MRP3, a new ATP-binding cassette protein
localized to the canalicular domain of the hepatocyte
Daniel F.
Ortiz1,
Shaohua
Li1,
Ramachandran
Iyer1,
Xingming
Zhang1,
Phyllis
Novikoff2, and
Irwin M.
Arias1
1 Department of Physiology, Tufts University
School of Medicine, Boston, Massachusetts 02111; and
2 Department of Pathology, Albert Einstein
College of Medicine, Bronx, New York 10461
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ABSTRACT |
Bile secretion in liver is
driven in large part by ATP-binding cassette (ABC)-type proteins that
reside in the canalicular membrane and effect ATP-dependent transport
of bile acids, phospholipids, and non-bile acid organic anions.
Canalicular ABC-type proteins can be classified into two subfamilies
based on membrane topology and sequence identity: MDR1, MDR3, and SPGP
resemble the multidrug resistance (MDR)
P-glycoprotein, whereas MRP2 is similar in structure and
sequence to the multidrug resistance protein MRP1 and transports similar substrates. We now report the isolation of the rMRP3 gene from
rat liver, which codes for a protein 1522 amino acids in length that
exhibits extensive sequence similarity with MRP1 and MRP2. Northern
blot analyses indicate that rMRP3 is expressed in lung and intestine of
Sprague-Dawley rats as well as in liver of Eisai hyperbilirubinemic
rats and TR
mutant rats, which are deficient in MRP2
expression. rMRP3 expression is also transiently induced in liver
shortly after birth and during obstructive cholestasis.
Antibodies raised against MRP3 recognize a polypeptide of
190-200 kDa, which is reduced in size to 155-165 kDa after treatment
with endoglycosidases. Immunoblot analysis and immunoconfocal
microscopy indicate that rMRP3 is present in the canalicular membrane,
suggesting that it may play a role in bile formation.
MRP; bile duct ligation; TR
rat; apical
hepatocyte domain; glycoprotein
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INTRODUCTION |
BILE SECRETION is an essential route for excretion and
recirculation of drugs, vitamins, and lipids as well as endogenous and
exogenous toxins. Biliary constituents such as bile acids, phospholipids, and glutathione and glucuronide conjugates exhibit concentrations in bile that are manyfold higher than those found in
cytoplasm, indicating that secretion is an active process. Various
ATP-binding cassette (ABC)-type proteins, which are sorted to the
apical domain of the hepatocyte, promote bile formation by mediating
ATP-dependent translocation of biliary components across the
canalicular membrane. SPGP effects ATP-dependent transport of
taurocholate (3, 7), generating a large fraction of the osmotic
gradient that drives bile flow. MDR3 mediates flipping of
phosphatidylcholine in the canalicular membrane, thereby fostering its
release into bile (28, 33). MRP2 (also called cMOAT and cMRP) is
primarily responsible for non-bile acid-dependent bile flow, effecting
biliary secretion of glutathione, sulfate, and glucuronide conjugates,
which include conjugated bilirubin (2, 12, 31). MDR1 is associated with
transport of a wide variety of small, hydrophobic, cationic drugs into
bile (16) and flipping of short-chain phospholipids in the canalicular
membrane (41).
A number of inheritable human disorders are associated with mutations
in genes that code for canalicular transporters. Mutations in the SPGP
gene are associated with subtype II of progressive familial
intrahepatic cholestasis (PFIC) (36). PFIC III and the Dubin-Johnson
syndromes have been linked with mutations in the MDR3 (5) and MRP2 (17,
32, 42) genes, respectively. Single nucleotide changes in the MRP2 and
MDR3 mRNAs introduce nonsense codons that interrupt the protein coding
sequences and result in destabilization of the mutant transcripts.
Animal models have been generated or identified for some of these
diseases. For example, mdr2
/
knockout mice (35) exhibit a
pattern of cholestasis and cirrhosis similar to that observed in PFIC
III patients. TR
mutant rats (2, 31) and Eisai
hyperbilirubinemic rats (EHBR) (12) were identified as
hyperbilirubinemic mutants deficient in biliary secretion of non-bile
acid organic anions and were shown to harbor point mutations in
the MRP2 gene that severely reduce RNA and protein levels. Lith mice,
which manifest cholesterol cholelithiasis, overexpress SPGP and display
altered taurocholate levels in bile (43).
Canalicular ABC-type transporters can be classified into two major
subtypes based on sequence and structure. ABC-type proteins characteristically contain one or two highly conserved
nucleotide-binding domains (NBD) associated with hydrophobic regions
capable of spanning the membrane multiple times. SPGP, MDR1, and MDR3
share extensive amino acid sequence similarity and exhibit a
symmetrical 6+6 arrangement of transmembrane helices in the hydrophobic
domains linked to the NBDs. MRP2 and its homologue MRP1 are
asymmetrical in structure, display an 11+6 organization of the
membrane-spanning domains (18, 19), and share little amino acid
sequence similarity with the MDR subfamily outside of the conserved NBD
regions. We now report identification in rat liver of another member of
the MRP subfamily, named rMRP3, which shares substantial sequence and
structural similarities with MRP2 and MRP1. rMRP3 expression is induced
by obstructive cholestasis and in mutant rats deficient in MRP2
expression. We also show that the rMRP3 protein is present in the
canalicular membrane of the hepatocyte.
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MATERIALS AND METHODS |
Cloning.
The amino acid sequence of the yeast bile acid transporter BAT1p (29)
was aligned with the proteins most closely resembling it in yeast and
mammals by using the CLUSTALW algorithm. Sequence motifs that were
conserved in MRP1, YHLOp, YK83p, and YCF1p, but absent in the
canalicular ABC-type transporters MDR1 and MDR3, were identified and
used to design degenerate oligonucleotide primers. The primers were
used for RT-PCR of 1 µg of poly(A)+ RNA prepared from
human liver and TR
rat liver; the latter represents a
rat mutant deficient in MRP2 mRNA accumulation. The sense primer
(AAAGAATTCGGIATIGTIGGIC/AGIACIGG) represented amino acid sequence
GIVGRT, and the antisense primer (AAAGAATTCAA/GICC/TG/ATGIGCIATIGT) was
derived from amino acid sequence TIAHRI. An EcoR I site
preceded by three random nucleotides was incorporated on the 3'
terminus of each primer. PCR parameters were 3× 95°C, 40°C,
72°C and 30× 95°C, 55°C, 72°C; all steps were for 1 min. PCR
products 470-520 bp in length were purified from agarose gels and
cloned into the pCR2 vector (Invitrogen). Clones were classified by
restriction mapping, Southern blot analysis, and nucleotide sequencing.
cDNA inserts were isolated from human liver (Stratagene) and rat
duodenum (a gift of Dr. Andrew Leiter) lambda cDNA libraries by
hybridization with radiolabeled human and rat RT-PCR inserts purified
from pCR2. cDNA inserts of 3.0 kb and 1.5 kb were isolated for rat and
human, respectively. Multiple rounds of 5' rapid amplification of cDNA
ends (RACE)-PCR of human and TR
rat liver
poly(A)+ RNA were performed with a kit obtained from Life
Technologies and according to the manufacturer's instructions.
A full-length cDNA was constructed for each gene by combining as much
as possible of the cDNA inserts obtained from lambda cDNA library and
cDNAs generated by PCR. The 5' portion of the rat and human cDNAs were
amplified from liver poly(A)+ RNA by RT-PCR using the
proofreading thermostable polymerase Pfu (Stratagene).
Oligonucleotide primers were designed based on sequence information
obtained from the furthermost 5' RACE products. The human gene primers
were TATGCGGCCGCTCGCCTTCCTTGCAGCC (sense) and
AGATCTAGAGTGTCAAAGAAGGACTGTGG (antisense), which included a Not
I and Xba I, respectively, on the 3' terminus. For the rat cDNA, primers were CTTCTAGCTGGGGTTGAG (sense) and GTCCCTGGTCCAGAAGGAG (antisense). The nucleotide sequences of the human and rat cDNAs were
obtained by automated DNA sequencing of double-stranded plasmid DNA.
Northern blot analysis.
Tissues were removed from euthanized rats and frozen immediately in
liquid nitrogen. Total RNA was prepared using the Trizol reagent system
(LifeTechnologies). Poly(A)+ RNA was prepared from total
RNA using the FastTrac 2.0 kit (Invitrogen). Poly(A)+ RNA
(5 µg/lane) was loaded and separated by electrophoresis on denaturing
formaldehyde-1% agarose gels. RNA was immobilized on GeneScreen plus
(DuPont) membranes after capillary transfer. DNA fragments were
radioactively labeled by random primer extension (Boehringer Mannheim).
The rMRP3 probe was a 1.3-kb BamH I fragment obtained from
rMRP3 cDNA. The rMRP2 probe was a 1.7-kb Nco I-Xho I
fragment obtained from a partial rMRP2 cDNA. Hybridization at 42°C
was done in 50% formamide, 6× standard saline citrate (SSC), 10×
Denhardt's, 0.5% SDS, and 100 µg/ml denatured salmon sperm DNA in a
Robbins Scientific hybridization oven. The blots were washed
sequentially with 2× SSC, 0.5% SDS at 25°C, 1× SSC, 0.25% SDS
at 42°C, and 0.2× SSC, 0.05% SDS at 65°C and exposed to film.
Bile duct ligation.
Male Sprague-Dawley rats 250-300 g in weight were anesthetized by
intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt).
The abdominal cavity was opened aseptically, and the common bile duct
was ligated at two locations and cut between the ligatures.
Sham-operated animals underwent the same procedure in which the common
bile duct was exposed but not ligated or cut. Livers were extracted at
the times indicated and processed for subcellular fractionation,
RNA preparation, or immunocytochemistry. Sinusoidal membrane vesicles
(SMV) and canalicular membrane vesicles (CMV) were prepared as
described (11).
Antibodies and immunoblot analysis.
The "linker" domain of MRP3, which resides immediately downstream
of the first NBD, exhibits little or no homology with other proteins in
the database. A 216-bp DNA fragment encoding amino acids 876-948
of human MRP3 was ligated in frame with the glutathione S-transferase (GST) coding region of pGEX5-3x
(Pharmacia). The construct was transformed into Escherichia
coli, and a GST-MRP3 fusion protein was overexpressed and purified
by affinity chromatography on GSH-Sepharose (Pharmacia). Antisera
raised against the GST fusion protein recognized an
isopropyl-
-D-thiogalactopyranoside inducible peptide of relative molecular mass 39 kDa in extracts of
E. coli expressing the GST-MRP3 fusion. IgG was purified from rabbit sera by affinity chromatography on protein A-Sepharose (Pharmacia). Anti-GST antibodies were cleared from IgG by passage through a GST-Sepharose column (Pierce). GST-MRP3 was bound to Affigel-10 (Bio-Rad) following the manufacturer's instructions and
used to purify MRP3-specific IgG by affinity chromatography. Immunoblot
analysis was performed as described (29). Briefly, 50 µg of protein
were incubated for 10 min at 65°C in 2.5% SDS, 0.1 M
Tris · HCl, pH 6.8, 1 M urea, and 5 mM
dithiothreitol and separated by SDS-PAGE. Proteins were
electrotransfered to nitrocellulose and incubated with antibody diluted
in 0.25% (wt/vol) gelatin, 0.5% (wt/vol) BSA, 50 mM
Tris · HCl, pH 8.0, 150 mM NaCl, and 0.05% (vol/vol) Tween
20. Secondary antibodies were horseradish peroxidase-conjugated goat
anti-rabbit IgG and anti-mouse IgG (Bio-Rad). Antibody staining was
visualized by enhanced chemiluminescence (Pierce)
Immunocytochemistry.
Liver fragments (5 × 5 × 2 mm) were immersed in 0.25 M
sucrose-PBS for 30 min at 0°C, submerged in optimum cutting
temperature (OCT) compound (Tissue-Tek), and frozen on dry
ice. Sections (6-8 µm) cut with a cryostat were incubated in
blocking buffer [PBS containing 3% (wt/vol) BSA, 2% fetal calf
serum, 0.02% (vol/vol) Triton X-100, and 0.1 M glycine] at 37°C for
1 h. Slides were washed in PBS at room temperature for 5 min and
incubated for 2 h with affinity-purified anti-MRP3 IgG (50 µg/ml) at
37°C. After washing with PBS for 5 min, the slides were immersed in
3% (vol/vol) formaldehyde-PBS at 0°C for 2 min, washed with three
changes of PBS for 15 min, and incubated in blocking buffer at room
temperature for 15 min. The sections were stained with 10 µg/ml C219
IgG (Signet) for 2 h at room temperature, washed with PBS for 5 min,
and incubated with Texas red-labeled goat anti-rabbit IgG and
Cy2-labeled goat anti-mouse IgG (Jackson ImmunoResearch) at 15 µg/ml in blocking buffer for 45 min at room temperature. Slides were
washed for 5 min with three changes of PBS at room temperature, mounted
with Crystalmount (Biomeda), and observed at ×400 magnification by confocal microscopy.
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RESULTS |
An MRP-like protein exhibits differential expression in liver of
TR
rats, EHBR, and normal rats.
RT-PCR of poly(A)+ RNA from TR
liver
identified a number of MRP-like sequences that differed from MRP2, the
canalicular multiple organic anion transporter. The primers were
degenerate oligonucleotides capable of hybridizing to the NBD2 coding
domains of MRP-type subfamily members but not to the MDR-type
subfamily. Northern blot analysis indicated that probes derived from
one of the PCR fragments hybridized to a 5500-nt transcript present in
lung and intestine of normal Sprague-Dawley rats and liver of mutant
EHBR. The mRNA was also elevated in liver of TR
mutant
rats but exhibited very low levels in normal Sprague-Dawley rat liver
(Fig. 1). A full-length cDNA for the gene
was assembled from lambda phage inserts and an RT-PCR fragment
generated by a proofreading thermostable polymerase. Nucleotide
sequence analysis indicated that the cDNA contained a single long open
reading frame (ORF) capable of encoding a polypeptide 1522 amino acids
in length that bears the hallmarks of an ABC-type protein (Fig.
2). The ORF contains two hydrophobic
polytopic membrane-spanning regions and two nucleotide-binding domains
(NBD1 and NBD2) harboring Walker A and B nucleotide-binding motifs and
the C moiety characteristic of ABC-type proteins.

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Fig. 1.
Expression of rMRP3 gene in rat tissues. Northern blot of RNA prepared
from Sprague-Dawley (normal) male rats and liver from an Eisai
hyperbilirubinemic rat (EHBR) mutant. Each lane contains 5 µg of
poly(A)+ RNA. The blot was hybridized to a radioactively
labeled 480-bp DNA fragment exhibiting homology to MRP subfamily
proteins. The probe recognizes a 5500-nt transcript in intestine, lung,
and EHBR liver and faint signals in other tissues. The blot was
stripped and rehybridized to a glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) probe to show comparable loading of RNA in each lane.
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Fig. 2.
Amino acid sequence deduced from a full-length rMRP3 cDNA. Analysis of
the nucleotide sequence obtained from a 5103-bp cDNA indicated the
presence of a 1522-amino acid open reading frame, which initiates with
a methionine codon that resides within a Kozak consensus sequence. Two
highly conserved nucleotide-binding domains (NDB) characteristic of
ATP-binding cassette (ABC)-type proteins can be identified that contain
consensus Walker A and B motifs as well as the ABC C signature moieties
(shown in boldface in sequence and labeled as A, B, and C). Analysis of
the amino acid sequence with the TMAP, TmPred, and PHD transmembrane
prediction algorithms identified 10-12 transmembrane domains in
the amino-terminal region and 4-6 membrane-spanning segments in
the carboxy region, which have been indicated in the sequence with a
stippled underline. PROSITE analysis identifies 9 possible
asparagine-linked glycosylation sites. Six of the sites reside within
the NBD1 and NDB2 domains and are probably not decorated with sugars.
The remaining putative receptor asparagine residues have been
circled.
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A blast search of the GenBank databases indicated the partial human
expressed sequence tag (EST) sequence MRP3 (21), so named
for its similarity to MRP1 and MRP2, is 80% identical to the rat
nucleotide and protein sequences. This level of identity is equivalent
to that observed between rat and human homologues of MRP2 and MDR3.
Accordingly, the new gene has been named rMRP3. Outside of
hMRP3, the protein most similar to rMRP3 is MRP1 (55% identity). Other
proteins exhibiting significant similarity are the canalicular multiple
organic anion transporter MRP2 (45%), EBCR (42%), Caenorhabditis
elegans MRP1 and MRP2 (39%), the sulfonylurea receptor SUR1
(31%), and Saccharomyces cerevisiae YCF1 (37%) and BAT1
(31%). After we had completed this analysis, the full-length sequences
for human MRP3 (20) and the rat MLP2 (9), which is identical to rMRP3,
were published.
Like other members of the MRP subfamily of ABC-type proteins, rMRP3 is
asymmetric and has an amino-terminal polytopic transmembrane domain
that is significantly larger than the corresponding region downstream
of the NBD. Computer-aided analysis of the deduced amino acid sequence
by Kyte-Doolittle hydropathy plot and algorithms that predict
transmembrane segments (TmPred, TMAP, and PHD) suggested that rMRP3
contains 10-12 putative membrane-spanning helices in the amino
terminus and 4-6 transmembrane segments in the carboxy region. The
hydropathy plots of the rMRP3 and MRP1 are almost identical, and
structural studies reveal that MRP1 exhibits a 6+5 arrangement of
membrane-spanning helices upstream of the first NBD and six
transmembrane helices between the two NBDs (18). The amino terminus of
MRP1 is extracellular and glycosylated (8); PROSITE analysis of the
rMRP3 sequence indicated that it contains a potential N-linked
glycosylation site in the amino terminus and a second site downstream
of the first NBD that match the position of glycosylation sites in
MRP1. Based on the amino acid sequence analysis and similarity with
MRP1, a putative membrane topology for rMRP3 is diagrammed in Fig.
3.

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Fig. 3.
Hypothetical membrane topology of rMRP3. A: Kyte-Doolittle
hydropathy plots of rMRP3 and MRP1 highlight the marked similarity in
structure between the 2 proteins. B: diagram
indicating location of putative structural features on
the rMRP3 amino acid sequence. Transmembrane segments are indicated by
dark bars and nucleotide-binding domains are indicated as shaded boxes
labeled NBD1 and NBD2. C: a model suggesting a hypothetical
membrane topology for rMRP3 that is based on the structural analysis
performed on MRP1. These studies indicated an extracellular amino
terminus, 6+5 transmembrane segments in the amino-terminal polytopic
membrane-spanning domain, and 6 membrane helices in the carboxy
terminus. Putative polysaccharide decorations on consensus asparagine
glycosylation sites exposed to the extracellular milieu are
indicated.
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rMRP3 mRNA levels change upon birth and during obstructive
cholestasis.
Little or no rMRP3 transcript was detected in livers of 18- or
20-day-old rat fetuses. Levels of rMRP3 RNA increased postpartum, peaked at 5-7 days after birth, and then decreased slowly to
levels observed in adults (Fig.
4A). The MRP2 RNA species was
also low in fetuses and increased postpartum. However, the rate of
increase in MRP2 mRNA, when compared with levels present in adults, was markedly slower than was observed for rMRP3.

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Fig. 4.
rMRP3 mRNA levels increase in liver early in development and after bile
duct ligation. Autoradiograph of Northern blots containing 5 µg of
poly(A)+ RNA prepared from 20-day-old rat fetuses and rat
pups at different times after birth (A) and RNA derived from
livers and intestine of rats that have undergone ligation of the common
bile duct (BDL) for 24 h and 72 h and from sham-operated
animals. (B) Equivalent amounts of RNA from
livers of control and TR rats were also included. Blots
were hybridized with a radioactively labeled rMRP3 probe, stripped, and
rehybridized to an MRP2 probe. The different mRNA species that
hybridize to the MRP2 probe are generated by alternative processing of
the 3'-untranslated region of the primary transcript (13).
Hybridization to a GAPDH probe was used to show comparable loading of
RNA. These results are representative of several experiments.
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rMRP3 expression is markedly increased in liver of MRP2-deficient
TR
rats and EHBR, suggesting that accumulation in the
hepatocyte of a compound normally secreted into bile may be responsible
for the increase in rMRP3 mRNA levels. An initial test of this
hypothesis is the response of rMRP3 to ligation of the common bile
duct. Northern blot analysis revealed that there was a severalfold
increase in rMRP3 mRNA 24 h after bile duct ligation (Fig.
4B). The high levels were maintained after 72 h, 5 days, or 7 days of bile duct ligation (not shown). This effect appears to be
specific to the liver; no change in rMRP3 was observed in intestine or
lung of bile duct-ligated rats, the two other rat tissues that display significant expression of rMRP3.
rMRP3 is present in the canalicular membrane.
Antibodies were raised against residues 876-948 of rMRP3, which
reside immediately downstream of the first NBD and represent the
variable linker region of rMRP3. This region is 62% identical in rat
and human MRP3 but exhibits little similarity with the MRP1-,
MRP2-, SPGP-, or MDR-encoded
P-glycoproteins. The antisera recognized a polypeptide with a relative
molecular mass of 190-200 kDa in immunoblots of CMV proteins
purified from rat liver. No detectable cross-reactive material was
detected in SMV preparations (Fig.
5A). Antibody staining of the
200-kDa protein was blocked by the GST-MRP3 fusion peptide but not by
GST alone. The c219 monoclonal antibody, which cross-reacts with
MDR-encoded P-glycoproteins, stains a 170-kDa band in CMV
proteins that is not detected in SMV. c219 staining was not affected by
GST-MRP3 or GST. The molecular mass of 169 kDa predicted for rMRP3 from
the deduced amino acid sequence is smaller than the apparent molecular
mass of 200 kDa observed in SDS-PAGE. Treatment of CMV proteins with
peptide N-glycosidase F (PNGaseF), which cleaves glycoprotein
asparagine-linked oligosaccharides (39), decreased the apparent
molecular mass of rMRP3 to a size more in agreement with that deduced
for the cDNA sequence (Fig. 5B). The 190- to 200-kDA signal
was also detected in CMV purified from TR
rats and rats
that had undergone bile duct ligation (Fig. 5C). Rat liver
subcellular fractions greatly enriched in lysosomal, mitochondrial,
endoplasmic reticulum, or cytosolic proteins exhibited no detectable
immunostaining with the MRP3 antibodies (not shown).

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Fig. 5.
Subcellular localization of rMRP3 in plasma membrane fractions
purified from rat liver. Immunoblots of canalicular membrane vesicle
(CMV) and sinusoidal membrane vesicle (SMV) proteins separated by
SDS-PAGE. A: affinity purified anti-MRP3 IgG (30 ng/ml)
recognizes a 190- to 200-kDa protein in CMV but not SMV (left).
Preincubation of the antibody with 0.5 µg/ml of purified glutathione
S-transferase (GST)-MRP3 essentially abrogates binding of the
antibody to the 200-kDa peptide (middle). Substitution of GST
alone for the fusion peptide has no effect on antibody binding (not
shown). The blot was stripped and incubated with the c219 monoclonal
antibody that recognizes MDR-encoded P-glycoproteins
(right). B: immunoblot stained with anti-MRP3 IgG
showing that incubation of SDS-solubilized CMV proteins with peptide
N-glycosidase F for 4 h (+PNGaseF) results in a 30- to 40-kDa reduction
in the apparent molecular mass of rMRP3 relative to untreated (control)
or mock treated ( PNGaseF) CMV. C: immunoblot stained with
anti-MRP3 IgG showing CMV and SMV proteins derived from
TR mutant rats, bile duct-ligated, and sham-operated
animals. In all cases, the 200-kDa protein is detected in CMV, but not
SMV, fractions. In all blots, CMV and SMV lanes contained 50 µg of
protein.
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Fluorescence immunomicroscopy of rat liver cryosections indicated that
rMRP3 colocalizes with canalicular ABC-type proteins recognized by the
c219 antibody (see Fig. 6). Strong staining of rMRP3 was observed in the canaliculus of TR
and bile
duct-ligated rats; less intense staining was detectable in liver
sections derived from sham-operated animals. Sparse lateral staining
was observed with anti-MRP3 antibodies, particularly in tissues derived
from bile duct-ligated animals; however, the strongest signal
originated from the canaliculus. No canalicular staining was observed
in liver sections incubated with preimmune IgG or when anti-MRP3 IgG
was preincubated with the GST-MRP3 fusion protein. Preincubation with
GST alone had no effect on immunostaining, and c219 staining was
unaffected by GST-MRP3 or GST.

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Fig. 6.
rMRP3 resides primarily in the canalicular membrane of hepatocytes.
Immunofluorescence confocal microscopy images of frozen rat liver
sections prepared from TR rats, animals that had
undergone bile duct ligation for 72 h (BDL), or animals that had been
sham operated (control). Sections were stained with affinity-purified
anti-MRP3 IgG and c219 monoclonal antibody, which recognizes MDR
P-glycoproteins. Bound antibodies were labeled with goat anti-rabbit
IgG-Texas red (rMRP3; left) and goat anti-mouse
IgG-Cy2 (c219; middle) and were visualized by fluorescence
confocal microscopy. Superimposition of the 2 images (rMRP3 + c219;
right) indicates colocalization of rMRP3 and P-glycoproteins in
yellow.
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DISCUSSION |
The rMRP3 gene encodes a protein that exhibits significant amino acid
sequence similarity with the glutathione conjugate pumps MRP1 (4) and
MRP2 (2, 12, 31) of mammals and YCF1 (37) and Atmrp1 (25) of yeast and
plants, respectively. Analysis of the rMRP3 primary sequence outlines a
putative membrane topology similar to that determined empirically for
MRP1, suggesting that rMRP3 has an extracellular amino terminus, 11 membrane-spanning helices in the amino-terminal region, and 6 transmembrane domains between the two NBDs. rMRP3 contains several
consensus N-linked glycosylation sites that match the location of sites
empirically identified in MRP1 (8). Treatment of membrane extracts with endoglycosidase reduces the apparent molecular mass of the protein that
reacts with anti-MRP3 antibodies, suggesting that rMRP3 undergoes a
pattern of decoration with oligosaccharides similar to that of MRP1.
The rMRP3 transcript is present at low levels in normal adult liver but
is elevated in TR
rats and EHBR, suggesting that
accumulation of an MRP2 substrate, or an event secondary to this
increase, regulates rMRP3 expression. Supporting this hypothesis is the
observation that rMRP3 mRNA levels increase rapidly during obstructive
cholestasis. Many changes occur in hepatocytes after bile duct ligation
that may induce the rMRP3 RNA increase. However, bile duct ligation is
accompanied by accumulation in the hepatocyte of MRP2 substrates, a
reduction of MRP2 RNA levels in liver, and almost complete
disappearance of MRP2 from the canalicular membrane (40). The
connection between the developmental patterns of MRP2 and rMRP3
expression in newborn liver is less clear but still consistent with the
hypothesis. rMRP3 RNA accumulates rapidly after birth and then declines
to adult levels by day 15. The MRP2 transcript undergoes a
slow, steady increase in accumulation that has not reached adult levels by day 15. However, the increase in MRP2 function by the
seventh day after birth may be sufficient to clear inducing substrates from the hepatocyte and lead to decreased rMRP3 expression.
Immunofluorescence confocal microscopy and immunoblot analyses of
subcellular liver membrane fractions indicate that rMRP3 is present in
the canalicular membrane, suggesting that it may play a role in bile
formation. However, the function of rMRP3 in liver is unknown.
Eukaryotes harbor multiple homologues of MRP-like proteins. A complete
inventory of S. cerevisiae ABC-type proteins identifies six
different genes (6), Arabidopsis thaliana contains at least
three (34), and mammals express eight or more (MRP1 through
MRP6, SUR1, and SUR2). Some of
the MRP subfamily members exhibit broad substrate specificities and
transport a wide variety of amphiphylic organic anions; MRP1 (22) and
MRP2 (15, 26) transport many glutathione and glucuronide conjugates, as
do the yeast YCF1 (23) and plant Atmrp1 (25) and Atmrp2 (24). However,
the homologues do not behave identically; MRP1 and MRP2 display
different affinities for specific substrates (15), as do the plant
homologues Atmrp1 and Atmrp2 (24). MRP2-deficient TR
rats and EHBR secrete compounds into bile that are related to MRP2
substrates and include unconjugated bilirubin (30), sulfate-conjugated drugs (38), and others (14). rMRP3 may transport one or more of these compounds.
Not all MRP subfamily members transport glutathione and glucuronide
conjugates. The yeast MRP-like protein BAT1p mediates ATP-dependent transport of bile salts that include taurocholate, glycocholate, and taurochenodeoxycholate (29). Bile salt secretion is
the principal force driving bile flow, and there is evidence suggesting
that the canalicular membrane contains a bile salt transporter in
addition to the SPGP taurocholate transporter. Isolated CMV display
robust ATP-dependent transport of glycocholate; however, insect cells
heterologously expressing SPGP exhibit no detectable glycocholate
transport (7). Moreover, the polarized WIF-B hepatoma-fibroblast fusion
cell line (10) exhibits vectorial transport and accumulation of
fluorescently labeled glycocholate in the canalicular vacuoles (1) that
form between adjacent cells and that contain canalicular transport
ATPases (27). However, WIF-B cells contain no detectable SPGP mRNA but
exhibit high levels of rMRP3 mRNA (data to be published elsewhere).
rMRP3 is similar in sequence and structure to the yeast bile acid
transporter BAT1p and may mediate transport of a subset
of bile acids that are poor substrates for SPGP.
 |
NOTE ADDED IN PROOF |
While this study was in press, Konig et al. (Hepatology 29:
1156-1163, 1999) reported localization of human MRP3 to the
basolateral hepatocyte domain. Further studies are required to
investigate possible species differences and specificity of the
immunologic observation.
 |
ACKNOWLEDGEMENTS |
We thank Fernado Agarraberes for help and suggestions in staining
and visualization of immunoconfocal fluorescence images.
 |
FOOTNOTES |
This work was supported by the American Liver Foundation Bobby Banks
Scholar Award and National Institutes of Health Grants DK-51005 and
DK-35652.
Address for reprint requests and other correspondence: D. F. Ortiz,
Dept. of Physiology, Tufts School of Medicine, 136 Harrison Ave.,
Boston, MA 02111 (E-mail: dortiz{at}opal.tufts.edu).
Received 22 December 1998; accepted in final form 15 February
1999.
 |
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