1 Division of Tumor Biochemistry and 2 Division of Cell Biology, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany
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ABSTRACT |
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Absence of a functional multidrug resistance protein 2 (MRP2; symbol ABCC2) from the hepatocyte canalicular membrane is the molecular basis of Dubin- Johnson syndrome, an inherited disorder associated with conjugated hyperbilirubinemia in humans. In this work, we analyzed a relatively frequent Dubin-Johnson syndrome mutation that leads to an exchange of two hydrophobic amino acids, isoleucine 1173 to phenylalanine (MRP2I1173F), in a predicted extracellular loop of MRP2. HEK-293 cells stably transfected with MRP2I1173F cDNA synthesized a mutant protein that was mainly core-glycosylated, predominantly retained in the endoplasmic reticulum, and degraded by proteasomes. MRP2I1173F did not mediate ATP-dependent transport of leukotriene C4 (LTC4) into vesicles from plasma membrane and endoplasmic reticulum preparations while normal MRP2 was functionally active. Human HepG2 cells were used to study localization of MRP2I1173F in a polarized cell system. Quantitative analysis showed that GFP-tagged MRP2I1173F was localized to the apical membrane in only 5% of transfected, polarized HepG2 cells compared with 80% for normal MRP2-GFP. Impaired protein maturation followed by proteasomal degradation of inactive MRP2I1173F explain the deficient hepatobiliary elimination observed in this group of Dubin-Johnson syndrome patients.
multidrug resistance protein 2; ATP-dependent transport; deficient protein maturation; protein trafficking
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INTRODUCTION |
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HUMAN DUBIN-JOHNSON SYNDROME (DJS) is an autosomal recessive disorder characterized by chronic conjugated hyperbilirubinemia (5, 33, 38). Absence of a functionally active multidrug resistance protein 2 (MRP2; gene symbol ABCC2) from the canalicular membrane of hepatocytes has been identified as the molecular basis of this disorder, accounting for the impaired secretion of conjugated bilirubin and other anionic conjugates into bile (18, 21, 31, 41). MRP2 is an integral membrane glycoprotein of ~190 kDa, localized to the apical membrane of polarized epithelia, including hepatocytes (2, 21, 23, 24, 30-32, 41), where it functions as an ATP-dependent export pump for both conjugated and unconjugated amphiphilic anions (4, 6, 17, 23).
Established mutations in the MRP2 gene leading to DJS are predominantly found in the 3'-proximal half of the mRNA and particularly in the exons encoding both nucleotide-binding domains (22, 31, 40-42). So far, however, no MRP2 protein was detectable in liver samples obtained from DJS patients (18, 21, 31, 41). A clear relationship between the site of the mutation in the MRP2 gene and the mechanisms contributing to the absence of a functional MRP2 protein from the canalicular membrane of hepatocytes has not been established. Some DJS mutations may lead to rapid degradation of the mutant mRNA, whereas others may affect chaperone interaction, protein stability, protein maturation, apical sorting, or function of a correctly localized protein. We recently demonstrated that the absence of the MRP2 protein from the canalicular membrane in a DJS patient carrying a two-amino-acid deletion within the second nucleotide-binding domain of MRP2 (41), results from impaired protein maturation, accumulation of the mutant protein within the endoplasmic reticulum (ER), and subsequent degradation by proteasomes (19).
In extension of our previous work (19), we analyzed
in the present study the consequences of two DJS-associated mutations described recently (27). On the basis of current topology
predictions, the corresponding amino acid exchange is located in an
extracellular loop of the third transmembrane-spanning domain of MRP2,
as indicated by the transmembrane-hidden Markov model (TMHMM) program
(36). An AT substitution in nucleotide 3517 in exon 25 leads to a conservative exchange of two hydrophobic amino acids,
isoleucine (I) 1173 to phenylalanine (F). The other DJS-associated
mutation results in a conservative exchange of two basic amino acids,
arginine (R) 1150 to histidine (H). Both mutations are more frequent in
the Iranian-Jewish and Moroccan-Jewish populations and comprise the largest groups of DJS patients known to date (27, 35).
Recent work has indicated that the MRP2I1173F mutation affects membrane insertion in the nonpolarized cell line HEK-293 (27). Because apical sorting of mutant MRP2 proteins can only be analyzed in a polarized cell system, we emphasize in the present study the use of polarized human hepatoblastoma G2 (HepG2) cells to study the consequences of the MRP2I1173F mutation. We demonstrate that GFP-tagged MRP2I1173F (MRP2I1173F-GFP) was predominantly retained in the ER of polarized HepG2 cells; however, a small amount of mutant protein apparently overcame the ER quality control mechanisms and reached the apical membrane. In contrast to normal MRP2, MRP2I1173F did not mediate ATP-dependent transport of the high-affinity substrate leukotriene C4 (LTC4) into isolated ER or plasma membrane vesicle preparations. Thus even when a small amount of mutant protein reaches the canalicular membrane of hepatocytes of patients carrying the MRP2I1173F mutation, transport activity is impaired, explaining the conjugated hyperbilirubinemia observed in these patients (35).
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MATERIALS AND METHODS |
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Materials.
[14,15,19,20-3H]LTC4 (4.2 TBq/mmol) and
17-D-glucuronosyl [6,7-3H]estradiol (1.5 TBq/mmol) were from New England Nuclear Life Science Products (Boston,
MA). Unlabeled LTC4 was from Amersham Biosciences
(Piscataway, NJ). Nitrocellulose filters (pore size 0.2 µm) were from
Schleicher & Schuell (Dassel, Germany). Leupeptin, pepstatin, protein
standard mixture (Mr 26,000 to 180,000),
agarose, and cell culture media were from Sigma (St. Louis, MO).
Geneticin (G418) was from Calbiochem (San Diego, CA). Endoglycosidase H and Fugene 6 transfection reagent were from Roche Molecular
Biochemicals (Indianapolis, IN). Pfu-DNA polymerase was from
Stratagene (Cedar Creek, TX). Restriction endonucleases
SfiI, SacII, and T4-DNA ligase were
from Promega (Madison, WI). All other chemicals were either from Sigma
or Merck (Darmstadt, Germany).
Antibodies.
Polyclonal EAG5 and MLE antisera were raised in rabbits against
the carboxy and amino terminals of human MRP2, respectively (1,
2, 4). The monoclonal mouse antibodies against protein disulfide
isomerase (anti-PDI) and against dipeptidyl peptidase IV
(DPPIV) were from Affinity Bioreagents (Golden, CO) and Ancell (Bayport, MN), respectively. The secondary goat anti-mouse and goat
anti-rabbit antibodies either coupled to Cy3 or Cy2 were from Jackson
Immunoresearch (West Grove, PA). The monoclonal mouse M2III-6 antibody was from Alexis Biochemicals (San Diego,
CA). The monoclonal mouse anti-vimentin antibody was provided by Progen (Heidelberg, Germany), and the monoclonal mouse anti--tubulin antibody was from Sigma. All antibodies were applied as described recently (19).
Cloning of the human MRP2I1173F, MRP2I1173F-GFP, and
MRP2R1150H-GFP constructs.
Cloning was performed as described previously (19).
Briefly, the substitution of adenine 3517 to thymine was introduced into the human MRP2 cDNA sequence (GenBank/EBI data
bank accession no. X96395) by PCR. To generate the mutation, three
independent PCRs were performed. PCR1 primer pairs were: sense
primer, 5'-CAGTGGATGCTCATGTAGG-3' (bases 2369-2387); antisense
primer, 5'-GGCACGGAAAACTGGCAAACC-3' (bases 3525-3505,
AT substitution underlined). PCR2 primer pairs were: sense primer,
5'-GGTTTGCCAGTTTTCCGTGCC-3' (bases 3505-3525, A
T
substitution underlined). The antisense primer for the MRP2I1173F construct was 5'-CCGCGGCTAGAATTTTGTGCTGTTCAC-3'
(bases 4638-4618 of the MRP2 sequence) containing a
SacII restriction site (italic) and a stop codon (bold), the
antisense primer for the MRP2I1173F-GFP construct was
5'-TGGTCCGCGGGAATTTTGT-3' (bases 4635-4627 of the MRP2
sequence) containing a SacII restriction site (italic) but
lacking the stop codon. PCR3 was run with the sense primer of PCR1 and
either one of the antisense primers of PCR2 using MRP2-GFP
(19) as the template. Fragments were subcloned between the
SfiI and SacII restriction sites of MRP2-GFP that had been subcloned into the mammalian expression vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA). For transfection into HepG2 cells, MRP2-GFP
chimeras were generated. GFP was inserted into constructs lacking a
stop codon using two SacII restriction sites. GFP-tagged MRP2R1150H (MRP2R1150H-GFP) was generated using the QuikChange site-directed mutagenesis kit (Stratagene), MRP2-GFP as the template, and the following primer pair: sense primer,
5'-CCAGCTGAGGCATCTGGACTCTGTCAC-3' (G
A substitution
underlined); antisense primer,
5'-GTGACAGAGTCCAGATGCCTCAGCTGG-3'. Successful cloning was
verified by sequencing.
Cell culture and transfection. HepG2 and HEK-293 cells were maintained in RPMI and minimum essential medium, respectively, as described (19). Stably MRP2-transfected HEK-293 cells (4) were cultured with the addition of G418 (0.8 mM). Fugene 6 transfection reagent was used according to the manufacturer's instructions. After transfection with MRP2I1173F cDNA, cells were selected with G418 for 3 wk. Single G418-resistant colonies were screened for MRP2I1173F protein expression by immunoblot analysis and immunofluorescence microscopy. HepG2 cells were transiently transfected with MRP2-GFP, MRP2I1173F-GFP, or MRP2R1150H-GFP as described (28). Expression of MRP2 constructs was enhanced by the addition of sodium butyrate (4) 24 h before cell harvesting (4, 19).
Preparation of crude membranes, immunoblot analysis, and deglycosylation. Preparation of crude membrane fractions, separation by SDS-PAGE, and immunoblotting have been described (4, 19). In some experiments, cells were treated for 24 h before being harvested with tunicamycin (1 µg/ml), which prevents formation of N-linked oligosaccharides, thus leading to synthesis of unglycosylated proteins. Membrane preparations of MRP2- and MRP2I1173F-transfected HEK-293 cells were also subjected to endoglycosidase H digestion as described (19). Core-glycosylated, immature proteins are sensitive to endoglycosidase H digestion, whereas proteins that have passed beyond the ER are insensitive to endoglycosidase H treatment.
Immunofluorescence and confocal laser scanning microscopy.
Immunofluorescence microscopy of stably transfected HEK-293 cells and
HepG2 cells transiently transfected with either GFP construct was
carried out by using the following antibody dilutions: 1:150 for EAG5,
anti-PDI, and MLE; 1:100 for anti-vimentin; 1:200 for anti--tubulin;
and 1:2,000 for anti-DPPIV (19).
Quantitative analysis of the subcellular localization of
MRP2-GFP, MRP2I1173F-GFP, and MRP2R1150H-GFP in polarized HepG2 cells.
HepG2 cells transiently transfected with MRP2-GFP,
MRP2I1173F-GFP, or MRP2R1150H-GFP were induced with 5 mM butyrate for 24 h and fixed with methanol (20°C, 1 min)
48 h after transfection. Immunostaining with the anti-DPPIV
antibody (1:300) and quantitative analysis were carried out as
described (28). At least 100 transfected (as observed by
GFP fluorescence) and polarized (as observed by ring-like DPPIV
fluorescence) cells were counted for each transfection. Localization of
the respective MRP2-GFP protein was analyzed for each transfected and
polarized cell and was classified into one of three categories:
1) apical localization, in which GFP and DPPIV fluorescence
merged in ring-like, microvilli-lined structures between adjacent
cells, i.e., the apical membrane; in addition, GFP fluorescence may
also be present in intracellular structures; 2) localization
in vesicles, in which the respective MRP2-GFP protein was absent from
the apical membrane but present in vesicles, sometimes additionally in
reticular structures; and 3) ER localization, an exclusive
reticular fluorescence with absence of GFP fluorescence from the
DPPIV-stained apical membrane and from vesicles. Six independent
transfections were analyzed, and the percentage of each localization
was calculated for each transfection.
Preparation of plasma membrane and ER vesicles and transport
studies.
Plasma membrane vesicles from control HEK-293 cells and HEK-293 cells
stably expressing MRP2 and MRP2I1173F, respectively, were prepared as
described (4, 20, 26). Briefly, cells (~3 × 109) were harvested from the cell culture by centrifugation
(1,200 g, 10 min, 4°C) and washed twice in ice-cold
phosphate-buffered saline (150 mM NaCl, 5 mM sodium phosphate, pH 7.4).
After centrifugation (1,200 g) the pellet (~5 ml) was
diluted 40-fold with hypotonic buffer (0.5 mM sodium phosphate, pH 7.0, 0.1 mM EDTA) supplemented with protease inhibitors (0.1 mM PMSF, 1 µM
leupeptin, and 0.3 µM aprotinin), gently stirred on ice for 1.5 h, and subsequently centrifuged at 100,000 g (40 min at
4°C). The pellet was resuspended in 20 ml hypotonic buffer,
homogenized with a Braun potter S 886 (500 rpm, 4°C, 300 strokes, 2 strokes/min), diluted with incubation buffer (250 mM sucrose, 10 mM
Tris · HCl, pH 7.4) and centrifuged at 12,000 g for 10 min at 4°C. The resulting postnuclear supernatant was stored on ice, and the corresponding pellet was resuspended in 20 ml incubation buffer supplemented with proteinase inhibitors, homogenized by 20 strokes, and centrifuged (12,000 g, 10 min, 4°C). Both postnuclear supernatants were combined and
centrifuged (100,000 g, 40 min, 4°C). The pellet was
resuspended in 20 ml incubation buffer, homogenized manually by 50 strokes with a tight-fitting Dounce B (glass/glass) homogenizer on ice,
and subsequently diluted with 10 ml incubation buffer and layered over
38% sucrose in 5 mM HEPES/KOH, pH 7.4. After centrifugation at 280,000 g for 2 h at 4°C in a swing-out rotor, the
interphases were collected, diluted in 20 ml incubation buffer, and
homogenized by 30 strokes with a tight-fitting Dounce B homogenizer on
ice. The suspension was centrifuged at 100,000 g (40 min,
4°C), the pellets were diluted in 1 ml incubation buffer, and
vesicles were formed by passing the suspension 20 times through a
27-gauge needle with a syringe. ER vesicles were obtained by using the
same procedure as for the plasma membrane vesicles, but instead of
collecting the interphases after centrifugation of the homogenized
membrane fraction over 38% sucrose, we retrieved the pellet from this
preparation step and diluted it in 20 ml of incubation buffer.
Formation of membrane vesicles and transport of
[3H]LTC4 (100 nM; 0.34 TBq/mmol) and
17-D-glucuronosyl [3H]estradiol (1.5 µM;
0.15 TBq/mmol) into the vesicles was performed as described (4,
20, 26). For transport measurements into plasma membrane
vesicles and ER vesicles, 50 µg and 500 µg of protein were used,
respectively, in a final volume of 110 µl.
Inhibition of proteasome activity. Proteasome function was inhibited in HEK-293 cells by the addition of MG132 at a final concentration of 2 µM 12 h before immunofluorescence microscopy (19).
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RESULTS |
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Immunoblot detection and immunolocalization of MRP2 and MRP2I1173F
in HEK-293 cells.
Synthesis of the MRP2 and MRP2I1173F proteins was studied in HEK-293
cells by immunoblot analysis using the MLE antiserum and the
M2III-6 antibody, which recognize the amino terminus and the carboxy terminus of human MRP2, respectively. Both antibodies detected MRP2 as a mature glycoprotein of 190 kDa together with a less
glycosylated form of ~175 kDa (Fig. 1, A and
D). The same molecular masses
were observed for the MRP2I1173F protein (Fig. 1B); however,
the intensities of these bands differed from those seen for MRP2.
Whereas MRP2 was predominantly present in the fully glycosylated
190-kDa form, the MRP2I1173F protein was mainly detected at 175 kDa, representing a core-glycosylated form of the protein. Tunicamycin prevented the formation of N-linked
oligosaccharide residues leading to unglycosylated MRP2I1173F
visualized at 170 kDa (Fig. 1B). The MRP2I1173F-GFP chimera
showed only one band at 205 kDa, characteristic of an immature,
partially glycosylated form of MRP2-GFP, which has an apparent
molecular mass of 220 kDa (Fig. 1C). In the presence of
tunicamycin, the chimeric protein had a mass of ~200 kDa.
Endoglycosidase H digestion decreased the molecular mass of the 175-kDa
form of both MRP2 and MRP2I1173F, whereas the bands at 190 kDa remained
unaffected (Fig. 1D). The detection signal in all MRP2I1173F
crude membrane preparations was less intense than in MRP2-containing
membranes, although the same amount of total protein (50 µg) was
used. We chose longer film exposure times for MRP2I1173F-containing
membranes to achieve a similar intense detection signal for MRP2 and
MRP2I1173F. These results suggest that a small fraction of mutant
MRP2I1173F protein matures to the fully glycosylated 190-kDa form,
whereas the larger amounts of MRP2I1173F and MRP2I1173F-GFP remain in
full length, yet immature, core-glycosylated, ER-resident forms.
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Immunofluorescence and confocal laser scanning microscopy of
MRP2-GFP, MRP2R1150H-GFP, and MRP2I1173F-GFP in HepG2 cells.
To analyze localization of MRP2I1173F-GFP in a polarized cell system we
used human HepG2 cells, which retain hepatic polarity and form apical
vacuoles corresponding to the bile canaliculus (37). To
distinguish the recombinant proteins from endogenous MRP2 (3, 13,
25), GFP chimeras were used. Imaging for GFP fluorescence in
MRP2-GFP transfectants showed ring-like structures, representing the circumference of apical vacuoles, in addition to some
vesicular structures. Apical localization of MRP2-GFP was confirmed by
overlay of the GFP fluorescence with the DPPIV-immunoreactive fluorescence as a marker protein for the apical membrane of hepatocytes (10) (Fig. 2,
A-C).
MRP2I1173F-GFP was predominantly localized in reticular structures
within the cytoplasm (Fig. 2, D-F) that were
identified as cisternae of the ER using the anti-PDI antibody as
described (19). In addition to its ER accumulation,
MRP2I1173F-GFP was occasionally found in vesicles, or, rarely, the
mutant protein was visualized within the apical membrane, where it
colocalized with the DPPIV-immunoreactive fluorescence (Fig. 2,
G-I). In contrast, MRP2R1150H-GFP was
predominantly localized to the apical membrane of polarized HepG2 cells
(Fig. 2, J-L). We then quantified the distribution of the mutant proteins among the different cellular compartments (Table 1). Normal MRP2-GFP
reached the apical membrane in 80% of polarized HepG2 cells, whereas
it remained in intracellular structures in 20% of polarized cells. In
contrast, mutant MRP2I1173F-GFP localized to the ER in 83% of
transfected and polarized cells without reaching the apical membrane
(representative image in Fig. 2F). Apical localization of
MRP2I1173F-GFP, in addition to its ER accumulation, was found in only
5% of polarized HepG2 cells (representative image in Fig.
2I). Mutant MRP2R1150H-GFP reached the apical membrane in
85% of polarized and transfected HepG2 cells (representative image in
Fig. 2L).
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Immunodetection of wild-type and mutant MRP2I1173F in
plasma membrane and ER vesicle preparations.
Immunoblot analysis of the different membrane preparations (Fig.
3A,
inset) using the EAG5 antiserum demonstrated the large amount of MRP2 in the plasma membrane fractions of
MRP2-transfected HEK-293 cells and a low level of MRP2 in
the ER fractions from these cells. MRP2I1173F was mainly detected in
the ER fractions. For the chosen film exposure time, the fully
glycosylated form of MRP2I1173F was not visible in the plasma membrane
preparations. No signal was obtained in vector-transfected HEK-293
cells.
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Transport of [3H]LTC4 and
17-D-glucuronosyl [3H]estradiol by
MRP2 and MRP2I1173F.
Transport of [3H]LTC4 into ER and plasma
membrane vesicles from HEK-293 cells, stably transfected with
MRP2I1173F cDNA, was studied over a 3-min period and
compared with transport rates measured with vesicles from MRP2
transfectants and vesicles from vector-transfected control cells.
ATP-dependent transport of [3H]LTC4 by plasma
membrane vesicles from MRP2I1173F-transfected and
vector-transfected control cells was 13.8 ± 2.7 pmol/mg protein over 3 min and 18.7 ± 1.7 pmol/mg protein over 3 min,
respectively (mean ± SD, n = 7) (Fig.
3A). The plasma membrane vesicles from MRP2-transfected cells showed a more than fivefold higher
ATP-dependent transport with 95.7 ± 13.5 pmol/mg protein over 3 min (n = 9) (Fig. 3A).
Inhibition of proteasome function leads to the formation of
aggresomes.
It has been described that inhibition of proteasomal activity may
result in a paranuclear accumulation of proteins that are bound for the
ubiquitin-proteasome degradation pathway (7, 15, 43). To
investigate the degradation of the MRP2I1173F protein in HEK-293 cells,
we inhibited proteasome function with 2 µM MG132 added 12 h
before analysis by immunofluorescence microscopy (29). We
detected MRP2I1173F in paranuclear, aggresome-like structures with both
the EAG5 antiserum directed against the carboxy terminus of MRP2 (Fig.
4A), as well as with the MLE
antiserum (Fig. 4B), recognizing the amino terminus of MRP2,
indicating that degradation of the mutant protein had not yet occurred.
The normal organization of microtubules, radiating from the central microtubule organization center was distorted by the aggresome formation (Fig. 4C). Microtubule distribution in the
periphery remained unaffected. Redistribution of the intermediate
filament vimentin, surrounding the aggresome structure, was also seen
after MG132 treatment (Fig. 4D).
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DISCUSSION |
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Impaired secretion of conjugated bilirubin and other amphiphilic anions across the hepatocyte canalicular membrane into bile in patients with DJS is caused by the absence of functionally active MRP2 protein from this apical membrane (for reviews see Refs. 22 and 23). The lack of MRP2 in DJS may be the consequence of rapid degradation of the mutant MRP2 mRNA or a defect in synthesis, stability, or sorting of the mutant MPR2 protein.
Established DJS mutations have been summarized (22, 23), and five of the eight reported mutations were either found within or in splice sites adjacent to both nucleotide-binding domains. Currently known DJS mutations include a nonsense mutation in exon 23 leading to a premature termination codon (31, 41), a missense mutation within the ABC family signature of the first nucleotide-binding domain (40, 42), and mutations affecting splice donor sites (16, 40, 42). In addition, a two-amino-acid deletion in the second nucleotide-binding domain (41) and two missense mutations in exon 25 (27) have been described. Rapid degradation of mutant MRP2 mRNA by a process termed "nonsense-mediated decay" (39) may explain the absence of MRP2 protein in DJS mutations that lead to premature termination codons (18, 21, 31, 41). Other mutations in the MRP2 gene may affect protein stability, trafficking, or transport function of the protein. Which of these mechanisms contribute to the DJS phenotype can only be determined by expression of mutated MRP2 cDNA in a polarized human cell line. We previously analyzed the six-nucleotide deletion in exon 30 (19, 41). Impaired maturation, ER retention, and subsequent degradation of the mutant protein by proteasomes account for the absence of MRP2 from the patient's liver (19).
The aim of our present study was to identify the consequences of the
DJS mutation MRP2I1173F on a molecular and cellular level using a
polarized cell system. This mutation was identified in 22 patients of
Iranian-Jewish origin and results from an AT substitution at
nucleotide position 3517 in exon 25 (27). Computational
topology analysis locates this mutation to an extracellular loop
between the transmembrane helices 14 and 15 of the third
transmembrane-spanning domain of MRP2 (36). The same
region contains the MRP2R1150H mutation that has been associated with
DJS in five patients of Moroccan-Jewish origin (27).
Our observation that most of the MRP2I1173F and MRP2I1173F-GFP exhibit an electrophoretic mobility corresponding to core-glycosylated, immature proteins remaining sensitive to endoglycosidase H digestion (Fig. 1) indicates that most of the mutant proteins fail to advance from the ER through the Golgi apparatus. Immunofluorescence microscopy confirmed that most of the mutant MRP2I1173F protein accumulated in the ER of polarized HepG2 cells without reaching the apical membrane (Fig. 2). Similarly, MRP2I1173F was present in the ER of nonpolarized HEK-293 cells (Fig. 1 and Ref. 27). However, a small amount of the mutant MRP2I1173F protein apparently progressed through the Golgi complex and reached full glycosylation (Fig. 1), which is consistent with our finding that MRP2I1173F-GFP was present in the apical membrane of a small fraction of polarized HepG2 cells (Fig. 2, Table 1). Degradation of cytoplasmic proteins, as well as of mutant membrane proteins, is carried out by proteasomes (8, 14). Inhibition of this pathway leads to an accumulation of misfolded mutant proteins in a structure near the nucleus and centriole that has been termed the aggresome (15). After inhibition of proteasomal activity with MG132, we detected full-length MRP2I1173F protein within aggresome-like structures indicating that degradation of the protein had not yet occurred (Fig. 4). A similar proteasome-mediated degradation was observed for the DJS mutation lacking two amino acids from the second nucleotide-binding domain (19).
Because neither plasma membrane nor ER vesicle preparations containing
MRP2I1173F mediated ATP-dependent transport of LTC4 and
plasma membrane vesicles mediated that of
17-D-glucuronosyl estradiol, which represent
high-affinity substrates for MRP2 (4), MRP2I1173F
is most likely functionally not active even when it reaches the apical
membrane. Despite the low amount of normal MRP2 protein in the ER
vesicle preparation, a low rate of ATP-dependent transport of
LTC4 was detected in the ER preparations from normal MRP2-expressing HEK-293 cells (Fig. 3). Thus the transport assay was
sensitive enough to measure even low transport rates. Impaired transport activity of MRP2I1173F has been indicated by using a carboxyfluorescein efflux assay in whole cells (27).
However, this assay requires a protein being present in the plasma
membrane and cannot monitor transport activity of ER-resident forms of a protein. By contrast, with transport measurements using ER vesicle preparations as described in this study, transport activity of ER-resident forms of a protein can be assayed.
Interestingly, the mutant MRP2R1150H-GFP protein reached the apical membrane of polarized HepG2 cells to the same extent as did MRP2-GFP (Table 1). So far, localization studies have not been carried out in liver biopsies of patients carrying this mutation (27). If canalicular localization were observed, MRP2R1150H would be the first DJS mutation described leading to a MRP2 protein correctly localized in human hepatocytes but deficient in transport function. Impaired function of MRP2R1150H has been indicated by the use of a carboxyfluorescein efflux assay in nonpolarized HEK-cells (27).
Substrate specificity and transport characteristics of rat and human MRP2 may be altered when amino acid residue changes are introduced within or close to transmembrane segments by site-directed mutagenesis (11, 12, 34). MRP2R1150H and MRP2I1173F are the first DJS-associated mutations that have been tested to be functionally inactive (Fig. 3 and Ref. 27), and are predicted to be located in an extracellular loop of MRP2 when using the TMHMM program for topology analysis (36). Although extracellular loops of export pumps are most likely not involved in substrate binding, amino acid residues in extracellular loops may be critical for overall stability and conformation of the protein, thus contributing to proper function. Disease-associated mutations located to extracellular loops of the CFTR protein (gene symbol ABCC7), another ABC transporter that shares 27% amino acid identity with MRP2, were correctly sorted to the plasma membrane but seriously compromised chloride channel activity (9).
In conclusion, a mutant MRP2I1173F protein is synthesized in HEK-293 and in polarized HepG2 cells. Most of the mutant protein is retained within the ER and subsequently degraded by proteasomes. However, a small amount of mutant yet functionally inactive protein apparently escapes the ER quality control machinery and reaches the apical membrane. Impaired maturation of the inactive MRP2I1173F protein explains the deficient hepatobiliary elimination of amphiphilic anions resulting in the DJS phenotype of patients carrying this relatively frequent mutation.
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ACKNOWLEDGEMENTS |
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The authors thank Drs. Gabriele Jedlitschky, Jörg König, Yunhai Cui, and Jürgen Kartenbeck, Heidelberg, for their advice and support during this research project.
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FOOTNOTES |
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This work was supported, in part, by the Deutsche Forschungsgemeinschaft through Grant SFB352.
Address for reprint requests and other correspondence: A. T. Nies, Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg (E-mail: a.nies{at}dkfz.de).
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.
October 2, 2002;10.1152/ajpgi.00362.2002
Received 26 August 2002; accepted in final form 30 September 2002.
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