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:
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.
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