Nies1, Jo¨rg Ko¨nig1, Yunhai Cui1, Manuela Brom1, Herbert Spring2and Dietrich Keppler1 1 Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany;2Division o
Trang 1Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2)
Anne T Nies1, Jo¨rg Ko¨nig1, Yunhai Cui1, Manuela Brom1, Herbert Spring2and Dietrich Keppler1
1
Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany;2Division of Cell Biology,
Deutsches Krebsforschungszentrum, Heidelberg, Germany
The human multidrug resistance protein 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 apical 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 generated MRP2 constructs
mutated and stepwise truncated at their C-termini These
constructs were fused via their N-termini to green fluorescent
protein (GFP) and were transiently transfected into
polar-ized, liver-derived human HepG2 cells Quantitative analysis
showed that full-length GFP–MRP2 was localized to the apical membrane in 73% of transfected, polarized cells, whereas it remained on intracellular membranes in 27% of cells Removal of the C-terminal TKF peptide and stepwise deletion of up to 11 amino acids did not change this pre-dominant apical distribution 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 data indicate that a deletion of at least 15 C-terminal amino acids impairs the localization of MRP2 to the apical membrane of polarized cells
Keywords: epithelial polarity; green fluorescent protein; multidrug resistance protein 2; protein trafficking
Members of the multidrug resistance protein (MRP) family
are integral membrane glycoproteins which mediate the
ATP-dependent export of amphiphilic anions across the
plasma membrane [1] MRP1, the first cloned member of
the MRP family [2], is present in the plasma membrane of
several cell types [3–5] After transfection of MRP1 cDNA
in polarized cells, MRP1 is localized to the basolateral
membrane [6] Several MRP 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, such as hepatocytes and
renal proximal tubule cells [1,11,12] MRP2 was initially
cloned from 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 conjugassoci-ated hyperbilirubinemia
Epithelial cell polarity is a result of the domain-specific sorting of proteins Neither apical nor basolateral trafficking seems to follow a ÔdefaultÕ pathway, rather, specific signals
or interactions are required for inclusion of proteins into apically or basolaterally destined transport vesicles within the trans Golgi network (TGN; reviewed in [25]) Basolat-eral sorting signals are most often tyrosine- or dileucine-based motifs in the cytoplasmic domains of proteins [26], however, other basolateral sorting signals have been also identified [27,28] Several mechanisms have been described for apical sorting These 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 be achieved is the binding of membrane proteins via their C-termini to PDZ domain-containing proteins The latter recognize a consen-sus sequence (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 membrane 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-mail: a.nies@dkfz.de
Abbreviations: GFP, green fluorescent protein; MRP2, multidrug
resistance protein 2 (human genome nomenclature symbol ¼ ABCC2);
PDZ, PSD-95/DlgA/ZO-1-like.
(Received 23 January 2002, accepted 6 February 2002)
Trang 2proteins [33] PDZ domain-containing proteins either bind
directly or via adaptor proteins to the cytoskeleton [33]
Present knowledge on the mechanisms by which MRP
isoforms are targeted to their respective membrane domain
in polarized cells is limited We recently showed that a
six-nucleotide deletion within the human MRP2 gene causes
Dubin–Johnson syndrome [24,34] This mutation, leading to
the loss of two amino acids from the second
nucleotide-binding domain [24], results in defective MRP2 maturation
and retention of MRP2 in the ER, so that sorting of MRP2 to
the apical membrane is impaired [34] The aim of the present
study was to identify structural determinants required for
apical sorting of human MRP2 Because MRP2 has 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 of
polarized Madin–Darby canine kidney (MDCK) cells [36]
This result may, however, be misleading because MRP2 was
tagged at the C-terminus with GFP and interaction with PDZ
domain-containing proteins may be disrupted by the
addi-tion of amino acids to the C-terminal PDZ-interacting motif
[37,38] In addition, human proteins may localize differently
in canine cells In the present work, we therefore used human
MRP2 tagged with GFP at the N-terminus, thus leaving the
C-terminus free for possible binding of interacting proteins
With this experimental setup, 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 by more than 15 amino acids resulted in
impaired delivery of MRP2 to the apical membrane
M A T E R I A L S A N D M E T H O D S
Materials and antibodies
Fetal bovine serum and agarose were from Sigma (St Louis,
MO, USA) Pfu DNA polymerase, restriction enzymes,
ligase, and modifying enzymes were from Stratagene (La
Jolla, CA, USA) or Promega (Madison, WI, USA)
Lysozyme and ampicillin were from Roche Molecular
Biochemicals (Indianapolis, IN, USA)
Rhodamine-conju-gated concanavalin A was from Vector Laboratories
(Burlingame, CA, USA) All other chemicals were of
analytical grade and obtained either from Merck
(Darm-stadt, Germany) or Sigma
The polyclonal rabbit antibody directed against the
C-terminus of human MRP2, EAG5, has been 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 the mouse monoclonal
antibody to protein disulfide isomerase (PDI; clone RL90)
was purchased from Affinity Bioreagents (Golden, CO,
USA) The mouse monoclonal anti-villin Ig was from
Transduction Laboratories (Lexington, KY, USA) Rat
anti-(ZO-1) Ig was from Chemicon (Temecula, CA, USA)
Goat anti-(rabbit IgG) Ig coupled to Alexa Fluor546 or
Alexa Fluor488 were from Molecular Probes (Eugene, OR, 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 chimeric MRP1/2 protein (Fig 1) was constructed by generating a XbaI restriction site in the cDNA sequence of human MRP1 in a PCR-based approach In detail, a MRP1 cDNA fragment was amplified using the MRP1 cDNA, inserted into the vector pcDNA3.1(+), as template and the T7 vector primer as forward primer The reverse primer ochimrp1.rev was used
to generate the XbaI restriction site in the MRP1 cDNA It has the sequence 5¢-AGAGGGGATCATCTAGAAG GTA-3¢ (position 2386–2365) and has three base-pair substitutions when compared with the MRP1 wild-type sequence: 2370Gfi A, 2371A fi G, and 2373G 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 55 °C annealing and 120 s 72 °C elongation, 30 cycles with 45 s 94°C denaturation, 45 s at
65°C annealing, and 120 s at 72 °C elongation, followed by
10 min at 72°C The fragment was subcloned into the vector pCR2.TOPO (Invitrogen, Carlsbad, CA, USA) 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 restricted with NotI/XbaI and the MRP1 cDNA fragment from the pmrp1/XbaI.topo plasmid obtained by NotI/XbaI restriction was inserted, thus generating the plasmid pmrp1/2chim.31 The correct sequence of the fragment and the cloning sites were verified
by sequencing and restriction analysis
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 proteins, so that the GFP moiety was in the lumen of 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 expression [40], was cloned into the BamHI and NotI restriction sites of the expression vector pcDNA3.1(+) (pGFP) GFP was PCR-amplified using the sense-primer 5¢-AGATCTGCCACCATGGTGAGC AAG-3¢, which introduced a BglII site (bold), and the antisense primer 5¢-CCGCGGCCGCTTGTATAGCTCGTCCATGCCG AG-3¢, which introduced a SacII (underlined) and a NotI site (bold), at the same time removing the stop codon and the BsrGI site at the 3¢ end of the GFP coding sequence PCR-amplified GFP was cloned into the pDisplay vector (Invi-trogen) using the BglII and the SacII sites (plumGFP) pMRP2 was digested with NotI and BsrGI, and the fragment was replaced with a PCR-fragment that enabled
Trang 3the in-frame insertion of GFP at the N-terminus of MRP2
(pMRP2.1) The sense primer for this PCR reaction was
5¢-GCGGCCGCTCATGCTGGAGAAGTTCTG-3¢ (NotI
site in bold) and the antisense primer was 5¢-GTGCCACA
GAGTATCGAG-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 of C-terminal deletion constructs, a
2346-bp DNA fragment encoding the C-proximal part of
MRP2 was generated by PCR with ApaI and SacII sites
added at the 3¢ end during amplification Primers used
were 5¢-AGCGGATCAGCCTGG-3¢ (sense primer) and
5¢-GGGCCCGCGGCTAGAATTTTGTGCTGTTCAC-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 generated
by cloning PCR-amplified fragments into the Bsu36I and
the SacII sites 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¢
GGGCTGCCGC-3¢ (MRP2D25), 5¢-CCGCGGCTATTC
D25MAKE), 5¢-CCGCGGCTACAGCCTGTGGGCGA
TGG-3¢ (MRP2D50), 5¢-CCGCGGCTACAGCAGCTG
CCTCTGGC-3¢ (MRP2D100), 5¢-CCGCGGCTAGAAT
TTTGCGCTGTTCACATTC-3¢ (MRP2T1543 A), and
5¢-CCGCGGCTAGAATTTTGTAAAGTAAAAGGGT
CCAGGG-3¢ (MRP2D15TKF) GFP 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 Human hepatoma HepG2 and MDCK cells (strain II) 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 100-mm and dishes at a density of 5· 105and 5· 106 cells per dish, respectively, 24 h prior to transfection HepG2 cells were transfected with the FuGENE 6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer’s instructions using 5 and 25 lL transfection reagent and 1.5 and 7.5 lg DNA per 35- and 100-mm dish, respectively MDCKII cells were transiently or stably [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 °C for 1 min and rehydrated in NaCl/Pi Cells were incubated with the primary antibody for 60 min at room temperature, washed three times with NaCl/Pi, incubated with the secondary antibody for 60 min, and then washed again three times with NaCl/Pi Cover slips were mounted in Moviol (Hoechst, Frankfurt, Germany) and observed on a confocal laser scanning microscope (LSM 510, Carl Zeiss, Jena, Germany) using the excitation wavelengths of the argon ion (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 at
Fig 1 Predicted topology models (A) and localization of MRP2 (B,C) and chimeric MRP1/2 (D,E) in polarized MDCKII cells The chimeric MRP1/2 consists of the MRP1 sequence followed by the sequence of MRP2 starting at amino acid 791 For MRP2, only four transmembrane segments are predicted between both nucleotide-binding domains (NBD1 and NBD2 [43]), whereas six trans-membrane segments are predicted for MRP1 [44] MDCKII cells stably synthesizing MRP2
or chimeric MRP1/2 were immunostained with the EAG5 antibody directed against MRP2 (green in B–E) Both proteins were localized to the apical membrane as observed
in the x–y plane (B,D) and the x–z plane (C,E) Nuclei were stained with propidium iodide (red in B–E) Bar, 10 lm.
Trang 4the following dilutions: anti-(ZO-1) Ig (1 : 100), EAG5
(1 : 200), anti-PDI Ig (1 : 400), anti-DPPIV Ig (1 : 500),
and the respective secondary antibodies at 1 : 300 For
staining of lysosomes, LysoTracker Red (Molecular Probes)
was used according to the manufacturer’s instructions For
staining of the apical membrane of MDCKII cells,
rhod-amine-labeled concanavalin A was added to the apical
chamber of a Transwell filter insert at 5 lgÆmL)1according
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 cells were transiently transfected and
immuno-stained with the anti-DPPIV Ig as 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 Zeiss, Jena, Germany) For each transfected and polarized cell, the localization of the respective GFP–MRP2 protein was analyzed and classified into one of three categories as follows: when GFP and DPPIV fluorescence merged in ring-like, microvilli-lined structures between adjacent cells, i.e the apical membrane [42], the localization was defined as ÔapicalÕ, irrespective of additional intracellular GFP fluorescence When GFP fluorescence was absent from these ring-like structures in polarized cells, but observed in vesicular structures, local-ization was defined as ÔvesicularÕ When DPPIV fluorescence was present in the ring-like structures and GFP fluorescence appeared exclusively reticular, localization was defined as endoplasmic reticulum (ER) Localization of the respective GFP–MRP2 in the ER was confirmed by colocalization with an antibody against an ER marker protein, protein disulfide isomerase (data not shown), as described previ-ously [34] For each GFP–MRP2 construct, the percentage
of each localization was calculated At least four indepen-dent transfections were analyzed in this way For analysis of the steady-state distribution of GFP–MRP2 proteins, cells were induced with 5 mMbutyrate for 24 h [16] and observed
48 h after start of transfection For analysis of the time-course of GFP–MRP2 protein localization, cells were observed after 1, 2, 3, and 4 days post-transfection without 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 were counted on a fluorescence microscope (Axioskop)
R E S U L T S 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 and the apically localized isoform MRP2 [1] hampers the identification of apical sorting signals in the MRP2 sequence by direct comparison of both sequences We therefore constructed a cDNA encoding a MRP1/2 chimeric protein and immunolocalized this chi-meric protein in MDCKII cells (Fig 1) The chichi-meric 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 sequence alignment of the C-terminal ends of human MRP1, MRP2, MRP3, and MRP6 (Fig 2) shows that the apical MRP2 has a seven amino-acid extension in compar-ison to the basolateral family members MRP1, MRP3, and MRP6 Recombinant MRP1 was localized to the basolat-eral membrane in polarized porcine cells [6] MRP3 and MRP6 are endogenously synthesized in polarized cells such
Fig 2 Alignment of the C-termini of members of the human MRP
family (A) and predicted topology models of MRP2, GFP–MRP2, and
lumGFP (B) According to the prediction of the TMHMM program [45],
and experimentally confirmed [16], the N-terminus of MRP2 has an
extracellular location Therefore, a cDNA was constructed which
encoded a fusion protein of GFP and MRP2 with the GFP moiety
targeted to the lumen of the ER, followed by the complete sequence of
human MRP2 (GFP–MRP2) Expression of GFP from the pDisplay
vector (lumGFP for Ôlumenal GFPÕ) resulted in a GFP which was
targeted to the lumen of the ER because of a murine Ig j-chain leader
sequence ([47]; black box) at the N-terminus of GFP and which was
anchored in the plasma membrane due to the platelet-derived growth
factor receptor transmembrane domain at the C-terminus of GFP
([48]; cross-hatched box).
Trang 5as hepatocytes and localized in the basolateral membrane
[7–10] Because the extension of MRP2 might represent a
signal for apical localization of MRP2, we generated
MRP2, which was mutated or stepwise 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 in these cells,
we constructed cDNAs coding for fusion proteins of MRP2
and GFP Because a ÔfreeÕ C-terminus may be necessary for
proper apical sorting of MRP2, e.g by binding of
interact-ing proteins, GFP was fused to the N-terminus of MRP2
The N-terminus of MRP2 is located on the extracellular side
[16], therefore a cDNA was constructed which led to
translation of a GFP inserted into the lumen of the ER by
the murine Ig j-chain leader sequence, a sequence described
to target proteins to the secretory pathway [47], followed by
the sequence of MRP2 (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 GFP–MRP2 constructs (Fig 2) LumGFP was equally
distributed in the apical and the basolateral membrane of
polarized HepG2 cells, and, in addition, in intracellular
vesicular structures (Fig 3) indicating that neither the
murine Ig j-chain leader sequence nor the PDGFR
transmembrane domain contained a specific signal for
apical localization To exclude an effect of GFP on MRP2
targeting, the distribution of GFP in polarized HepG2 cells
was analyzed (Fig 3) The soluble GFP was present within
the cells without any localization in the plasma membrane
As a control, GFP–MRP2 was also observed in
MDCKII cells where it localized to the apical membrane
(Fig 4) The polarity of the MDCKII cells was confirmed
by immunostaining with an antibody detecting the
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 acids 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 amino acids of the human MRP2
sequence (TKF, Fig 2) have been reported to interact with
a PDZ domain-containing protein [35] and may thus be
necessary for apical sorting of MRP2 We therefore deleted
the C-terminal three amino acids or substituted threonine
with alanine at position 1543 The respective, mutated
GFP–MRP2 was observed in polarized HepG2 cells For
quantitative analysis, localization of GFP–MRP2 proteins
were classified into one of three categories as shown in the
representative images of Fig 5 and described in Materials
and methods
Because apical vacuoles form between adjacent HepG2 cells as vesicle-like structures lined with microvilli [49], they can be stained 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 for polarity, HepG2 cells were double-stained for DPPIV and MRP2 The majority (98.9%) of DPPIV-positive, microvilli-lined ring-like structures were also positive for MRP2 (540 apical vacuoles counted) Similarly, 99.6% of villin-positive, microvilli-lined ring-like structures were also positive for MRP2 (535 apical vacuoles counted) This result indicates that staining for all three proteins, villin, DPPIV, and MRP2, can be used as marker for cell polarity in HepG2 cells
Fig 3 Localization of GFP–MRP2, lumGFP, and GFP in polarized HepG2 cells HepG2 cells were transiently transfected with GFP– MRP2 (A,B) or lumGFP (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 visualized by fluorescence microscopy (E) or by phase-contrast (F) In GFP–MRP2-transfected cells (A), fluorescence was observed in ring-like structures, i.e the apical (vacuolar) membrane, and, in addition, in intracellular vesicular structures of varying size In contrast, lumGFP (C) was observed in the basolateral and in the apical membrane in equal amounts, and, additionally, in intracellular vesic-ular structures, most likely vesicles of the secretory pathway GFP (E) was distributed throughout the cells without localization to the plasma membrane Asterisks mark the lumen of apical vacuoles Bars, 10 lm.
Trang 6In 73% of transfected and polarized HepG2 cells GFP– MRP2 reached the apical membrane (Table 1) In the remaining 27% of transfected and polarized cells, GFP– MRP2 did not reach the apical membrane, but was present
in intracellular compartments, such as vesicular structures and the ER Deletion of the C-terminal three amino acids TKF or substitution of threonine with 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 localized 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 apical membrane was visualized by rhod-amine-conjugated concanavalin A added to the upper chamber of the Transwell insert 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 the basolateral membrane
Localization of C-terminally truncated GFP–MRP2 proteins
Because the PDZ-interacting motif was not necessary for apical sorting of MRP2, the C-terminus of GFP–MRP2 was further truncated Truncation of the C-terminus by seven or 11 amino acids led to proteins 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 transfected with GFP–MRP2 were fixed
48 h after transfection and immunostained with an antibody against
the tight-junctional protein ZO-1 (C,D), or with the EAG5 antibody
(G,H) which is directed against the 15 C-terminal amino acids of
human MRP2 [11,12] The GFP fluorescence (A,B,E,F) shows that
GFP–MRP2 is localized to the apical membrane, as observed 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 dots in the vertical section (D) EAG5 fluorescence (G,H)
was identical to the GFP fluorescence (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 in polarized HepG2 cells as quantified in
Tables 1–3 When GFP fluorescence (A) and
DPPIV fluorescence (B) merged to yellow in
the apical membrane (C) the localization of
the GFP–MRP2 construct 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 remained in reticular structures, i.e.
the ER (G), and no GFP fluorescence of the
apical vacuolar membrane (H) was observed
(I) Bars in A–I, 10 lm Asterisks mark apical
vacuoles.
Trang 7GFP–MRP2 (Table 2) However, delivery to the apical
membrane was largely impaired when GFP–MRP2 was
C-terminally truncated by 15, 20, 25, 50 or 100 amino acids
The percentage of polarized and transfected cells in which
the respective protein reached the apical membrane
MRP2D20), 8% (GFP–MRP2D25), and 1% (GFP–
accumulation of the proteins in intracellular compartments,
such as the ER and intracellular vesicles (Table 2) Because
deletion of the tetrapeptide MAKE, i.e amino acids 1531–
1534, resulted in a shift in the percentage of cells with an apical (GFP–MRP2D11) to an intracellular localization (GFP–MRP2D15), this sequence might be involved in the apical delivery of MRP2 However, addition of this tetrapeptide onto GFP–MRP2D25, which had an intracel-lular localization in most of the cells, did not increase the number of cells in which GFP–MRP2D25MAKE reached the apical membrane This result indicates that it is not the co-linear sequence of the tetrapeptide that is required for apical delivery of MRP2 The intracellular vesicles contain-ing the respective GFP–MRP2 construct were not lyso-somes as shown by the 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 15–25 amino acids may be due to a delay in intracellular transport 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 the respective GFP–MRP2 protein over time
D I S C U S S I O N MRP2 is the only MRP isoform known so far which localizes to the apical membrane of polarized cells [1,10] Recently, the C-terminal three amino acids (TRL) of the cystic fibrosis transmembrane conductance regulator (CFTR), which comprise a PDZ-interacting motif, were identified as a signal for apical localization [51] Because CFTR is a member of the MRP (ABCC) family with 27% amino-acid identity to MRP2 [1], we investigated whether the C-terminal tail of MRP2 is also involved in apical sorting Interaction of a PDZ domain-containing protein with the C-terminal three amino acids of MRP2 (TKF, Fig 2) has been described previously [35]
The epithelial MDCKII cell line is often used to study the polarized sorting of proteins to different plasma membrane domains, however, some proteins are sorted differently in the canine MDCKII cells as compared to polarized kidney cells from other species [52], therefore sorting of 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 constructs in polarized HepG2 cells Data are percentages of cells in which the respective localization of recombinant protein was observed as described in Materials and methods Cells were observed 2 days after transfection Data are means ± SD of six transient transfections using butyrate-induced cells as described under Materials and methods.
Fig 6 Localization of GFP–MRP2 (green in A,B), GFP–MRP2D3
(green in C,D), and GFP–MRP2-T1543A (green in E,F) in polarized
MDCKII cells MDCKII cells grown on Transwell filter membranes
were transiently transfected with the respective construct and fixed
24 h after transfection The apical membrane was visualized by
staining with rhodamine-conjugated concanavalin A (red
fluores-cence) In the x–y planes (A,C,E), the GFP signals of all three
con-structs give a pattern typical for apical localization The intense yellow
color in the x–z planes, due to merging of the green GFP and the red
concanavalin A fluorescence, shows that GFP–MRP2, GFP–
MRP2D3, and GFP–MRP2-T1543A are almost exclusively localized
in the apical membrane Bars, 10 lm.
Trang 8synthesize MRP2 [42,46], we used GFP-tagged MRP2 to
distinguish between endogenous and recombinant MRP2
Although MRP2 tagged with GFP at its C-terminus
localized correctly to the apical membrane in polarized
HepG2 cells [1,34] we constructed MRP2 tagged with GFP
at the N-terminus in order to leave the C-terminus free for possible binding of interacting proteins Interaction of the C-terminal PDZ-interacting motif with PDZ domain-con-taining proteins seems to require a free C-terminus [37,38]
A comparable approach of N-terminal GFP-tagging was taken for the identification of apical localization signals in the C-termini of CFTR [51] and of the type IIb Na+/Pi co-transporter [53]
In contrast to CFTR [51] and the type IIb Na+/Pi 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 the murine Ig j-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 vector, was targeted to the apical and to the basolateral membrane in equal 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 membrane of polarized HepG2 cells whereas GFP was not (Fig 3)
With this experimental setup, the effect of C-terminal mutations and truncations on apical sorting of MRP2 was investigated In contrast to our expectations, neither the
Table 2 Quantitative analysis of the subcellular localization of C-terminal deletion constructs in polarized HepG2 cells Data are percentages of cells
in which the respective localization of recombinant protein was observed as described in Materials and methods Cells were observed 2 days after start of transfection Data are means ± SD of n ¼ 6 (GFP–MRP2D25MAKE, GFP–MRP2D50, GFP–MRP2D100, n ¼ 4) transient trans-fections using butyrate-induced cells as described in Materials and methods.
Construct % Apical % Vesicles % ER C-Terminal sequence (1510–1545)
Fig 7 Localization of GFP–MRP2 constructs in vesicular structures in
polarized HepG2 cells HepG2 cells transiently synthesizing GFP–
MRP2 (green in A,B) were incubated with LysoTracker Red to stain
lysosomes (red in A), or immunostained with an antibody against
DPPIV to stain DPPIV-containing vesicles (red in B) Absence of
colocalization indicates that GFP–MRP2 is neither present in
lyso-somes nor in DPPIV-containing vesicles Bars, 2.5 lm.
Table 3 Quantitative analysis of the subcellular distribution of GFP–MRP2, GFP–MRP2D15, and GFP–MRP2D25 at different times after trans-fection in polarized HepG2 cells Data are percentages of cells in which the respective localization of recombinant protein was observed as described
in Materials and methods Data are means ± SD of four transient transfections Experiments were performed without butyrate induction.
Trang 9PDZ-interacting motif TKF nor the seven-amino-acid
extension of 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 with the type
IIb Na+/Pico-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+/Pico-transporter
[53] Similarly, mutants of the basolateral GABA
trans-porter lacking the PDZ-interacting motif were still targeted
to the basolateral membrane [56] Although the C-terminal
PDZ-interacting motif of MRP2 is not required for apical
sorting, it may be necessary for linking additional regulatory
proteins to MRP2 or for clustering of MRP2 in 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 the 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 apical membrane (Table 2)
Because MRP2 is still observed in the apical membrane in a
very low percentage of cells, MRP2 is at least in part
delivered into apically-destined vesicles A truncation of 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 membrane or for
stabilization of MRP2 within the apical membrane
More-over, a MRP2 protein truncated by at least 15 amino acids
may alter the conformation of the transport protein to such
an extent that the misfolded protein is retained in the ER A
single leucine residue was recently shown to be part of a, yet
unidentified, motif required for delivery of the type IIb
Na+/Pico-transporter to 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 they were not retained, but
internal-ized into an endosomal recycling compartment
When the present work was in progress, a study was
published describing the PDZ-interacting motif as a signal
for apical localization of MRP2 [36]; deletion of the
C-terminal three amino acids resulted in localization of
MRP2 predominantly to the basolateral membrane of
MDCK cells These observations are in disagreement with
our results However, the differences may be attributable to
the expression in the canine MDCK cells of unspecified
origin and to tagging of MRP2 at the C-terminus [36] rather
than expression of N-terminally tagged MRP2 in human
HepG2 cells (Tables 1 and 2) or polarized MDCKII cells
(Fig 6) as described in the present study
In conclusion, the C-terminal 11 amino acids of human
MRP2, including the PDZ-interacting motif, are not
required for apical 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 either because part of a
motif required for apical sorting is lost or because of a
conformational change in the transport protein impairing
MRP2 maturation
A C K N O W L E D G E M E N T S
We thank Dr Tobias Cantz for contributions to this work and helpful discussion, Dr Blanche Schwappach for helpful discussions on GFP tagging, Dr Wolfgang Hagmann for MRP1 cDNA, and Marion Pfannschmidt for excellent technical assistance This work was supported in part by grants from the Deutsche Forschungsgemein-schaft through SFB 352/B3.
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