From the Molecular Genetics Group, Division of
Molecular Medicine, John Curtin School of Medical Research, Australian
National University, Canberra ACT 0200, Australia, the
§ Department of Medical Biochemistry, Graduate School of
Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka 812-8582, Japan, and ¶ Biotron Limited, Innovations Building, Australian
National University, Canberra ACT 0200, Australia
Received for publication, November 22, 2000, and in revised form, February 26, 2001
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ABSTRACT |
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The human canalicular multispecific organic anion
transporter (cMOAT), known as the multidrug resistance-associated
protein 2 (MRP2), is normally expressed in the liver and to a lesser
extent in the kidney proximal tubules. In these tissues MRP2
specifically localizes to the apical membrane. The construction of MRP2
fused to the green fluorescent protein, and subsequent site-directed mutagenesis enabled the identification of a targeting signal in MRP2
that is responsible for its apical localization in polarized cells. The
specific apical localization of MRP2 is due to a C-terminal tail that
is not present in the basolaterally targeted MRP1. Deletion of three
amino acids from the C-terminal of MRP2 ( The canalicular multispecific organic anion transporter
(cMOAT)1 is a member of the
ATP binding cassette (ABC) transporter family of membrane proteins
(designated ABCC2). cMOAT is a member of a subfamily of transporters
called multidrug resistance-associated proteins (MRPs) (recently
reviewed in Refs. 1 and 2). There are six known members, MRP1 to MRP6,
of which cMOAT is known as MRP2. These proteins function as export
"pumps" and extrude a broad range of compounds from the cell. MRP1
was the first (3) and most extensively characterized member and has
49% sequence identity with MRP2 (4-7). The function of MRP2 was
initially characterized and shown to be distinct from MRP1 by the use
of MRP2-deficient rats GY/TR The polarization of cells, such as epithelial cells lining the bile
duct or the kidney proximal tubules, is created largely by the
differential localization of specific proteins. To use the hepatocyte
as an example, the proteins required for the excretion of products are
concentrated solely in the membrane that creates the canalicular border
of the cells. To create the polarization of cells, proteins destined
for specific membranes have been shown to contain molecular targeting
signals. Such signals include glycosylation, phosphorylation,
transmembrane domains, or cytoplasmic motifs (12). The recent
identification of a PDZ-interacting domain in the cystic fibrosis
transmembrane regulator (CFTR) is the first report of a targeting motif
in mammalian ABC transporters (13). This domain interacts with PDZ
domain containing proteins, which are commonly involved in scaffolding
and stabilization at the plasma membrane (14). The signals responsible
for the differential targeting of the MRP proteins to particular
domains have not been determined.
Initially we aimed to further characterize the function of MRP2 by
expressing the protein in a hematopoietic cell line. However, these
transfected cells did not show MRP2 function due to intracellular accumulation of the protein and minimal cell membrane localization. Similar results have been reported by others (15). In contrast, MRP1
shows total cell membrane localization in these cells. We were
therefore interested in the sorting signals responsible for the
difference in localization of these two proteins in both epithelial cell lines and cells with a hematopoietic lineage. Using green fluorescent protein (GFP) fusion proteins and site-directed mutagenesis we have been able to identify a sequence motif responsible for exclusive apical localization of MRP2 in polarized MDCK cells. Deletion
of the motif results in lateral localization of MRP2 in polarized MDCK
cells and allows cell membrane localization in L1210 cells. The mutated
protein was able to transport 2,4-dinitrophenyl glutathione (DNP-GS), a
known substrate of MRP2.
Green Fluorescent Protein Fusion Constructs--
GFP was fused
to the C-terminal of MRP1 and MRP2 and utilized to detect the
localization of the fusion proteins. Human MRP2 cDNA was amplified
by polymerase chain reaction using PfuTurbo DNA
polymerase (Stratagene) to remove the stop codon and introduce restriction enzyme sites suitable for cloning. The cDNA was
amplified using the sense primer (5'-AGCGCTAGCGATGCTGGAGAAGTTCTGCAAC),
which adds an NheI site immediately adjacent to the start
codon, and the antisense primer
(5'-TACGGTACCGGTGCGAATTTTGTGCTGTTCACATTC), which adds an
AgeI site after the final codon and removes the stop codon.
The polymerase chain reaction product was digested with
NheI/AgeI and ligated into the
NheI/AgeI-digested EGFP-N1 vector
(CLONTECH). Human MRP1 cDNA was cloned from
HL60ADR cells and ligated into EGFP-N1
(SacII/AgeI) using the same polymerase chain
reaction method. The MRP1 sense primer
(5'-GCGGCCGCGGATGGCGCTCCGGGGCTTC) introduces a SacII site
immediately adjacent to the start codon, and the antisense primer
(5'-TACGGTACCGGTGCCACCAAGCCGGCGTCTTTGG) adds an AgeI site
and removes the stop codon of MRP1.
The constructs were transiently transfected into MDCK cells and L1210
cells using a LipofectAMINE transfection kit (Life Technologies, Inc.) using 1 µg of DNA. Transfections of MDCK cells were carried out
using Transwell plates (Costar, 24 mm × 3 µm polycarbonate membrane) to enable cell polarization. Cells were imaged using a Nikon
TE300 inverted microscope linked to a Radiance 2000 Laser Scanning
System for confocal microscopy and Lasersharp 2000 imaging software (Bio-Rad).
Site-directed Mutagenesis--
All mutations were achieved using
the QuikChange site-directed mutagenesis kit (Stratagene). Successful
mutagenesis was confirmed by sequencing through the site of the
mutations using the Big Dye Terminator Cycle Sequencing Kit
(Applied Biosystems). Two clones from separate mutagenesis
reactions were selected for all mutants and transfected separately to
eliminate the chance that an unintended mutation that would interfere
with the targeting of the protein would be incorporated during the
amplification reaction.
2,4-Dinitrophenyl Glutathione Transport--
DNP-GS was
generated in L1210 cells by exposure to 1-chloro-2,4-dinitrobenzene and
its efflux determined as described previously (16).
Immunofluorescence--
Detection and localization of
untagged mutant MRP2 (
Detection of P-glycoprotein was achieved using the antibody MRK16
(Kamiya Pty. Ltd.). 2 × 105 cells were washed with
PBSF and incubated with the primary antibody (2 µg) for 1 h at
room temperature then washed two times with PBSF. The cells were
incubated with fluorescein isothiocynate-conjugated F(ab') (1:400) for
30 min, washed three times, and resuspended in PBSF ready for
immediate confocal microscopy.
Alignments and Molecular Modeling--
The protein sequence of
the C-terminal cytoplasmic domains of 37 ABC transporters from the
P-glycoprotein and MRP subfamilies were aligned with the histidine
permease (HisP) sequence using the ClustalW alignment program. The
multiple sequence alignment was used with the coordinates of the HisP
crystal structure (17) to generate a homology model of the C-terminal
cytoplasmic domain from MRP1 and MRP2 using
BioNavigator at the ANGIS Internet site (BioNavigator
by eBioinformatics Pty. Ltd.). The models were generated using
the Rigorous Models software (18) and presented using Swiss PdbViewer (v3.6b3) (19).
MRP2-gfp and MRP1-gfp Localization in MDCK Cells--
To
conveniently detect the localization of the proteins under
investigation, we constructed GFP fusion proteins and used confocal microscopy to visualize the fluorescent product. Using an MRP2-specific antibody, native MRP2 was previously shown to localize to the apical
membrane of MDCK cells (15, 20). In the present study, human MRP2 with
GFP fused to its C terminus localized to the apical membrane in
polarized MDCK cells, consistent with the localization of the native
protein (Fig. 1). The apical membrane of
polarized MDCK cells grown on Transwell membranes is the surface facing the media as opposed to the surface adhering to the membrane
(basolateral).
MRP1 has been previously immunolocalized to the basolateral membrane of
a pig kidney epithelial cell line (LLC-PK1) (21). In the present study,
human MRP1 with GFP fused to its C terminus also demonstrated
basolateral localization in polarized MDCK cells (Fig.
2). These studies establish that fusion
of MRP1 and MRP2 to GFP does not interfere with the normal targeting of
these proteins to the basolateral and apical membranes.
Expression of MRP2-gfp Alanine Mutants in MDCK Cells--
To further
characterize the TKF motif of Expression of MRP2 in L1210 Cells--
In initial unpublished
studies we found little evidence for the transport of DNP-GS by MRP2
expressed in the mouse leukemia cell line L1210 (data not shown). This
lack of function suggests that the protein did not localize to the cell
membrane in these cells. To confirm this observation, MRP2-gfp was
expressed in L1210 cells and was found to localize predominantly in
intracellular vesicles with only minor membrane localization (Fig.
5A, (i)). Since
2,4-Dinitrophenyl Glutathione Transport--
L1210 cells are
nonadherent and nonpolarized and could be potentially used as a
convenient cell line for assessing the transport function of MRP2.
Although deletion of the TKF motif allowed expression of MRP2 in the
L1210 cell membrane, it was not clear if the motif was also necessary
for its transport function. As shown in Fig. 6, L1210 cells stably expressing Alignments and Homology Modeling--
To gain an overview of the
potential differences in structure of the MDR and MRP proteins targeted
to apical membranes, an alignment was made of the sequences encoding
the C-terminal cytoplasmic domains of 37 ABC transporter proteins. Part
of the alignment is represented in Fig.
7, which shows that those MRP proteins that localize to the apical membrane (MRP2 from four species) have a
C-terminal extension when compared with MRP1, MRP3, MRP5, and MRP6,
which are targeted to the basolateral membrane. MRP4 also appears to
have a potential PDZ-interacting domain at its C terminus, but its site
of expression has not been described.
The structural coordinates for the ATP binding subunit of histidine
permease from Salmonella typhimurium (17) and the full ABC
transporter sequence alignment allowed the construction of homology
models of the equivalent regions of MRP1 and MRP2 (Fig. 8). Since the C-terminal motif of MRP2
extends beyond the alignment with HisP, the exact position of the TKF
residues cannot be predicted. However, the models predict that the TKF
motif is positioned on the outside of the protein, away from the ATP
binding cassette and regions involved in the cytoplasmic subunit
interface. In addition, the external position of the motif would favor
interactions with other proteins involved in the targeting process.
Human MRP2 specifically localizes to the apical membrane of
polarized epithelial cells in the liver and kidney. This localization can be replicated experimentally in MDCK cells (15, 20) and LLC-PK1 cells (23, 24), and we demonstrate in this study that an
MRP2-gfp fusion protein also localizes to the apical membrane (Fig. 1).
This allowed us to undertake mutational analysis to determine targeting
signals for apical localization. Deletion of the three amino acids from
the C terminus of MRP2 ( To further characterize the motif, alanine was introduced into the
position of each residue separately, and an additional mutant was made
in which all three residues were replaced by alanine. The T1543A mutant
did produce a change in targeting compared with the native protein,
allowing both basolateral and apical targeting, i.e.
nonpolarized targeting, and also an increased accumulation in vesicles,
suggesting some instability in the targeting mechanism. This is
consistent with the motif being a PDZ-interacting domain characterized
by the motif Ser/Thr-X-hydrophobic residue, where X represents any amino acid (26). This was consistent with
the results obtained by the TKF-AAA mutant. The F1545A mutant did not
alter normal targeting, suggesting that alanine is a sufficiently hydrophobic residue. The canonical PDZ domain is reported to tolerate any residue (X) at the The deletion of the TKF motif increases the sequence similarity
of MRP2 to MRP1 and results in the same basolateral targeting. To
investigate the tertiary structure of the subunit and the position of
the motif, homology models of both MRP1 and MRP2 were created based the
crystal structure of HisP. Comparisons of the homology models clearly
show the difference in length of the C terminus of MRP1 and MRP2. It is
not clear whether it is the interaction with the PDZ-interacting domain
that is solely responsible for the apical localization or whether it is
the spatial arrangement of the extension and the predicted motif that
allows binding/modification to another part of the protein. From the
homology model of MRP2 it appears likely that the motif is available
for interaction and not buried within the subunit. Also, the position
of the C terminus in this model suggests that the motif does not
interact with functionally significant areas such as the ATP binding
sites. This is further supported by the ability of the deletion mutant There is a notable difference in the sorting of MRP2 in L1210
cells compared with the MDCK cells. When expressed in the L1210 cells,
MRP2 transport function was minimal (results not shown). Immunofluorescence studies of cells expressing MRP2 and confocal imaging of cells expressing MRP2-gfp both confirm the intracellular localization of the protein in L1210 cells (Fig. 5). Deletion of the
apical targeting motif (the PDZ-interacting domain) from MRP2 and
MRP2-gfp allows the mutant protein to be expressed in the plasma
membrane. Therefore it is the apical targeting motif that excludes the
native MRP2 from the membrane. The stability of the protein in the
membrane also differs between MDCK cells and L1210 cells. In MDCK cells
The attachment of GFP to the C terminus of MRP1 or MRP2 did not
interfere with correct targeting in this study nor in a previous study
of MDR1 (27). Based on the homology models in Fig. 8, the
position of the C-terminal helix indicates that GFP would sit
on the outside of the subunit. The position of GFP therefore would
suggest that the PDZ-interacting domain does not need to be freely
exposed and carboxylated to function. In support of this, rabbit MRP2
has a predicted PDZ-interacting domain in the same position as human,
mouse, and rat MRP2 but is followed by a further 21 amino acids (Fig.
7).
The GFP fusion proteins were expressed at consistent levels under the
CMV promoter of the EGFP-N1 vector. MRP2-gfp localized apically in the
majority of polarized MDCK cells as represented in Fig. 1. However, a
minority of cells had very high levels of MRP2-gfp expression and
exhibited both apical and lateral localization. This situation could
occur in vivo in cancer cells where a number of
multidrug-resistant proteins are up-regulated and thus
"over"expressed. MRP2 has been found to be expressed in ovarian
cancer cells lines (28), renal clear cell carcinomas (29), lung,
gastric, and colorectal cancer cells (25). Depending on the
expression levels of MRP2 in these cells, the localization may not be
solely apical but may also be lateral and basolateral. Interestingly,
P-glycoprotein is specifically apical in polarized cells but can also
be expressed in the membrane of hematopoietic cells without the
intracellular accumulation observed with MRP2 in L1210 cells (results
not shown). Furthermore, Pgp is normally expressed in polarized cells
of the gastrointestinal tract but is expressed in MDR tumors in other tissue types that are not necessarily polarized. This indicates a
difference in the targeting signals between MRP2 and
P-glycoprotein.
This study demonstrates that the PDZ-interacting domain of MRP2
functions as the apical targeting signal in MDCK cells yet excludes the
protein from the cell membrane in L1210 cells. This motif is not
present in the basolaterally targeted MRP proteins 1, 3, 5, and 6.
MRP2) causes the protein to
be localized predominantly in the basolateral membrane in polarized
Madin-Darby canine kidney cells. Interestingly, MRP2 expressed
in a mouse leukemia cell line (L1210 cells) predominantly accumulates
intracellularly with minimal cell membrane localization. In contrast,
MRP2 was shown to predominantly localize in the cell membrane in
L1210 cells. Increased transport of 2,4-dinitrophenyl glutathione from
L1210 cells expressing
MRP2 showed that the re-targeted protein
retains its normal function.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(8-10) and EHBR (11). Many
studies have shown that MRP1 and MRP2 have similar substrates, which
include glutathione conjugates, glucuronide conjugates, reduced
glutathione, and chemotherapeutic drugs. Although these two proteins
have similar functions, their tissue distribution would indicate that
they have different roles. MRP1 is found throughout the body in many
tissues, including the hematopoietic system, the blood brain barrier,
lungs, and at lower expression levels in the liver and kidneys. In
contrast, MRP2 is only found at significant levels in the liver and to
a lesser extent in the kidneys. In these two tissues, where both
proteins are expressed, they differ in their specific cellular
localization. MRP1 is found in the basolateral (sinusoidal) membrane
and thus may serve to redirect potential excretion products back into
the bloodstream. Conversely, MRP2 is solely found in the apical
membrane, and this defines its function as an export pump of compounds
destined for terminal excretion from the body. Although both proteins
can be found in the hepatocyte, higher expression levels of MRP2 than MRP1 create the vectorial transport of excretion products from the
blood into bile.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MRP2 Function--
A construct that expressed MRP2 with the
C-terminal TKF motif deleted (
MRP2) and without the GFP tag
was prepared using the QuikChange site-directed mutagenesis kit. A
construct of the MRP2 cDNA in the mammalian expression vector
pRc/CMV (Invitrogen) (7) was used as the template for site-directed
mutagenesis. The sense primer (5'-GAATGTGAACAGCTAGCAGAAGGCC) and
antisense primer (5'-GGCCTTCTGCTAGCTGTTCACATTC) deleted the nine
nucleotides that coded for the amino acid residues Thr1543, Lys1544, and
Phe1545. Successful mutagenesis of two clones from separate
reactions was confirmed by sequencing. Stable transfectants in L1210
cells were selected for further study.
MRP2) was achieved by immunofluorescence using
the antibody M2III6 (Kamiya Pty. Ltd). 2 × 105 cells were washed with PBSF (phosphate-buffered saline
supplemented with 2.5% fetal bovine serum). The cells were
permeabilized using digitonin (5 µg/ml) and incubated at room
temperature for 15 min. The cells were then washed three times with
PBSF and then incubated with the primary antibody (2 µg) for 1 h
at room temperature before being washed twice with PBSF. The cells were
incubated with fluorescein isothiocynate-conjugated F(ab')2 (Silenus,
Hawthorn, Victoria, Australia) (1:80 dilution) for 30 min at room
temperature. Finally the cells were washed three times and resuspended
in PBSF ready for immediate confocal microscopy.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A representative MDCK cell expressing
MRP2-gfp in a confluent monolayer of cells. Fluorescence is
evident throughout the cell in the top down view. However, in
cross-section, the XZ view reveals specific apical (AP)
localization and minimal basolateral (BL) targeting. The
coverslip is detected as a line on the apical surface of the cells due
to autofluorescence. All scale bars = 5 microns.
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Fig. 2.
Confluent MDCK cells expressing MRP1-gfp have
a ringed appearance in the top down view due to fluorescence in the
(baso)lateral (BL) membrane. In the XZ view the
lateral targeting of MRP1-gfp is confirmed with the cell to cell
membranes being defined. AP, apical.
MRP2-gfp in MDCK Cells--
Recently a specific
apical targeting signal was identified in CFTR (13). The signal
consisted of a three-residue PDZ-interacting motif at the C terminus.
The alignment of C-terminal sequences of MRP1 and MRP2 revealed a
similar C-terminal motif in MRP2 (TKF) that was absent in MRP1 and has
recently been shown to function as a PDZ-interacting domain (22). To
determine whether the TKF motif influenced the apical localization of
MRP2, we constructed
MRP2-gfp in which the three C-terminal residues
were deleted. When expressed in polarized MDCK cells,
MRP2-gfp was
found to localize predominantly in the lateral membranes rather than
the apical localization of MRP2-gfp (Fig.
3).
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Fig. 3.
Confluent MDCK cells expressing
MRP2-gfp appear ringed in the top down view with a
similar appearance to MRP1-gfp (Fig. 2). In the XZ view
MRP2-gfp shows definite lateral localization with the cell to cell
membrane outlined by fluorescing protein. Apical (AP)
targeting is minimal compared with native MRP2 fused to GFP (Fig. 1).
BL, basolateral.
MRP2-gfp and to determine the
relative importance of each residue, individual alanine mutations were
introduced into each of the residue positions 1543-1545 of the
MRP2-gfp construct. Since residues 1542-1544 (STK) form a predicted
phosphorylation site, residue 1542 was also mutated to alanine. Fig.
4, A
E, show the localization of each of
these mutants in MDCK cells. The effects of the substitutions were
determined by visualizing the change in localization of the mutant
compared with the native protein. The T1543A and K1544A mutants had
both apical and basolateral targeting (nonpolarized distribution) with an increase in protein accumulation in intracellular vesicles. The
F1545A mutant did not have altered targeting. Mutation of all three
residues to alanines caused the protein to be distributed in a
nonpolarized manner.
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Fig. 4.
Mutations of the targeting motif by
site-directed mutagenesis. A, the T1543A mutation
produced a nonpolarized distribution of the fusion protein.
Fluorescence was detected in both the apical (AP) and
basolateral (BL) membranes giving a ringed appearance from
the top down view, but the XZ view reveals the non polarized
distribution. The intracellular fluorescence is due to background
autofluorescence and not GFP. B, the K1544A mutation also
lost polarized distribution of the protein with the protein detected in
the apical and basolateral membranes. C, F1545A, mutation of
the C-terminal residue to alanine, did not alter the apical targeting
of the protein. D, TKF-AAA, mutation of all three residues
in the motif, results in the ringed effect from the top down view.
However, in the XZ view there is distribution of the protein in both
the apical and basolateral (shallow cell) membranes, indicating
nonpolarized distribution. E, S1542A, mutation of the serine
produced a less distinct distribution. The plasma membrane was outlined
by the fluorescence of the protein, but on closer inspection in the XZ
view, the fluorescence appears to be in submembrane vesicles.
MRP2-gfp localized basolaterally in MDCK cells, we were interested
to determine whether it was able to localize in the L1210 cell
membrane. In contrast to MRP2-gfp,
MRP2-gfp expressed in L1210 cells
almost exclusively localizes to the cell membrane (Fig. 5A,
(ii)). To confirm the localization, we studied cells stably
expressing MRP2 and
MRP2 without the GFP tag by immunofluorescence. MRP2 was detected intracellularly and had a vesicular localization within the cell (Fig. 5B, (i)), the same
distribution as in Fig. 5A.
MRP2 was detected in the cell
membrane (Fig. 5B, (ii)), the same localization
as
MRP2-gfp shown in Fig. 5A, (ii).
These results suggest that the deletion of the TKF motif from MRP2
allows the successful targeting of the protein to the membrane of
nonpolarized L1210 cells. However, some L1210 cells expressing
MRP2
or
MRP2-gfp demonstrated a degree of intracellular accumulation,
suggesting that localization of
MRP2 in the cell membrane of L1210
cells is not as stable as in MDCK cells.
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Fig. 5.
A, L1210 cells were transiently
transfected with MRP2-gfp and MRP2-gfp. i, the majority
of MRP2-gfp accumulated in intracellular vesicles with minimal plasma
membrane localization. ii, in contrast, the majority of
MRP2-gfp localized to the cell membrane. B, an antibody
to MRP2 (M2III6) was used to detect MRP2 and
MRP2
localization in stably transfected L1210 cells. i, MRP2 was
detected in intracellular vesicles surrounding the nucleus
(N). This localization is consistent with MRP2-gfp
localization. ii,
MRP2 was detected in the cell membrane
confirming the effects of the TKF motif deletion found with
MRP2-gfp.
MRP2
demonstrated significantly higher efflux of DNP-GS compared with
control L1210 cells or L1210 cells expressing native MRP2.
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Fig. 6.
Efflux of DNP-GS into the supernatant by
L1210 cells was determined at specific time intervals by
spectrophotometry (16). The control L1210 cells and those
transfected with wild type MRP2 had the same rate of efflux. The L1210
cells expressing MRP2 had increased transport of the DNP-GS into the
extracellular medium. The background transport of DNP-GS is due to
constitutive MRP1. Results are the mean of three separate
experiments ± the S.D.
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Fig. 7.
Alignment of the C terminus of MRP proteins
from a number of species with the HisP protein. This alignment is
derived from an alignment of the entire C-terminal cytoplasmic domain
of 37 ABC transporters. The MRP2 homologues have a distinct C-terminal
extension when compared with the basolaterally targeted proteins MRP1,
MRP3, and MRP6.
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Fig. 8.
The homology model of the C-terminal domain
of MRP1 and MRP2, based on the crystal structure of HisP. The view
is looking down on the subunit from the membrane into the cytoplasm.
The lower face is the C-terminal helix. The tail on the end of this
helix in MRP2 is clearly longer than MRP1. The TKF motif sits at the
end of the tail close to helix 4.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MRP2-gfp) caused a dramatic change in the
targeting of the protein to the basolateral membrane in MDCK cells. The
mutant's localization in a polarized cell now mimics MRP1 (Fig. 2),
which does not have the deleted motif. This observation indicates that
the three-amino acid motif targets MRP2 to the apical membrane and
dominates any (as yet unidentified) basolateral targeting signals.
Conversely, the motif could exclude the protein from the basolateral
domain thus only allowing apical localization. Targeting and/or
exclusion are usually mediated by a secondary protein(s) responsible
for the recognition of such sorting signals. Interestingly, the motif responsible for apical targeting of MRP2 is a predicted PDZ-interacting domain and has been reported to associate with a PDZ protein, PDZK1
(22). This is an analogous mechanism to that of CFTR, which is also
targeted apically by a PDZ-interacting domain at its C terminus (13,
30). CFTR was shown to associate
with EBP-50, a PDZ protein, and both EBP-50 and PDZK1 were shown to localize specifically to the apical domains in polarized cells (13,
22). Whether PDZK1 and EBP-50 are responsible for sorting and targeting
of their respective ABC transporters is yet to be determined.
Alternatively these PDZ proteins may be responsible for stabilizing
transporters such as CFTR and MRP2 in the membrane, which could also
explain their localization solely at the apical domain.
1 position, but the K1544A caused
nonpolarized targeting, suggesting some flexibility in the constraints
determining functional PDZ domains. Interestingly, the serine residue
at position 1542 forms a predicted phosphorylation site, and mutation
of the serine caused the fusion protein to localize in subapical
vesicles. This suggests that the serine may be phosphorylated and could regulate recruitment into the apical membrane.
MRP2 to transport 2,4-dinitrophenyl glutathione when expressed in
L1210 cells (Fig. 6). Alignment of MRP2 with the basolateral MRP
proteins MRP1, MRP3, MRP5, and MRP6 shows the absence of the motif in
the basolateral transporters. Based on this alignment the MRP2 residues
1539-1545 may play a role in the targeting mechanism as this is the
full length of the extension of the C terminus compared with the
basolateral proteins. If this motif is the sole targeting signal, then
one could predict that MRP4 will be found to be apically targeted in
polarized cells. The P-glycoprotein homologues MDR2 (or MDR3) and sPgp
(all apically targeted in polarized cells) also have a difference in
length compared with the basolaterally localized proteins (alignment
not shown). However their overall similarity to MRP2 is very low and
does not allow a prediction based on the MRP2 motif. Further studies
are required to determine the targeting signals of the Pgp subfamily.
MRP2-gfp has basolateral localization in the majority of cells. In
L1210 cells, the majority of cells have intracellular localization of
MRP2-gfp or
MRP2 with the remainder of the cells having cell
membrane targeting. This suggests an instability of the mutant protein
in the membrane, and it is possible that an additional protein
responsible for stabilizing MRP2 in the membrane is not present in
L1210 cells.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Dan Liu for initial discussions and to Professor David Jans for advice on the confocal microscopy. We thank Dr. W. Hagmann for the MRP1 cDNA.
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FOOTNOTES |
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* This work was supported by Biotron Limited and was also supported in part by a grant from the Canberra Hospital Private Practice Trust Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Molecular Genetics
Group, Division of Molecular Medicine, John Curtin School of Medical
Research, Australian National University, Canberra ACT 0200, Australia.
Tel.: 61-2-61254714, Fax: 61-2-61254712; E-mail: Philip.Board@anu.edu.au.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M010566200
2 B. D. Moyer, M. Duhaime, C. Shaw, J. Denton, D. Reynolds, K. H. Karlson, J. Pfeiffer, S. Wang, J. E. Mickle, M. Milewski, G. R. Cutting, W. B. Guggino, M. Li, and B. A. Stanton, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: cMOAT, canalicular multispecific organic anion transporter; ABC, ATP binding cassette; MRP, multidrug resistance-associated protein; CFTR, cystic fibrosis transmembrane regulator; GFP, green fluorescent protein; MDCK, Madin-Darby canine kidney; DNP-GS, 2,4-dinitrophenyl glutathione; HisP, histidine permease.
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