmid obtained b y NotI/XbaI restriction was inserted, thus gener ating the plasmid pmrp1/2chim.31. T he correct sequence of the fragment a nd the cloning sites w ere verified by sequencing and restriction a nalysis. Generation of green fluorescent protein (GFP)–MRP2 constructs Normal and C -terminally mutated GFP–MRP2 constructs were generated in the mammalian expression vector pcDNA3.1(+) (Invitrogen). After translation, GFP was attached to the N-terminus of the p roteins, so that the GFP moiety was in the lumen o f the ER or on the extracellular side (Fig. 2). Constructs were restriction-mapped and sequenced to verify correctness of the fragments. GFP, optimized for maximal fluorescence [39] and mam- malian e xpression [40], was cloned into the Bam HI and NotI restriction sites of the expression vector pcDNA3.1(+) (pGFP). GFP was P CR-amplified using the sense-primer 5¢-AGATCT GCCACCATGGTGAGC AAG-3¢,which introduced a BglII site (bold), and the antisense primer 5¢- CCGCGGCCGCTTGTATAGCTCGTCCATGCCG AG-3¢, which introduced a SacII (underlined) and a NotIsite (bold), a t t he same time removing the stop codon and the BsrGI site at the 3¢ end of the GFP coding sequence. PCR- amplified GFP w as cloned into the pDisplay vector (Invi- trogen) using the BglII and the SacII sites (plumGFP). pMRP2 was digested with NotIandBsrGI, a nd the fragment was replaced with a PCR-fragment that enabled Ó FEBS 2002 Apical sorting of human MRP2 (Eur. J. Biochem. 269) 1867 the in-frame insertion of GFP a t the N-terminus of MRP2 (pMRP2.1). The sense primer for this PCR reaction was 5¢-GCGGCCGCTCATGCTGGAGAAGTTCTG-3¢ (NotI site in b old) a nd the antisense primer was 5 ¢-GTGCCACA GAGTATCGA G-3¢. plumGFP vector was digested with HindIII and NotI, and the resulting GFP-encoding frag- ment including the murine Ig j-chain leader sequence was cloned into HindIII/NotI-digested pMRP2.1 (pGFP- MRP2). For generation o f C-terminal deletion constructs, a 2346-bp DNA fragment encoding the C -proximal part o f MRP2 was generated by PCR w ith ApaIandSacII sites added at the 3 ¢ end during a mplification. Primers u sed were 5 ¢-AGCGGATCAGCCTGG-3¢ (sense primer) and 5¢-GGGC CCGCGGCTAGAATTTTGTGCTGTTCAC-3¢ (antisense primer, ApaI site bold, SacII site underlined). This PCR fragment was ligated into ApaI-digested pMRP2 (pMRP2.2). C-Terminal deletion constructs were gen erated by cloning PCR-amplified fragments into the Bsu 36I and the SacII s it es of pMRP2.2. For these PCR reactions, the sense primer was 5¢-CCTGTTCTCTGGAAGCC-3¢ and the antisense primers were 5 ¢-CCGCGGCTAGCTGTTC ACATTCTCAATG-3¢ (MRP2D3), 5¢-CCGCGGCTACT CAATGCCAGCTTCCTT-3¢ (MRP2D7), 5¢-CCGCGG CTATTCCTTAGCCATAAAGTAAAA-3¢ (MRP2D11), 5¢-CCGCGGCTAAAAGTAAAAGGGTCCAGGG-3¢ (MRP2D15), 5¢-CCGCGGCTAAGGGATTTGTAGCA GTTCT-3¢ (MRP2D20), 5¢-CCGCGGCTATTCTTCA GGGCTGCCGC-3¢ (MRP2D25), 5 ¢-CCGCGGCTATTC CTTAGCCATTTCTTCAGGGCTGCCGC-3¢ (MRP2 D25MAKE), 5¢-CCGCGGCTACAGCCTGTGGGCGA TGG-3¢ (MRP2D50), 5¢-CCGCGGCTACAGCAGCTG CCTCTGGC-3¢ (MRP2D100), 5¢-CCGCGGCTAGAAT TTTGCGCTGTTCACATTC-3¢ (MRP2T1543 A), and 5¢-CCGCGGCTAGAATTTTGTAAAGTAAAAGGGT CCAGGG-3¢ (MRP2D15TKF). G FP constructs were gen- erated by digesting pGFP-MRP2 with HindIII/BsrGI and by cloning this fragment into the respective HindIII/BsrGI- digested deletion construct. Cell culture and transfection HumanhepatomaHepG2andMDCKcells(strainII)were maintained in Dulbecco’s modified Eagle’s medium (Sig- ma), supplemented with 10% (v/v) fetal bovine serum, penicillin (100 UÆmL )1 ) and streptomycin (100 lgÆmL )1 ). For transient transfections, cells were seeded into 35-mm and 1 00-mm and dishes a t a density of 5 · 10 5 and 5 · 10 6 cells per d ish, respectively, 24 h prior to transfection. HepG2 cells were transfected with the FuGENE 6 transfec tion reagent (Roche Molecular Biochemicals) according to the manufacturer’s instructions using 5 and 25 lLtransfection reagent and 1.5 and 7.5 lg DNA per 35- and 100-mm dish, respectively. MDCKII cells were transiently or s tably [16] transfected using calcium phosphate precipitation or the FuGENE transfection reagent. Immunofluorescence microscopy HepG2 or MDCKII cells grown on glass cover slips were fixed with methanol at )20 °Cfor1minandrehydratedin NaCl/P i . C ells were incubated w ith the primary antibody for 60 min at room temperature, washed three times with NaCl/P i , incubated with the secondary antibody for 60 min, andthenwashedagainthreetimeswithNaCl/P i . C over slips were mounted in Moviol (Hoechst, F rankfurt, Germany) and observed on a confocal laser scanning microscope (LSM 510, Carl Zeiss, Jena, Germany) using t he excita tion wavelengths of t he argon i on (488 nm) and the helium/neon laser (543 nm). Prints were taken of optical sections of 0.8-lm thickness. Antibodies were diluted in NaCl/P i at Fig. 1. Predicted topology models (A) and localization of MR P2 (B,C) and chimeric MRP1/2 (D,E) in p olarized MDCKII cells. The chimeric MRP1/2 consists of the MRP1 sequence followed by the sequence of MR P2 starting at am ino acid 791. For MRP2, only four tr ansmembrane s egments are predicted between both nucleotide-bindin g domains (NBD1 and NBD2 [43]), whereas six trans- membrane segments are predicted for MRP1 [44]. M DCKII cells stably synthesizing MRP2 or chimeric MRP1/2 were immunostained with the EAG5 a ntibody directed against MRP2 (green in B– E). Both p roteins were localized to the apical membrane as observed in the x–y plane (B,D) an d the x–z plane (C,E). Nuclei were stained with propidium iodide ( red in B–E). Bar, 1 0 lm. 1868 A. T. Nies et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the following dilutions: anti-(ZO-1) Ig (1 : 100), EAG5 (1 : 200), anti-PDI Ig (1 : 400), anti-DPPIV Ig (1 : 500), and t he respective secondary antibodies at 1 : 300. For staining of lysosomes, LysoTracker Red (Molecular Probes) was used according to the manufacturer’s i nstructions. For staining of th e apical membrane of M DCKII cells, r hod- amine-labeled concanavalin A was added to the apical chamber o f a Transwell filter insert at 5 lgÆmL )1 according to a method described recently [41]. Live HepG2 cells expressing GFP were observed as described previously [42]. Quantitative analysis of the subcellular localization of C-terminally mutated and truncated GFP-MRP2 proteins in polarized HepG2 cells HepG2 c ells were transiently transfected and immuno- stained with the anti-DPPIV Ig a s described above. For each transfection, at least 100 transfected (as observed by GFP fluorescence) and polarized (as observed by ring-like DPPIV fluorescence) cells were counted on a fluorescence microscope (Axioskop ; Carl Z eiss, Jena, Germany). For each transfected and polarized cell, the localization of t he respective GFP–MRP2 protein was analyzed and classified into one of three categories as follows: when GFP and DPPIV fluorescenc e merged in ring-like, microvilli-li ned structures between adjacent c ells, i.e. the apical membrane [42], the localization was defined as ÔapicalÕ, i rrespective of additional i ntracellular G FP fluorescence. When G FP fluorescence was absent from these r ing-like structures i n polarized cells, but observed in v esicular structures, l ocal- ization w as defined as Ôvesicular Õ. When DPPIV fluorescence was present in the ring-like structures and GFP fluorescence appeared exclusively reticular, localization w as defined as endoplasmic reticulum (ER). Localization of t he respective GFP–MRP2 in the E R was confirmed by colocalization with an antibody against an ER marker protein, p rotein disulfide isomerase (data not shown), as described previ- ously [34]. F or each GFP–MRP2 construct, the percentage of each localization was calculated. At least four indepen- dent transfections were analyzed in this way. For analysis o f the s teady-state distribution of GFP–MRP2 proteins, cells were induced with 5 m M butyrate for 24 h [16] and observed 48 h a fter start o f transfection. For a nalysis of t he time- course of GFP–MRP2 protein localization, cells were observed after 1, 2, 3, and 4 days post-trans fection w ithout prior induction with butyrate. For assessment of polarity, HepG2 cells were double- labeled with anti-DPPIV Ig (1 : 100) and EAG5 (1 : 100), or anti-villin Ig (1 : 100) and EAG5 (1 : 100), and the respective secondary antibodies as described above. Apical vacuoles staining positive for DPPIV and MRP2 or villin and MRP2 w ere counted on a fluorescence microscope (Axioskop). RESULTS Apical localization of a MRP1/2 chimeric protein in polarized MDCKII cells The amino-acid identity of only 48% between the laterally localized isoform MRP1 a nd the a pically localized isoform MRP2 [1] hampers the identification of apical sorting signals in the MRP2 sequence by direct comparison of both sequences. W e therefore constructed a cDNA encoding a MRP1/2 chimeric protein and immunolocalized this chi- meric protein in MDCKII cells (Fig. 1). The chimeric MRP1/2 protein was localized in the apical membrane of polarized MDCKII cells as was full-length MRP2 (Fig. 1 ) suggesting that the C-proximal part of MRP2 contains information for apical sorting of MRP2. Apical localization of GFP–MRP2 in polarized HepG2 and MDCKII cells A s equence alignment of the C-terminal ends of human MRP1, MRP2, MRP3, and MRP6 (Fig. 2) shows th at the apical MRP2 has a seven amino-acid extension in compar- ison to the basolateral fa mily members M RP1, MRP3, a nd MRP6. Recombinant MRP1 was localized to the basolat- eral membrane in polarized porcine cells [6]. MRP3 and MRP6 are endogenously synth esized in polarized cells such Fig. 2. Alignment of t he C-termini of members of the human MRP family (A) a nd predicted topology models of MRP2, GFP–MRP2, and lumGFP (B ). According t o the pre diction of the TMHMM program [45], and experimentally confirmed [16], the N-terminus of MRP2 has an extracellular location. Th eref ore, a cDNA w as constructed which encoded a fusion protein of G FP and MRP2 with t he GFP moiety targeted to the lumen of the ER, followed by t he complete sequ ence of human MRP2 ( GFP–MRP2). Expression of GFP from the pD isplay vector (lumGFP for Ôlu mena l GFPÕ) resulted in a GFP which was targeted to the lumen of the ER b ecause of a murine Ig j-chain leader sequence ([47]; b lack box) at the N -terminus of GFP and which w as anchored in the plasma membrane due to th e platelet-derived growth factor receptor transmembrane domain a t the C-terminus of G FP ([48]; cross-hatched box). Ó FEBS 2002 Apical sorting of human MRP2 (Eur. J. Biochem. 269) 1869 as hepatocytes and localized in the basolateral membrane [7–10]. Because the extension of MRP2 m ight represent a signal for a pical localization of M RP2, we generated MRP2, which was mutated or stepw ise truncated at its C-terminus, and analyzed quantitatively the localization of these MRP2-derived proteins in polarized HepG2 cells. In order to distinguish between endogenous MRP2 in HepG2 cells [42,46] and C-terminally mutated MRP2 i n t hese cells, we constructed cDNAs c oding for f usion p roteins of M RP2 and GFP. B ecause a ÔfreeÕ C-terminus may b e necessary for proper apical sortin g o f M RP2, e.g. by binding of interact- ing p roteins, GFP was fused to the N-terminus of MRP2. The N -terminus of MRP2 is located on the extracellular s ide [16], therefore a cDNA was constructed which led to translation of a GFP inserted into t he lumen of the ER by themurineIgj-chain leader sequence, a s equence described to target proteins to the secre tory pathway [47], followed by the s equence of M RP2 (Fig. 2). This GFP–MRP2 fusion protein was localized to the apical membrane of polarized HepG2 cells (Fig. 3). When lumenal GFP (lumGFP) was expressed from the pDisplay vector, lumGFP was not secreted into the medium but anchored to the plasma membrane due to the platelet-derived growth factor recep- tor (PDGFR) transmembrane domain at the C-terminus of GFP (Fig. 2, [48]). This PDGFR domain is not present in the G FP–MRP2 constructs (Fig. 2). LumGFP was equally distributed in the apical and the basolateral membrane of polarized HepG2 cells, and, in ad dition, in intracellular vesicular structures (Fig. 3) indicating that neither t he murine Ig j-chain leader sequence nor the PDGFR transmembrane domain contained a specific signal for apical localization. To exclude an effe ct of GFP on MRP2 targeting, the distribution of GFP in polariz ed HepG2 cells was analyzed (Fig. 3). The soluble GFP was p resent within the cells without any localization in the plasma membrane. As a control, GFP–MRP2 was also observed in MDCKII cells w here it l ocalized to the apical membrane (Fig. 4 ). The polarity of the MDCKII cells was confirmed by immunostaining with an a ntibody detecting t he tight- junctional protein ZO-1 (Fig. 4), indicating that the MDCKII cells were polarized under our experimental conditions. MDCKII cells synthesizing GFP–MRP2 were also immunostained with the EAG5 antibody resulting in identical fluorescence as the GFP fluorescence (Fig. 4). Because the EAG5 antibody was raised against the 15 C-terminal amino a cids of human MRP2 [11,12], this result demonstrates that the observed GFP fluorescence reflects localization of a complete GFP–MRP2 protein. The C-terminal PDZ-interacting motif is not required for apical sorting of MRP2 The C-terminal three a mino acids of t he human MRP2 sequence ( TKF, Fig. 2) have been r eported to interact with a PDZ domain-containing protein [35] and may t hus be necessary for apical sorting of MRP2. We t herefore deleted the C-terminal three amino a cids or substituted threonine with alanine at position 1543. The respective, mutated GFP–MRP2 was observed i n polarized HepG2 cells. F or quantitative analysis, localizatio n o f GFP–MRP2 proteins were classified i nto one of three c ategories as shown in the representative images of Fig. 5 and described in Materials and methods. Because apical vacuoles form betw een adjacent HepG2 cells as vesicle-like structures lined with microvilli [49], they can be s tained with antibodies either to cytoskeletal proteins such as villin [49,50] or with antibodies to canalicular membrane proteins such as DPPIV and MRP2 [42]. To assess the validity of DPPIV as a marker f or polarity, HepG2 cells were double-stained for DPPIV a nd MRP2. The majority (98.9%) of DPPIV-positive, microvilli-lined ring-like structures w ere also positive f or MRP2 (540 apical vacuoles counted). Similarly, 99.6% of villin-positive, microvilli-lined ring-like structures were also p ositive f or MRP2 (535 apical vacuoles counted). This result indicat es that staining for all three proteins, villin, DPPIV, and MRP2, can be used as marker for cell polarity i n HepG2 cells. Fig. 3. Localization of GFP–MRP2, lumGFP, and GFP i n polarized HepG2 cells. HepG 2 c ells were transiently transfected with G FP– MRP2 (A,B) or l umGFP (C,D), fixed 48 h after transfection, and immunostained with an antibody against dipeptidylpeptidase IV (DPPIV) in order to visualize apical vacuoles (B,D). GFP-transfected cells ( E,F) were visualiz ed by fluor escence microscopy (E) or by p hase- contrast (F). In GFP–MRP2-transfected cells (A), fl uorescence was observed in ring-like structures, i.e. the apical (vacuolar) membrane, and, in addition, in intracellular vesicular structures of varying size. In contrast, l umGFP (C) was observed in the basolateral and in t he apical membrane in equal amounts, and, additionally, in intracellular vesic- ular structu res, m ost like ly v esicle s o f t he sec reto ry p athway . GF P ( E) was distributed throughout t he cells without l ocalization to the plasma membrane. Asterisks mark the l umen of apical vacuoles. Bars, 10 lm. 1870 A. T. Nies et al. (Eur. J. Biochem. 269) Ó FEBS 2002 In 73% of transfected and polarized He pG2 cells GFP– MRP2 reached the apical membrane (Table 1). In t he remaining 27% of transfected and polarized cells, G FP– MRP2 did not reach t he apical membrane, but was present in intracellular compartments, such as vesicular structures and t he ER. Deletion o f t he C-terminal three a mino acids TKF or substitution of threonine w ith alanine led to proteins that were as efficiently sorted to the apical membrane of polarized HepG2 cells as was full-length MRP2 (Table 1). Furthermore, GFP–MRP2D15 which was predominantly l ocalized in the ER was not ÔrescuedÕ from this localization by addition of the TKF motif (Table 1). Asacontrol,localizationofGFP–MRP2,GFP–MRP2D3, and GFP–MRP2-T1543A was also analyzed in MDCKII cells grown polarized on Transwell filter membranes (Fig. 6). The a pical m embrane w as visualized by rhod- amine-conjugated concanavalin A added to the upper chamber of t he Transwell i nsert. GFP–MRP2, GFP– MRP2D3, and GFP–MRP2-T1543A were almost exclu- sively present in the apical membrane with some GFP fluorescence also present in intracellular compartments. None of the three analyzed proteins were observed in t he basolateral membrane. Localization of C-terminally truncated GFP–MRP2 proteins Because t he PDZ-interacting motif was not n ecessary for apical sorting of MRP2, the C-terminus of GFP–MRP2 was further truncated. T runcation of the C-terminus by seven or 11 amino acids led to protein s that reached the apical membrane of polarized HepG2 cells as full-length Fig. 4. Localization of GFP–MRP2 in polarized MDCKII cells. MDCKII cells transiently t ransfected with GFP–MRP2 were fixed 48haftertransfectionandimmunostainedwithanantibodyagainst the t ight-junctional protein ZO-1 (C,D), or with the E AG5 antibody (G,H) w hich is directed against the 15 C-te rminal amino a cids of human M RP2 [11,12]. The GFP fluorescence (A,B,E,F) shows that GFP–MRP2 is localized to the apical membrane, a s o bserved in the x–y plane (A,E) and the x–z plane (B,F). ZO-1 staining lines the cells in the x–y view (C), however, ZO-1 is restricted to the tight-junctions appearing as d ots in the vertical section (D). EAG5 fluorescence (G,H) was i dentical to th e GFP fluoresce nce (E,F) showing synthesis of a complete GFP–MRP2 protein. Bars, 10 lm. Fig. 5. Representative fluorescence images of subcellular localization of GFP–MRP2 con- structs i n polarized He pG2 cells as quantified in Tables 1–3. When GFP fluorescence ( A) and DPPIV fluorescence (B) m erged to yellow in the apical m embrane (C) the l ocalization of the GFP–MRP2 c onstruct was designated as ÔapicalÕ. When the GFP–MRP2 construct was present in intracellular vesicles (D) without reaching the apical membrane (E), no yellow color was observed (F). Some GFP–MRP2 constructs rem ained in reticu lar structures, i.e. the ER (G), and no G FP fluoresc ence of the apical vacuolar membr ane (H) was observed (I). Bars in A–I, 10 lm. Asterisks mark a pical vacuoles. Ó FEBS 2002 Apical sorting of human MRP2 (Eur. J. Biochem. 269) 1871 GFP–MRP2 (Table 2). However, delivery to t he apical membrane was largely impaired when GFP–MRP2 was C-terminally truncated by 15, 20, 25, 50 or 100 amino a cids. The p ercentage of polarized and transfected cells in which the respective protein reached the apical membrane decreased to 16% (GFP–MRP2D15), 15% (GFP– MRP2D20), 8 % ( GFP–MRP2D25), and 1% (GFP– MRP2D50, GFP–MRP2D100) with a concomitant accumulation of the proteins i n intracellular compartments, such as the E R and intracellular vesicles (Table 2). Because deletion of the tetrapeptide MAKE, i.e. a mino acids 1531– 1534, resulted in a s hift in the percentage of cells with an apical (GFP–MRP2 D11) to an intracellular localization (GFP–MRP2D15), this sequence might be involved i n the apical delivery of MRP2. However, addition of this tetrapeptide onto GFP–MRP2D25, which had an intracel- lular localization in most of the c ells, did not increase the number o f cells in whic h GFP–MRP2D25MAKE reached the apical m embrane. This r esult indicates t hat i t is not the co-linear s equence of the tetrapeptide that is required for apical de livery of MRP2. The intracellular vesicles contain- ing the respective GFP–MRP2 c onstruct were n ot lyso- somes as shown by t he lack of colocalization with the lysosomal marker LysoTracker Red (Fig. 7). Similarly, GFP–MRP2 constructs were not present in intracellular vesicles that contained DPPIV (Fig. 7). These results suggest that GFP–MRP2 was present in endosomes of yet unidentified nature. Because intracellular accumulation of GFP–MRP2 trunc- ated by 15–2 5 a mino acids may be due t o a delay in intracellular t ransport to the apical membrane we analyzed localization of GFP–MRP2, GFP–MRP2D15, and GFP– MRP2D25 from 1 to 4 days after the start of transfection (Table 3). There was no difference in the intracellular distribution of t he respective GFP–MRP2 p rotein over time. DISCUSSION MRP2 is the only MRP isoform known so far which localizes t o t he apical membrane of polarized cells [1,10]. Recently, the C-terminal three a mino acids (TRL) of the cystic fibrosis transmembrane conductance r egulator (CFTR), which comprise a PDZ-interacting m otif, were identified as a signal for apical localization [51]. Because CFTR is a member of t he MRP (ABCC) family with 27% amino-acid identity to MRP2 [1], we investigated whether the C-terminal tail of MRP2 is also involved i n apical sorting. Interaction of a PDZ domain-containing protein with the C-terminal three a mino a cids o f M RP2 ( TKF, Fig. 2) has been described previously [35]. The epithelial M DCKII cell line is often used t o s tudy the polarized sortin g of proteins t o different plasma membrane domains, however, some proteins a re sorted differently in the canine MDCKII cells as compared to polarized kidney cells from other species [52], therefore sorting o f human proteins might be different in a canine cell line. We therefore used human hepatoma HepG2 cells that polarize after several days in culture and form apical vacuoles reminiscent of bile canaliculi [49]. Because HepG2 cells endogenously Table 1 . Quantitative analysis of the subcellular localization of C-terminally mutated GFP–MRP2 c onstructs in polarized HepG2 cells. Data a re percentages o f cells in which the respective localization of recombinant protein was observed as described in Materials and methods. Cells were observed 2 d ays after transfection. Data are means ± SD of six transient transfections using butyrate-induce d cells as described under Materials and methods. Construct % Apical % Vesicles % ER C-Terminal sequence (1516–1545) GFP–MRP2 73 ± 9 18 ± 9 9 ± 5 GSPEELLQIPGPFYFMAKEAGIENVNSTKF GFP–MRP2D3 64±9 13±5 23±9 GSPEELLQIPGPFYFMAKEAGIENVNS GFP–MRP2-T1543A 67 ± 6 16 ± 2 17 ± 6 GSPEELLQIPGPFYFMAKEAGIENVNSAKF GFP–MRP2D15 16 ± 7 17 ± 7 67 ± 14 GSPEELLQIPGPFYF GFP–MRP2D15TKF 21 ± 11 21 ± 7 58 ± 11 GSPEELLQIPGPFYFTKF Fig. 6. Loca lization of GFP–MRP2 (green i n A,B), GFP–MRP2D3 (green in C,D), and GFP–MRP2-T1543A (green in E,F) in polarized MDCKII cells. MDC KII cells grown on Transwell filter membranes were transiently transfected with the resp ective construct and fixed 24 h after transfection. The a pical membrane was visualized by staining with rhodamine-conjugated concanavalin A (red fluo res- cence). In the x–y planes (A,C,E), th e GFP signals of all three con- structs g ive a pattern typical for apical localization. The intense yellow color in the x–z planes, due to merging of the green GFP and th e red concanavalin A fluorescence, shows that G FP–MRP2, GFP– MRP2D3, a nd G FP–MRP2-T1543A are a lmost exclusively locali zed in the apical membrane. Bars, 10 lm. 1872 A. T. Nies et al. (Eur. J. Biochem. 269) Ó FEBS 2002 synthesize MRP2 [42,46], we used GFP-tagged MRP2 to distinguish between endogenous and r ecombinant MRP2. Although MRP2 t agged with G FP at its C-terminus localized correctly to the apical m embrane in polarized HepG2 cells [1,34] we constructe d MRP2 t agged with GFP at the N-terminus in order to leave the C-terminus free for possible binding of interacting proteins. Interaction of t he C-terminal PDZ-interacting motif with PDZ domain-con- taining proteins seems t o require a free C-terminus [37,38]. A comparable approach of N-terminal GFP-tagging was taken f or the identification of apical localization signals in the C-termini of CFTR [51] and of the type IIb Na + /P i co-transporter [53]. In contrast to CFTR [51] and the type IIb Na + /P i co-transporter [53], the N-terminus of MRP2 is located extracellularly [16]. Therefore, a GFP–MRP2 was con- structed in which the GFP moiety was extracellular due to themurineIgj-chain leader sequence preceding the GFP sequence [47]. This sequence does not function as a signal for apical localization because GFP, when expressed from the pDisplay v ector, was ta rgeted to th e apical a nd to the basolateral membrane in e qual amounts (Fig. 3). Synthesis of extracellular GFP was also reported for other signal sequences known to direct proteins to the lumen of the ER [54,55]. As expected, GFP–MRP2 was localized to the apical membr ane of polarized HepG2 cells whereas GFP was not (Fig. 3). With this experimental setup, the e ffect o f C-terminal mutations and truncations on apical sorting of MRP2 was investigated. I n c ontrast to our expectations, neither the Table 2. Q uantitative analysis o f the subcellular localization of C-terminal deletion constructs in polarized HepG2 c ells. D ata are percentages of cells in which t he respective localization of recombinant protein was observed as described i n M aterials and m ethods. C ells wer e observed 2 days after start o f transfection. Data are means ± SD of n ¼ 6(GFP–MRP2D25MAKE, GFP–MRP2D50, GFP–MRP2D100, n ¼ 4) tr ans ien t trans- fections using butyrate-induced cells as described in Materials and me tho ds. Construct % Apical % Vesicles % ER C-Terminal sequence (1510–1545) GFP–MRP2 73 ± 9 18 ± 9 9 ± 5 GKIIECGSPEELLQIPGPFYFMAKEAGIENVNSTKF GFP–MRP2D7 69±7 18±6 13±3 GKIIECGSPEELLQIPGPFYFMAKEAGIE GFP–MRP2D11 65 ± 7 20 ± 4 15 ± 4 GKIIECGSPEELLQIPGPFYFMAKE GFP–MRP2D15 16 ± 7 17 ± 7 67 ± 14 GKIIECGSPEELLQIPGPFYF GFP–MRP2D20 15 ± 7 64 ± 9 20 ± 4 GKIIECGSPEELLQIP GFP–MRP2D25 8 ± 3 59 ± 14 33 ± 15 GKIIECGSPEE GFP–MRP2D25 MAKE 9 ± 4 59 ± 5 32 ± 5 GKIIECGSPEEMAKE GFP–MRP2D50 1±1 64±8 35±6 GFP–MRP2D100 1±1 35±7 65±6 Fig. 7. Localization of G FP–MRP2 constructs in vesicular s tructures in polarized HepG2 cells. HepG2 cells transiently synthesizing GFP– MRP2 (green in A,B) were incubated with LysoTracker Red t o stain lysosomes(redinA),orimmunostainedwithanantibodyagainst DPPIV t o stain D PPIV-cont aining vesicles (red in B). Absenc e of colocalization indicates that GFP–MRP2 is neither present in lyso- somes nor in DPPIV-containing vesicles. Bars, 2.5 lm. Table 3. Q uantitative analysis of the subcellular d istribution of GFP–MRP2, GFP–MRP2 D15, and GFP–MRP2D25 at different times after trans- fection in polarized HepG2 cells. Data are p ercentages o f cells in which t he respective l ocalization o f recombinant protein w as observed as described in Materials and me tho ds. Data are means ± SD of four transient transfec tion s. Experiments were performed without butyrate induction. Construct Time (days) % Apical % Vesicles % ER GFP–MRP2 1 70 ± 6 21 ± 8 9 ± 4 281±48±411±3 3 77±3 11±6 12±4 471±37±122±3 GFP–MRP2D15 1 9 ± 2 55 ± 10 36 ± 10 2 8±1 47±7 45±6 3 7±3 54±11 38±9 4 5±3 47±9 47±8 GFP–MRP2D25 1 2 ± 1 78 ± 10 20 ± 9 2 2±1 69±6 28±7 3 2±1 69±8 29±7 4 3±1 63±3 35±2 Ó FEBS 2002 Apical sorting of human MRP2 (Eur. J. Biochem. 269) 1873 PDZ-interacting m otif TKF nor the seven-amino-acid extension o f MRP2, which is not present in basolaterally localized MRP family members (Fig. 2), was required for apical sorting of GFP–MRP2 in polarized HepG2 cells (Tables 1 and 2). A similar result was obtained w ith t he type IIb Na + /P i co-transporter, whose C-terminal three amino acids (TVF) strongly resemble a PDZ-interacting motif. However, deletion of these amino acids did not affect the apical localization of the type IIb Na + /P i co-transporter [53]. Similarly, mutants of the basolateral GABA t rans- porter lacking the PDZ-interacting motif were still tar geted to the basolateral membrane [56]. Although the C-terminal PDZ-interacting motif of MRP2 is not required for apical sorting, it may b e necessary for linking additional regulatory proteins to MRP2 or for clustering of MRP2 i n the apical membrane in order to modulate function, as recently discussed for CFTR [57]. In addition, interaction of PDZ domain-containing proteins with internal PDZ-interacting motifs within the MRP2 protein may occur [58,59]. Whereas t he C-terminal 11 amino acids were not required for apical sorting of MRP2, a C-terminal deletion of 15 or more amino acids markedly reduced the percentage of cells in which MRP2 reached the a pical membrane ( Table 2). Because MRP2 is still observed in the apical membrane in a very low percentage of cells, MRP2 i s at least in part delivered into apically-destined vesicles. A truncation o f the C-terminus of MRP2 by at least 15 amino acids may cause the loss of a motif required either for efficient fusion of MRP2-containing vesicles with the apical m embrane o r for stabilization of MRP2 within the apical membrane. More- over, a MRP2 protein truncated by at least 1 5 a mino acids may alter the c onformation of the transport protein to such an extent tha t th e misfold ed protein i s retai ned in t he ER. A single leucine residue was r ecently shown to be p art of a, ye t unidentified, motif required for delivery of t he type IIb Na + /P i co-transporter t o the apical membrane [53]. Stabi- lization of the GABA transporter in the basolateral membrane has been demonstrated to be mediated by a PDZ-interacting motif [56]. Whereas GABA transporters lacking the PDZ-interacting motif were still targeted to the basolateral membrane t hey w ere not retained, but internal- ized into an endosomal recycling compartment. When the pre sent work w as in p rogres s, a stu dy was published describing the PDZ-interacting m otif as a signal for apical localization o f MRP2 [36]; deletion of the C-terminal three amino acids resulted in l ocalization of MRP2 predominantly to the basolateral membrane of MDCK cells. These observations are in d isagreement with our results. However, the differences may b e attributable to the expression in the canine MDCK cells of unspecified origin and t o t agging of MRP2 at the C -terminus [36] rather than expression of N-terminally tagged M RP2 i n human HepG2 cells (Tables 1 and 2) or polarized MDCKII cells (Fig. 6 ) as described in the p resent study. In conclusion, the C -terminal 1 1 amino acids o f human MRP2, including the PDZ-interacting motif, are not required f or ap ical sorting in polarized HepG2 cells. However, a C-terminal deletion of at least 15 amino acids prevents efficient delivery of the conjugate export pump MRP2 to the apical membrane e ither because p art of a motif required for apical sorting is lost or because of a conformational change in the transport p rotein impairing MRP2 maturation. ACKNOWLEDGEMENTS We thank Dr Tobias Cantz for contributions to this work and helpful discussion, Dr Blanche S chwappach for helpful discussions on G FP tagging, Dr Wolfgang H agmann for MRP1 cDNA , a nd Marion Pfannschmidt for excellent technical assistance. This work was supported in part by grants from t he Deutsche Forschungsgemein- schaft through S FB 352/B3. REFERENCES 1. Ko ¨ nig,J.,Nies,A.T.,Cui,Y.,Leier,I.&Keppler,D.(1999) Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate s pecificity, and MRP2- mediated drug resi stance. Biochim. Biophys. Acta 1461, 377–394. 2. Cole, S.P.C., Bhardwaj, G., Gerlach, J.H., Mackie, J.E., Grant, C.E., A lmquist, K.C., Stewart, A .J., Kurz, E.U., Duncan, A.M.V. & D eeley, R.G. (1992) Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 258, 1650– 1654. 3. Hipfner, D.R., Gauldie, S.D., Deeley, R.G. & Cole, S.P.C. (1994) Detection of the M r 190,000 multidrug protein, MRP, with monoclonal antibodies. Cancer Res. 54, 5788–5792. 4. Flens, M.J., Izquierdo, M.A., Scheffer, G.L., F ritz, J .M., Meijer, C.J., S ch eper, R.J. & Za m an, G.J. (1994) Immunochemical detection of the multidrug resistance-associated protein MRP in human multidrug-resistant tumor cells by monoclonal antibodies. Cancer Res. 54, 4 557–4563. 5. Flens, M.J., Zaman, G.J.R., van der Valk, P., Izquierdo, M.A., Schroeijers, A.B., Scheffer, G .L., van der Groep, P ., de Haas, M., Meijer, C.J.L.M. & Scheper, R .J. ( 1996) Ti ssue distribution o f the multidrug resistance p rotein. Am. J. Pathol. 148 , 1237–1247. 6. Evers, R., Z aman, G.J.R., van Deemter, L., Jansen, H., Calafat, J., O omen, L .C.J.M., Oude Elferink, R .P.J., Bors t, P. & S chinkel, A.H. (1 996) Basolateral l ocalization and export activity of the human multidrug resistance-associated protein in p o larized pig kidney cells. J. Clin. Invest. 97, 1211–1218. 7. Ko ¨ nig, J., Rost, D., Cui, Y. & Keppler, D. (1999) Characterization of the human multidrug resistance protein isoform MRP3 loca- lized to the basolateral hepatocyte membrane. Hepatology 29 , 1156–1163. 8. Kool, M., van der Linden, M., de Haas, M., Scheffer, G.L., de Vree, J.M., Smith, A.J., Jansen, G., Peters, G.J., Ponne, N., Scheper, R.J., Elferink, R.P., Baas, F. & Borst, P. (1999) MRP3, an organic anion transporter able to transport anti-cancer d rugs. Proc. Natl Acad. Sci. USA 96, 6914–6919. 9. Madon, J., Hagenbuch, B., Landmann, L ., Meier, P.J. & Stieger, B. (2000) Transport function and hepatocellular localization of mrp6 in rat liver. Mol. Pharmacol. 57, 634–641. 10. Ke ppler, D., Ko ¨ nig, J . & Nies, A.T. (2001) C onjugate export pumps of the multidrug resistance protein (MRP) family in liver. In The Liver: Biology and Pathobiology (Arias, I.M., Boyer, J.L., Chisari,F.V.,Fausto,N.,Schachter,D.&Shafritz,D.A.,eds),pp. 373–382. Lippincott, Williams & Wilkins, New Y ork. 11. Bu ¨ chler, M., Ko ¨ nig, J., Brom, M., K artenbeck, J ., Spring, H., Horie, T. & Kepple r, D. (1996) c DNA cloning of the h ep atocyte canalicular isoform of the multidrug resistance p rotein, cMRP, reveals a novel conjugate export pump deficient in hyper- bilirubinemic mutant r ats. J. Biol. C hem. 271, 15091–15098. 12. Schaub, T.P., Kartenbeck, J., Ko ¨ nig, J., S pring, H., D o ¨ rsam, H., Sta ¨ hler, G ., Sto ¨ rkel, S ., Thon, W.F. & Keppler, D. (1999) Expression of the MRP2 gene-encoded conjugate export pump inhumankidneyproximaltubulesandinrenal-cellcarcinoma. J. Am. Soc. Nephrol. 10, 115 9–1169. 13. Paulusma, C.C., Bosma, P.J., Zaman, G.J.R., Bakker, C.T.M., Otter, M., Scheffer, G.L., Scheper, R.J., Borst, P. & Oude Elfer- ink, R.P.J. (1996) Congenital jaundice in rats with a mutation in a 1874 A. T. Nies et al. (Eur. J. Biochem. 269) Ó FEBS 2002 multidrug resistance associated-protein gene. Science 271, 1126– 1127. 14.Ito,K.,Suzuki,H.,Hirohashi,T.,Kume,K.,Shimizu,T.& Sugiyama, Y. (1997) Molecular cloning o f c analicular multi- specific organic anion transporter defect ive in EHBR. Am. J . Physiol. 272, G 16–G22. 15. Paulusma,C.C.,Kool,M.,Bosma,P.J.,Scheffer,G.L.,terBorg, F.,Scheper,R.J.,Tytgat,G.N.J.,Borst,P.,Baas,F.&Oude Elferink, R.P.J. (1997) A mutation i n the human c analicular multispecific organi c anion transporter gene causes th e Dubin – Johnson s yndrome. Hepatology 25, 1539–1542. 16. Cu i, Y., Ko ¨ nig, J., Buchholz , U., Spring, H., L eier, I. & Ke ppler, D. (1999) Drug resistance and ATP-dependent conjugate trans- port mediated by the apical m ultidrug resistance protein, MRP2, permanently e xpressed in human and canine cells. Mol. Pharma- col. 55, 9 29–937. 17. Taniguchi, K., Wada, M., Kohno, K., Nakamura, T., Kawabe, T., Kawakami, M., Ka gotani, K., Okumura, K., A kiya ma, S. & Kuwano, M. (1996) A human canalicular multispecific organic anion transporter (cMOAT) overexpressed in cisplatin-resistant human c ancer cell lines with decreased d rug accumulation. Cancer Res. 56, 4124–4129. 18. Ishikawa, T., Mu ¨ ller, M., K lu ¨ nemann, C., Schaub, T. & Keppler, D. (1990) ATP-dependent primary active transport of cysteinyl leukotrienes a cross live r c analicular m em brane: Role o f th e A TP- dependent transport system for glutathione S-conjugates. J. Biol. Chem. 265, 1 9279–19286. 19. Nies, A.T., C antz, T., Brom , M., Le ier, I. & Keppler, D. (1998) Expression of the apical conjugate export pump, Mrp2, in the p olarized hepatoma cell line, WIF-B. He pato logy 28, 1332– 1340. 20. Evers,R.,Kool,M.,vanDeemter,L.,Janssen,H.,Calafat,J., Oomen, L.C.J.M., Paulusma, C.C., O ude E lferink, R.P.J., Baas, F., S chinkel, A.H. & Borst, P. (1998) Drug export activity of the human canalicular multispecific organ ic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA. J. Clin. Invest. 101, 1310–1319. 21.Ito,K.,Suzuki,H.,Hirohashi,T.,Kume,K.,Shimizu,T.& Sugiyama, Y. (1998) Functional an alysis of a canalicular multi- specific organic anion transporter cloned from rat l iver. J. Biol. Chem. 273, 1 684–1688. 22. Kartenbeck, J., Leuschner, U., Mayer, R. & Keppler, D . (1996) Absence of t he canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin–Johnson syndrome. Hepatology 23, 1061–1066. 23. Keppler, D. & Kartenbeck, J. (1996) The canalicular c onjugate export pump encoded by the cmrp/cmoat gene. In Progress in Live r Diseases (Boyer, J .L. & Ockner, R.K., e ds), pp. 55–67. Saunders, Philadelphia, PA, USA. 24. Tsujii, H., K o ¨ nig, J., Ros t, D., S to ¨ ckel, B., Leuschner, U. & Keppler, D. (1999) Exon-intron organization of the human multidrug-resistance protein 2 (MRP2) gene mutated in Dubin – Johnson s yndrome. Gastroenterology 117, 653–660. 25. Ikonen, E. & Simons, K . (1998) P rotein and lipid sorting f rom the trans-Golgi network to the plasma membrane in p olarized cells. Semin. Cell Dev. Biol. 9, 503–509. 26. Matter,K.,Hunziker,W.&Mellman,I.(1992)Basolateralsort- ing of LDL receptor in MDCK ce lls: the cytoplasmic domain contains two tyrosine-dependent targeting determinants. Cell 71 , 741–753. 27. Le G all, A.H., Powell, S.K., Yeaman, C .A. & Rodriguez-Boulan, E. (1997) T he neural cell adhesion molecule expresses a tyro sine- independent b asolateral sorting signal. J. Biol. C hem. 272 , 4559– 4567. 28. Distel, B., Bauer, U., Le Borgne, R. & Hoflack, B. ( 1998) Baso- lateral sorting of the cation-dependent mannose 6-phosphate receptor in Ma din–Darby canine kidney cells. Identification of a basolateral determinant unrelated to clathrin-coated pit localiza- tion signals. J. Biol. Chem. 273, 186–193. 29. Dunbar, L.A. & Caplan, M.J. (2001) Ion pumps in polarized cells: sorting and regulation of the Na,K-and H,K-ATPases. J. Biol. Chem. 276, 2 9617–29620. 30. Simon s, K. & Ikonen, E. (1997) Functional raft s in cell mem- branes. Nature 387, 569–572. 31. Matter, K. (2000) Ep ithelial polarity: sorting out the sorters. Curr. Biol. 10, R 39–R42. 32. Songyang, Z., Fanning, A.S., Fu, C ., Xu, J., Marfatia, S.M., Chishti, A.H., Crompton, A., Chan, A.C., A nderson, J.M. & Cantley, L.C. (1997) Recognition of unique c arboxyl-terminal motifs by distinct PDZ domains. Science 275 , 73–77. 33. Fanning, A.S. & Anderson, J.M. (1999) PDZ domains: funda- mental b uilding blocks in the organization of p rotein complexes at the plasma membrane. J. Clin. Invest. 103, 7 67–772. 34. Keite l, V., K artenb eck, J., Nies, A.T., Spring, H., B rom, M. & Keppler, D. (2000) Impaired protein maturation of the conjugate export pu mp MRP2 as a c onsequen ce of a deletion mutation in Dubin–Johnson syndrome. Hepatology 32, 1 317–1328. 35. Kocher, O., Comella, N., Gilchrist, A., Pal, R ., Tognazzi, K., Brown, L.F. & Knoll, J.H. (1999) PDZK1, a novel PDZ domain-containing protein up-regulated in carci nomas and mapped to chromosome 1q21, interacts with cMOAT (MRP2), the multid rug resistan ce-asso ciated p rotein. Lab. Invest. 79 , 1161– 1170. 36. Harris, M.J., Kuwano, M., Webb, M. & Board, P.G. (2001) Identification of the apical membrane-targeting s ignal of the multidrug resistance-associated protein 2 (MRP2/MOAT). J. Biol. Chem. 276, 2 0876–20881. 37. Reczek, D. & Bretscher, A. (2001) Identification o f EPI64, a TBC/ rabGAP domain-containing microvillar protein that binds to the first PD Z domain of EBP50 and E3KARP. J. Cell Biol. 153, 191–206. 38. Muth, T.R., Ahn, J. & Caplan, M.J. (1998) Identification of sorting determinants in the C-te rminal cytoplasmic tails of the gamma-aminobutyric acid transporters GAT-2 and GAT -3. J. Biol. C hem. 273, 25616–25627. 39. Cormack, B.P., Valdivia, R.H. & Falkow, S. (1996) FACS-opti- mized mutants o f the green fl uorescent protein (GFP). Gene 173, 33–38. 40. Yang, T., Cheng, L. & Kain, S.R. (1996) Optimized codon usage and chromophore mutations p rovide enhanced sensitivity with the green fluorescent p rotein. Nucleic Acids Res. 24 , 4592–4593. 41. Rowe, J., Calegari, F., T averna, E., Longhi, R. & R osa, P. (2001) Syntaxin 1A is delivered to the apical and basolateral domains of epithelial cells: the role of munc-18 proteins. J. Cell Sci. 114, 3323– 3332. 42. Cantz,T.,Nies,A.T.,Brom,M.,Hofmann,A.F.&Keppler,D. (2000) MRP2, a human conjugate export pump, is present and transports Fluo-3 i nto apical vacuoles of HepG2 cells. Am. J. Physiol. 278, G 522–G531. 43. Kep pler, D. & Ko ¨ nig, J. (1997) Expression and l ocalization o f t he conjugate export pump encoded by the MRP2 (cMRP/cMOAT) gene in liver. FASEB J. 11, 509–516. 44. Bakos, E., Hegedus, T., Hollo, Z., Welker, E ., Tusnady, G.E., Zaman, G.J., Flens, M.J., Varadi, A. & Sarkadi, B. (1996) Membrane topology and glycosylation of the human multidrug resistance-associated protein. J. Biol. Chem. 271 , 12322–12326. 45. Sonnhammer, E.L., v on H eijne, G. & Krogh, A. (1998) A h idden Markov mod el for predicting transmembrane h elices in protein sequences. Proc.Int.Conf.Intell.Syst.Mol.Biol.6, 175–182. 46. Jedlitsc hky, G., Leier, I., Buchholz, U., Hummel-Eisenbeiss, J., Burchell, B. & Keppler, D . (1997) ATP-de pendent transport of bilirubin glucuronides by the m ultidrug resistance protein MRP1 and i ts hepatocyte canalicular isoform M RP2. Biochem. J. 327 , 305–310. Ó FEBS 2002 Apical sorting of human MRP2 (Eur. J. Biochem. 269) 1875 [...]... canine kidney and LLC-PK1 epithelial cells J Biol Chem 27 3, 26 8 62 26 869 53 Karim-Jimenez, Z., Hernando, N., Biber, J & Murer, H (20 00) Requirement of a leucine residue for (apical) membrane expres- 54 55 56 57 58 59 sion of type IIb NaPi cotransporters Proc Natl Acad Sci USA 97, 29 16 29 21 Blum, R., Stephens, D.J & Schulz, I (20 00) Lumenal targeted GFP, used as a marker of soluble cargo, visualises... and expression of a cDNA coding for the human platelet-derived growth factor receptor: evidence for more than one receptor class Proc Natl Acad Sci USA 85, 3435–3439 49 Sormunen, R., Eskelinen, S & Lehto, V (1993) Bile canaliculus formation in cultured HepG2 cells Lab Invest 68, 6 52 6 62 50 Zegers, M.M & Hoekstra, D (1997) Sphingolipid transport to the apical plasma membrane domain in human hepatoma... Caplan, M.J (20 01) The C-terminal tail of the metabotropic glutamate receptor subtype 7 is necessary but not sufficient for cell surface delivery and polarized targeting in neurons and epithelia J Biol Chem 27 6, 9133–9140 Perego, C., Vanoni, C., Villa, A., Longhi, R., Kaech, S.M., Frohli, E., Hajnal, A., Kim, S.K & Pietrini, G (1999) PDZ–mediated interactions retain the epithelial GABA transporter on the basolateral...Ó FEBS 20 02 1876 A T Nies et al (Eur J Biochem 26 9) 47 Coloma, M.J., Hastings, A., Wims, L.A & Morrison, S.L (19 92) Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction J Immunol Methods 1 52, 89–104 48 Gronwald, R.G., Grant, F.J., Haldeman, B.A., Hart, C.E., O’Hara,... epithelial GABA transporter on the basolateral surface of polarized epithelial cells EMBO J 18, 23 84– 23 93 Bezprozvanny, I & Maximov, A (20 01) PDZ domains: more than just a glue Proc Natl Acad Sci USA 98, 787–790 Cuppen, E., Gerrits, H., Pepers, B., Wieringa, B & Hendriks, W (1998) PDZ motifs in PTP-BL and RIL bind to internal protein segments in the LIM domain protein RIL Mol Biol Cell 9, 671–683 Hillier,... HepG2 cells J Cell Biol 138, 307– 321 51 Moyer, B.D., Denton, J., Karlson, K.H., Reynolds, D., Wang, S., Mickle, J.E., Milewski, M., Cutting, G.R., Guggino, W.B., Li, M & Stanton, B.A (1999) A PDZ-interacting domain in CFTR is an apical membrane polarization signal J Clin Invest 104, 1353– 1361 52 Roush, D.L., Gottardi, C.J., Naim, H.Y., Roth, M.G & Caplan, M.J (1998) Tyrosine-based membrane protein sorting. .. segments in the LIM domain protein RIL Mol Biol Cell 9, 671–683 Hillier, B.J., Christopherson, K.S., Prehoda, K.E., Bredt, D.S & Lim, W.A (1999) Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex Science 28 4, 8 12 815 . part of MRP2 contains information for apical sorting of MRP2. Apical localization of GFP–MRP2 in polarized HepG2 and MDCKII cells A s equence alignment of the C-terminal ends of human MRP1, MRP2,. PCR-fragment that enabled Ó FEBS 20 02 Apical sorting of human MRP2 (Eur. J. Biochem. 26 9) 1867 the in-frame insertion of GFP a t the N-terminus of MRP2 (pMRP2.1). The sense primer for this PCR reaction. 11±6 12 4 471±37± 122 ±3 GFP–MRP2D15 1 9 ± 2 55 ± 10 36 ± 10 2 8±1 47±7 45±6 3 7±3 54±11 38±9 4 5±3 47±9 47±8 GFP–MRP2D25 1 2 ± 1 78 ± 10 20 ± 9 2 2±1 69±6 28 ±7 3 2 1 69±8 29 ±7 4 3±1 63±3 35 2 Ó