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Division of Cancer Biology and Genetics (P.E.B., C.J.W., C.E.G., S.P.C.C., R.G.D.), and Departments of Pathology and Molecular Medicine (P.E.B., C.E.G., S.P.C.C., R.G.D.) and Biochemistry (C.J.W., R.G.D.), Queen's University Cancer Research Institute, Kingston, Ontario, Canada
Received January 28, 2008; accepted March 31, 2008
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Abstract |
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). MRP1 was discovered by virtue of its overexpression in the multidrug-resistant human small cell lung cancer cell line H69AR, and the presence of the protein in many types of cancers has since been correlated with poor prognosis (Cole et al., 1992; Deeley et al., 2006). MRP2 was initially identified by its similarity to MRP1 and subsequently shown to be defective in Dubin-Johnson syndrome, a rare congenital form of mild hyperbilirubinemia, as well as in naturally occurring strains of mutant rats that are defective in bile canalicular transport (Keppler et al., 1997; Paulusma et al., 1997).
MRP1 and MRP2 are 49% identical, and they have extensively overlapping substrate specificities, although their relative affinities for a given substrate often differ significantly. Both transport a wide range of glutathione- (GSH), glucuronate-, and sulfate-conjugated organic anions, as well as other unconjugated organic anions, such as methotrexate (Qian et al., 2001b; Leslie et al., 2005; Deeley et al., 2006). They are also capable of GSH-stimulated transport of various hydrophobic chemotherapeutic drugs, including anthracyclines and Vinca alkaloids (Deeley et al., 2006). However, MRP1 and MRP2 differ markedly with respect to their tissue distribution and subcellular location. MRP1 is expressed in a wide range of tissues and in general it traffics to basolateral membranes, whereas MRP2 is found exclusively in the apical membranes of secretory epithelia, primarily in the liver, gallbladder, kidney, small intestine, colon, placenta, and lung (Keppler et al., 1997; Van Aubel et al., 2000). Thus, although both proteins contribute to cellular efflux of a wide range of phase II metabolites, as well as unconjugated drugs and toxins, MRP2 is particularly important for the elimination of these compounds.
In addition to two membrane spanning domains (MSDs) and two cytoplasmic nucleotide binding domains characteristic of ABC transporters, some ABCC proteins, including MRP1and MRP2, have an additional NH2-terminal MSD (MSD0) (Hipfner et al., 1997; Tusnády et al., 1997). MSD0 is required for appropriate trafficking and targeting of MRP2, as well as other ABCC proteins, such as SUR1 (Babenko and Bryan, 2003) and the yeast MRP1 ortholog YCF1 (Mason and Michaelis, 2002). In contrast, human MRP1 lacking MSD0 traffics to basolateral membranes, and it is capable of transporting LTC4 and certain other substrates (Bakos et al., 1998; Westlake et al., 2003). This may be attributable to redundant trafficking determinants present in both MSD0 and the COOH-terminal region of MRP1 (Westlake et al., 2005). Unlike MSD0 from MRP2, MRP1 MSD0 traffics efficiently to the plasma membrane in the absence of the "core" of the protein (Bakos et al., 1998; Fernández et al., 2002; Westlake et al., 2005).
The COOH-terminal regions of both MRP1 and MRP2 have been implicated in targeting these proteins to their respective locations in the plasma membrane (Westlake et al., 2004). This region of MRP2 contains a putative PDZ binding motif that could target the protein to the apical membrane via interaction with scaffolding proteins such as radixin. In support of this suggestion, localization of MRP2 to the hepatocanalicular membrane is impaired in radixin knockout mice (Kikuchi et al., 2002). However, the COOH-terminal 70- to 75 aa of MRP1 and MRP2 can be exchanged without affecting localization of either protein (Westlake et al., 2004). Likewise, reciprocal exchange of MSD0 between the two proteins is not sufficient to alter polarized membrane targeting of the two proteins (Konno et al., 2003; Westlake et al., 2005). Thus, other regions of MRP1 and MRP2 must contribute to, or be essential for, determining their different subcellular localization.
Using hybrid proteins, we demonstrate that aa 1 to 319 of MRP2, which includes MSD0 and the cytoplasmic loop (CL) 3 connecting it to the remainder of the protein, contain all of the targeting elements necessary to redirect the core region of MRP1 from a basolateral location exclusively to the apical membrane. Within CL3 of MRP2, we identify a lysine-rich element between aa 294 and 303 that is unique among the MRPs and that is essential for apical targeting of MRP2. This element alone is sufficient to partially redirect MRP1 to an apical location. However, the integrity of the region spanning MSD0 and CL3 seems to be essential for targeting the protein exclusively to the apical membrane.
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Materials and Methods |
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, 1998), and a polyclonal rabbit anti-calnexin antibody (Sigma-Aldrich). The MRP2-specific mAb used was M2I-4 (mouse) (Alexis Laboratories). Conjugated antibodies used for immunofluorescent detection were as follows: Alexa 488 goat anti-mouse/rat and Alexa 594 goat anti-rabbit (Invitrogen, Carlsbad, CA). Hoechst 33342 nuclear stain was also obtained from Invitrogen. Goat-anti-mouse/rat/rabbit IgG (H+L) conjugated with horseradish peroxidase (HRP; Pierce Chemical, Rockford, IL) was used as a secondary antibody. Immobilon-P membranes and chemiluminescence HRP substrate (Millipore Corporation, Billerica, MA) were used for detection in immunoblotting. All restriction endonucleases were purchased from New England Biolabs (Ipswich, MA).
Generation of MRP1/MRP2 Chimeric or Truncated Constructs. pcDNA3.1(-) vectors for expression of full-length MRP1 and MRP2 have been described previously (Ito et al., 2001; Westlake et al., 2004). The strategy for constructing expression vectors for MRP1/MRP2 hybrids is summarized in Table 1. In brief, fragments corresponding to specific portions of MRP1 or MRP2 were generated by PCR amplification using primers with "tails" complementary to hybrid junctions listed in Table 1. Appropriate amplified DNA products were then mixed, denatured, reannealed, and used as a template to generate the required hybrid fragment using ProofStart enzyme (QIAGEN, Mississauga, ON, Canada). Each recombinant DNA fragment was then amplified by PCR using primers indicated in bold in Table 1 and inserted into the respective vector backbone via restriction digests as indicated (Table 1).
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MRP21-319 was generated by PCR amplification of the corresponding cDNA from pcDNA3.1(-)-MRP2 with the forward primer 5'-GCTCTCTGGCTAACTAGAGAACC-3' in pcDNA3.1 and the reverse primer 5'-CGGCCGCCTAGTAGAAAGTTTTGAACAG AGCC-3', which resulted in addition of a stop codon and a NotI site (underlined) following the codon for Tyr319. The PCR product was digested with NheI and NotI and cloned into pcDNA3.1(-). MRP11-203/MRP2189-319/MRP1323-1531 was generated by ligation of the small fragment from SacI-digested pcDNA3.1(-)MRP11-203/MRP2189-1546 (Westlake et al., 2005) to the SacI-digested hybrid construct pcDNA3.1-MRP21-319/MRP1323-1531 (described in Table 1). The cDNAs of hybrid molecules intended for viral infection of insect cells and vesicular transport assays were cloned into pFastBac vector after digestion with XbaI/KpnI. The fidelity of all constructs was confirmed by DNA sequencing.
Generation of Mutants in the MRP21-319/MRP1323-1531 Hybrid Construct. The QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used according the manufacturer's protocol to generate all mutant proteins. The codon for Lys296 in pcDNA3.1(-)MRP21-319/MRP1323-1531 was mutated to arginine with F:5'-GAAACGCAAGA AGTCTGGG-3'/R:5'-CCCAGACTTCTTGCGTTTC-3', to glutamine with F:5'-GAAA CAGAAGAAGTCTGGG-3'/R:5'-CCCAGACTTCTTCTGTTTC or to glutamate with F:5'-GAAAGAGAAGAAGTCTGGG-3'/R:5'-CCCAGACTTCTTCTCTTTC. Residues 294 to 303 in the MRP11-319 hybrid were also mutated to the corresponding MRP1 sequence in the same manner, with the following primers: F:5'-GATGTTGAAAAGGAGTGGAACCCCTCTCTG TTTAAGGTGGATGTTCCA-3'/R:5'-TGGAACATCCACCTTAAACAGAGAGGGGTT CCACTCCTTTTCAACATC-3'. The fidelity of all constructs was confirmed by DNA sequencing.
Expression and Localization of MRP1/2 Hybrid and/or Mutated Proteins in Mammalian Cells. MRP1/MRP2 hybrids were expressed in stably transfected MDCK-1 cells and transiently transfected LLC-PK1 cells. Additional mutant-hybrid constructs were expressed in transiently transfected MDCK-1 cells. Cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum that was supplemented with F-12 nutrient mix when used for LLC-PK1 cells. Transfections were performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according the manufacturer's instructions. Stably transfected MDCK-1 cells were obtained by selection in 800 µg/ml Geneticin (G-418; Invitrogen) for 2 weeks, followed by limited dilution to isolate clonal populations that were then maintained in G-418-supplemented medium. To assess MRP1 protein expression, cells were grown to confluence in 15-cm dishes, and then they were harvested in ice-cold phosphate-buffered saline containing Complete protease inhibitor (Roche Diagnostics, Mississauga, ON, Canada) by scraping the dish with a rubber policeman. Cells were washed and lysed by incubation on ice in buffer containing 10 mM Tris, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, and protease inhibitors for 10 min. Total cellular membranes were then isolated by centrifugation. Membrane proteins were resolved by 7.5% SDS-PAGE and blots were probed with mAb MRPm6 or mAb M2I-4. A mouse or rat IgG secondary antibody conjugated to HRP was applied, and antibody-protein interactions were detected using a chemiluminescent substrate for HRP.
For protein localization studies, cells were seeded onto glass coverslips and allowed to become confluent over 7 to 9 days at 37°C in 5% CO2. Cells were then fixed with 95% ethanol at 0-4°C, and proteins were detected with mAb MRPr1 or mAb M2I-4 and goat anti-rat Alexa Fluor 488 or goat anti-mouse Alexa Fluor 488, respectively. Cells were also stained with a rabbit polyclonal antibody to the endoplasmic reticulum marker calnexin, which was detected with goat anti-rabbit Alexa Fluor 594 as described previously (Westlake et al., 2003). Cell nuclei were stained with Hoechst 33342. Fluorescent antibodies were detected with a Leica TCS SP2 dual photon confocal microscope.
Viral Infection, Membrane Vesicle Preparation, and Immunoblotting. Generation of baculovirus from recombinant bacmids and methods for infection of Sf21 cells have been described previously (Gao et al., 1996). Membrane vesicles were prepared from infected insect cells by nitrogen cavitation at 200 psi for 5 min and followed by sucrose density centrifugation (Gao et al., 1996). Membrane proteins were resolved by 7.5% SDS-PAGE, and MRP levels were determined by immunoblotting and densitometry. Slot blots were performed with Bio-Dot SF microfiltration apparatus (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions.
ATP-Dependent Uptake of LTC4, E13SO4, and E217βG Transport by MRP1 and Hybrids across Sf21 Membranes. MRP-mediated uptake of various substrates by membrane vesicles from Sf21 cells was determined using a rapid filtration assay (Gao et al., 1996) at 23°C as described previously. In brief, Sf21 membrane vesicles (4 µg of total protein) were incubated at 23°C in transport buffer (50 mM Tris and 250 mM sucrose, pH 7.4) containing 50 nM [3H]LTC4 (10 nCi/reaction), 4 mM AMP or ATP, and 10 mM MgCl2. ATP-dependent transport was determined by subtracting the uptake in the presence of AMP from the uptake in the presence of ATP. ATP-dependent uptake of 300 nM [3H]E13SO4 (50 nCi/reaction) or 400 nM [3H]E217βG (25 nCi/reaction and 6 µg of protein) was measured at 37°C in a similar manner. For the transport of E13SO4, reactions were supplemented with 2 mM S-methyl-GSH (Qian et al., 2001a).
Synthesis of Azidophenacyl-[35S]GSH. Synthesis of azidophenacyl-[35S]GSH was carried out as described previously (Qian et al., 2001a). In brief, 100 µCi of stock [35S]GSH (1498 Ci/mmol) was extracted with ethyl acetate to remove dithiothreitol and diluted to 500 Ci/mmol with GSH (Sigma-Aldrich). This was added to a reaction mixture containing N2 saturated, degassed phosphate-buffered saline, 4 mM 4-azidophenacylbromine, 125 mU GSH-reductase, and 1 mM NADPH for 1 h at room temperature. Products were separated by silica-G thin layer chromatography using i-propanol/water/acetic acid [12:5:1 (v/v/v)]. Spots exhibiting the appropriate mobility on the thin layer chromatography plate (Rf 
0.6) were confirmed to contain azidophenacyl-[35S]GSH by autoradiography, and then they were scrapped off the plate and extracted 6 times with 400 µl of water. Extracts were concentrated under a stream of nitrogen.
Photolabeling of MRP1 and Hybrids with Azidophenacyl-[35S]GSH. Membranes (75 µg of total protein) from Sf21 cells expressing MRP1 and various hybrid proteins were incubated with azidophenacyl-[35S]GSH (0.5 µCi) at room temperature for 10 min in transport buffer (50 mM Tris and 250 mM sucrose, pH 7.4) and then UV-irradiated (312 nM) for 5 min on ice. Membrane proteins were analyzed by SDS-PAGE (10% acrylamide) after solubilization in Laemmli's buffer. Gels were treated with Amplify (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) and dried, and then autoradiography was performed at -70°C. Exposure times ranged from 1 to 3 days.
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). However, these studies also demonstrated the importance of parts of the cytoplasmic region (CL3) that links MSD0 and MSD1, both with respect to processing through the endoplasmic reticulum (ER) and subsequent trafficking of MRP1 to the basolateral membrane. Figure 1 shows an alignment of CL3 from human MRPs 1, -2, and -6 and the yeast MRP1 ortholog YCF1, with the cytoplasmic amino termini of human MRP4 and cystic fibrosis transmembrane conductance regulator. It also illustrates the relatively conserved N-proximal region of CL3 referred to above and the divergence of the C-proximal portions of the aligned regions (Westlake et al., 2005).
To investigate the possible importance of MRP2 CL3 for apical targeting, we constructed various MRP1/MRP2 hybrids or truncated proteins and we expressed them in polarized epithelial cells. The multiple sequence alignments of CL3 (Fig. 1) were used to identify points for connecting various fragments in the hybrid proteins, taking care to maintain predicted conserved secondary structures shown and to minimize loss or duplication of homologous sequences. The hybrids generated are illustrated in Fig. 2. Two polarized kidney epithelial cell lines that reportedly differ in their sorting mechanisms were used to compare trafficking of the hybrids with that of wild-type MRP1 and MRP2 (Roush et al., 1998). Figure 3 shows confocal micrographs of the wild-type proteins stably expressed in MDCK-1 (canine) and transiently expressed in LLC-PK1 (porcine) cells. As expected from previous studies, MRP1 and MRP2 localized exclusively to the basolateral or apical plasma membranes of both cell types, respectively. We next examined the polarized sorting of several MRP1/MRP2 hybrid molecules (constructs A, B, and C shown in Fig. 2) in stably transfected MDCK-1 cell lines (Fig. 4). We have shown that exchange of the first 280 aa of MRP1 for those of MRP2 resulted in a protein that was inactive when expressed in Sf21 insect cells and that failed to exit the ER in mammalian cells (Gao et al., 1998). Consequently, we initially extended the substituted region so that aa 1 to 289 of MRP2 replaced aa 1 to 290 of MRP1 (MRP21-289/MRP1291-1531) (Fig. 2B). However, this hybrid protein also remained trapped in the ER, as evidenced by colocalization with the ER marker calnexin (Fig. 4A, left). In contrast, substitution of the first 319 residues of MRP2, which includes essentially all of MSD0 and CL3, for aa 1 to 322 of MRP1 (MRP21-319/MRP1323-1531) (Fig. 2A) resulted in apical sorting of the hybrid (Fig. 4B, left). No evidence of the presence of the hybrid in basolateral membranes could be detected by confocal microscopy.
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To determine whether similar behavior was observed in LLC-PK1 cells, we generated transient transfectants using the same set of hybrids (Fig. 4, A-C, right). All of the constructs displayed the same sorting profiles observed in the MDCK-1 cells, including combined apical and basolateral localization of the MRP2286-319 hybrid. These findings indicate that the region encompassing residues 286 to 319 in MRP2 contains elements that are necessary for apical sorting of the protein. These elements are also sufficient to target a substantial fraction of MRP1 to the apical membrane, albeit with reduced efficiency compared with a hybrid containing all of MSD0 and CL3 from MRP2.
Functional Interaction of COOH-Proximal End of CL3 with Other Regions in CL3 or MSD0. To further investigate the role of the COOH-proximal end of MRP2 CL3 in apical sorting, stable MDCK-1 cell lines were created expressing one of four additional MRP1/MRP2 hybrids (Fig. 2, D-G). Polarized sorting was then examined as described above. First, we determined whether the region between aa 286 and 319 was essential for apical targeting of MRP2 by exchanging this region for aa 291 and 322 of MRP1 (MRP21-289/MRP1291-322/MRP2320-1546) (Fig. 2D). Confocal microscopy revealed that this substitution severely impaired apical trafficking (Fig. 5A, left). However, this hybrid did not colocalize with calnexin, indicating that it was able to exit the ER. Instead, the protein was apparently retained in an intracellular vesicular compartment. Thus, the results support the importance of the region between aa 286 and 319 of MRP2 for targeting to the apical plasma membrane.
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), we generated a hybrid (MRP21-187/MRP1204-290/MRP2286-319/MRP1323-1531) that contained all of MRP2 MSD0 (MRP21-187) together with MRP2286-319. However, this hybrid had a plasma membrane distribution that was indistinguishable from the hybrid containing only MRP2286-319 (Fig. 5B, left). Thus, despite the requirement of MSD0 for the trafficking of MRP2 to the plasma membrane, its inclusion did not detectably increase the apical location of the hybrid MRP1 protein compared with MRP11-290/MRP2286-319/MRP1323-1531 (Fig. 4C, left). To determine whether NH2-proximal regions of CL3 of MRP2 were also required for apical targeting, we constructed a hybrid in which all of CL3 of MRP1 was replaced with CL3 from MRP2 (MRP11-203/MRP2189-319/MRP1323-1531) (Fig. 5C, left). Like the construct containing MSD0 of MRP2 (Fig. 5B, left), the plasma membrane distribution of this hybrid was essentially unchanged from that of MRP11-290/MRP2286-319/MRP1323-1531 (Fig. 4C, left).
We and others have previously shown that MSD0 of MRP1 alone or connected to various segments of CL3 can traffic independently to basolateral membranes. In contrast, Fernández et al. (2002) reported that MRP21-305 was retained in an intracellular compartment in MDCK-II cells and that it failed to reach the plasma membrane. Having determined that MRP21-319 contained all the information necessary to direct MRP1 exclusively to the apical membrane, we determined whether MRP21-319 was independently capable of apical trafficking. However, this construct was found to colocalize predominantly with calnexin, indicating that it was retained in the ER (Fig. 5D, left). As with the initial set of hybrids, trafficking of these four constructs was also examined by transient expression in LLC-PK1 cells (Fig. 5, A-D, right). The distribution in LLC-PK1 cells did not differ from that observed in MDCK-1 cells, with the exception of MRP21-319, which had a punctate, intracellular, vesicular-like distribution, as opposed to being retained in the ER.
Overall, these results support the suggestion that a critical region for apical trafficking lies between aa 286 and 319 in MRP2, but they also indicate that additional interactions involving both MSD0 and the NH2-proximal half of CL3 may be required for full localization to, or retention in, the apical membrane. That MRP21-319 does not traffic independently also raises the possibility of a requirement for additional processing and trafficking signals present in the core regions of both MRP1 and MRP2 or that the presence of the core regions of the proteins is required for the correct folding of MRP21-319.
Impact of Mutations in CL3 on the Apical Localization of MRP21-319/MRP1323-1531 in Transiently Transfected MDCK-1 Cells. Sequence alignments of CL3 from several MRPs and related proteins (Fig. 1) reveal a unique polybasic motif from residues 294 to 303 in MRP2. This motif falls within the small region of MRP2 (MRP2286-319) shown above to be capable of causing partial redistribution of MRP1 to the apical membrane when introduced into that protein. Polybasic motifs, such as the one present in MRP2 CL3, are potential sites for membrane interaction (Heo et al., 2006). Consequently, we performed a series of mutations within this polybasic motif in the MRP21-319 hybrid, which we showed above contains all the necessary components to redirect MRP1 exclusively to the apical membrane in polarized cells. Lysine at position 296 of the hybrid protein was arbitrarily selected because it is located in the middle of a stretch of five lysine residues in the polybasic motif (KKKKKSGTKK; Fig. 1), and it was mutated to arginine, glutamine, or glutamate, and transiently expressed in MDCK-1 cells. In addition, we exchanged the whole basic motif (aa 294-303) for the most closely corresponding region in MRP1 (aa 299-308).
Confocal micrographs shown in Fig. 6 demonstrate that point mutations at Lys296 do not affect the apical trafficking of this hybrid regardless of the level of conservation of the mutation. In contrast, when the whole motif was mutated in the hybrid, a large proportion of the protein was localized in an (subapical) intracellular component. However, it was not retained in the ER, because it did not colocalize with calnexin (Fig. 6). Thus, the hybrid lacking the basic motif is able to exit the ER but either fails to be targeted to, or retained in, the apical membrane.
LTC4 and E217βG Transport by MRP1/MRP2 Hybrid Proteins. CL3 of MRP1 is important, not only for trafficking of the protein but also for the binding, transport, or both of substrates such as LTC4 and azidophenacyl-GSH (Gao et al., 1998; Qian et al., 2002). The region of CL3 required for activity extends from amino acid 208 to 260, whereas that necessary for efficient trafficking is somewhat longer and extends an additional 9 aa to 269 (Westlake et al., 2003). Given the functional importance of CL3 of MRP1 and the relatively low sequence identity of this region between MRP1 and MRP2 (30%), we investigated whether those hybrids in which various regions of CL3 had been exchanged retained transport activity when expressed in insect Sf21 cells (Fig. 7). Previous studies of mutant or hybrid forms of MRP1 have revealed that efficient processing and the ability to traffic to the plasma membrane does not necessarily indicate that the proteins are functional. Conversely, an inability to traffic appropriately in mammalian cells does not preclude the possibility that the protein may be capable of folding into an active conformation when expressed in insect cells.
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Data in Fig. 7 (D and E) are combined from two separate experiments, and they show time courses of LTC4 uptake by MRP1 and hybrid proteins at 23°C. Hybrids with MRP1 regions substituted for MRP2286-319 or MRP21-319 demonstrated normalized transport levels that were approximately 60 and 30% of wild-type MRP1, respectively (Fig. 7D). Although both MRP2286-319 and MRP21-319 hybrids retained some ability to transport LTC4, transport of E217βG could be detected only with the MRP2286-319 hybrid, but it was approximately 25% as active as wild-type MRP1 (Fig. 7C). Taken together, these findings suggest that different regions of CL3 may be important for the transport of these two substrates.
Although both MRP1 and MRP2 transport LTC4, previous kinetic analyses indicate that the apparent affinity of MRP2 for this substrate is approximately 10-fold lower than that of MRP1 (Cui et al., 1999). Consequently, we determined how the introduction of regions from MRP2 affected the kinetic parameters of LTC4 transport by the MRP2286-319 and MRP21-319 hybrids (Fig. 7E). The Km and Vmax values of wild-type MRP1 for LTC4 were determined to be 83 nM and 89 pmol/min/mg vesicle protein, respectively, and they were similar to our previously published values (Gao et al., 1996). Introduction of MRP2286-319 increased the Km value to approximately 220 nM without a detectable change in Vmax, whereas Km value of the hybrid containing the MRP21-319 substitution increased to approximately 710 nM. The normalized Vmax value of this construct was modestly increased to 120 pmol/min. Thus, introduction of the limited MRP2286-319 region decreased the apparent affinity for LTC4 approximately 2.5-fold, whereas exchange of the entire MSD0/CL3 region decreased the apparent affinity 8- to 9-fold.
GSH Binding and E13SO4 Transport by MRP1/MRP2 Hybrid Proteins. Because transport of the GSH-conjugated LTC4 was readily detectable with both hybrids, we evaluated the ability of the hybrid proteins to transport the GSH-dependent substrate E13SO4. We have shown that transport of E13SO4 by MRP1 is stimulated 5- to 10-fold in the presence of GSH or S-methyl GSH (Qian et al., 2001a). In contrast, this conjugate is not a substrate for MRP2, either alone or in the presence of GSH. ATP-dependent uptake of 300 nM E13SO4 into Sf21 membrane vesicles was measured after incubation at 37°C for 3 min in the presence of 2 mM S-methyl GSH (Qian et al., 2001a). Figure 8A shows that uptake by the hybrid containing MRP2286-319 was markedly reduced to approximately 10% that of wild-type MRP1 (8 pmol/mg/3 min), whereas transport by the MRP21-319 hybrid was not detectable.
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). Nothing is currently known of the elements in MRP2 that are required for interaction with GSH, which has been reported to be a low-affinity substrate for the protein (Km value of 19 mM) (Paulusma et al., 1999). To determine whether the reduction of E13SO4 transport by the hybrid proteins might be attributable to loss of GSH binding, we carried out a photolabeling study with azidophenacyl-[35S]GSH (Ciaccio et al., 1996), which has been established previously as a functional, non-reducing substitute for GSH (Qian et al., 2002). As shown in Fig. 8B, and as we have reported previously, wild-type MRP1 expressed in Sf21 membrane vesicles was clearly labeled with azidophenacyl-[35S]GSH (Qian et al., 2002). Photolabeling of the MRP1 hybrid containing MRP21-319 was barely detectable, whereas photolabeling of the MRP2286-319 substituted protein was only modestly affected. |
Discussion |
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, 2005). This redundancy explains, at least in part, the ability of MRP1 to traffic to the basolateral plasma membrane in the absence of MSD0. The regions involved in targeting MRP2 to apical membranes have been less well defined, but they clearly differ from those identified in MRP1. MRP2 accumulates in intracellular vesicles when expressed in the absence of MSD0, suggesting that this region may contain trafficking/targeting signals (Fernández et al., 2002). However, Konno et al. (2003) found that MRP1/MRP2 hybrids containing the NH2-terminal 240 aa from MRP1, which includes MSD0 and 37 aa of CL3, trafficked normally, whereas a hybrid in which the NH2-terminal 480 aa were exchanged failed to reach the apical membrane. These results suggest that a signal(s) essential but not necessarily sufficient for the apical location of MRP2 may be located between aa 240 and 480, which encompasses the C-proximal 79 aa of CL3 and transmembranes 6 to 9 of MSD1. The importance of the putative PDZ binding motif in the COOH-terminal region of MRP2 remains controversial, because this element can be deleted without altering the apical localization of the protein (Nies et al., 2002). Likewise, introduction of this region into MRP1 fails to change the protein's basolateral location (Westlake et al., 2005).
Rather than inferring the presence of targeting signals from a loss of targeting specificity, we sought to identify regions, elements, or both in MRP2 that were sufficient to redirect the core region of MRP1 from the basolateral to the apical membranes of two polarized kidney cell lines. We began by investigating the importance of MSD0 and CL3. The primary sequence conservation of MSD0 among the ABCC proteins is low and MSD0 of MRP2, unlike MSD0 from MRP1, is unable to traffic independently to the plasma membrane, although it is processed through the ER (Fernández et al., 2002; Westlake et al., 2005). Sequence conservation within CL is also relatively low. However, within CL3, several secondary structure algorithms predict the presence of two "-helical motifs that are conserved even in distantly related MRP orthologs and homologs (Fig. 1) (Bakos et al., 2000; Westlake et al., 2003). In MRP1, residues 208 to 269 of CL3, which include both conserved helices, are critical for ER processing (Bakos et al., 2000; Westlake et al., 2003).
We have shown previously that exchange of the first 280 aa of MRP1 for the comparable region of MRP2, results in a protein that fails to exit the ER in mammalian cells and is inactive when expressed in insect Sf21 cells (Gao et al., 1998). Extending the exchanged region to residue 289 did not prevent the hybrid from being trapped in the ER (Fig. 3), and this hybrid was also inactive in Sf21 cells (data not shown). In contrast, a MRP1 hybrid containing MRP21-319 was able to exit the ER and localized exclusively to the apical membrane in both MDCK-1 cells and LLC-PK1 cells (Fig. 4). Thus, MRP21-319, which includes MSD0 and the whole of CL3, contains all components necessary to efficiently redirect the core of MRP1 to the apical membrane. Furthermore, inclusion of the COOH-proximal region of MRP2 CL3 (aa 289-319) seems to be required for appropriate folding and processing of the hybrid protein through the ER.
The above-mentioned results suggested that aa 290 to 319 of MRP2 CL3 were involved in targeting the protein to the apical membrane. Consistent with this possibility, a substantial fraction of a MRP1 hybrid containing only these 20 aa localized to apical membranes (Fig. 4C). Although not sufficient to redirect MRP1 exclusively to the apical membrane, the reciprocal substitution of this region of MRP2 with the corresponding region of MRP1 (Fig. 5A), indicated that it was essential for apical targeting, either directly from the Golgi or via transcytosis. The lack of apical localization of the mutated MRP2 was not simply attributable to misfolding, because the protein was able to exit the ER, but it accumulated in an intracellular vesicular compartment rather than the apical membrane. Furthermore, no trace of the MRP2 mutant was detectable on the basolateral membrane, suggesting that a direct route is followed to the apical surface.
CL3 is critical not only for trafficking of MRP1 but also the binding of substrates, such as LTC4 and azidophenacyl-GSH (Gao et al., 1998; Qian et al., 2001a; Konno et al., 2003). Consequently, we determined how substitution of this region by fragments from MRP2 affected the function of the hybrid protein by examining transport of two well characterized organic anion substrates of MRP1 and MRP2 (LTC4 and E217βG) and a third substrate (E13SO4), which is transported only by MRP1. MRP21-319/MRP1323-1531 displayed a Vmax value for LTC4 transport very similar to that of MRP1, whereas the Km value of the hybrid was similar to that of MRP2 (i.e., 10-fold higher than that of MRP1). Thus, the relatively low sequence conservation of CL3 between MRP1 and MRP2 may contribute to the substantial difference in affinity that the two proteins display for this common substrate. Photolabeling with 35S-labeled azidophenacyl GSH was also markedly diminished, consistent with studies indicating that MRP1 CL3 is important for binding this GSH derivative, as well as LTC4 (Qian et al., 2001b, 2002).
We were unable to detect transport of either E217βGor E13SO4 by MRP21-319/MRP1323-1531 (Figs. 7C and 8A). Furthermore, exchange of only the COOH-proximal region of CL3 (aa 286-319) for the comparable region of MRP2 pro-foundly decreased transport of both conjugated steroids (75-90%) (Figs. 7C and 8A), but it had little effect on LTC4 transport (Fig. 7, D and E) or binding of 35S-labeled azidophenacyl-GSH (Fig. 8B). Consequently, this region of MRP1 CL3 seems to be critical for binding of the two steroid conjugates but not for interaction with azidophenacyl-GSH or the GSH conjugate LTC4. Both MRP1 and MRP2 display reciprocal competition between LTC4 and E217βG, suggesting common or mutually exclusive binding sites for these substrates. Our present findings suggest that CL3 is important for interaction with both substrates but that the regions involved are not identical.
Attempts to identify additional regions in MSD0 or CL3 that contributed to apical targeting of MRP21-319/MRP1323-1531 were unsuccessful (Fig. 5). MRP1 hybrids containing MSD0 and the COOH-terminal region of CL3 from MRP2, or just MRP2 CL3 localized to both basolateral and apical membranes, as observed with a construct containing only MRP2286-319. These and previous results using MRP1 hybrids in which MSD0 and only parts of CL3 had been exchanged, strongly suggest that the integrity of the entire region encompassing MSD0 and CL3 of MRP2 is required for efficient apical targeting. That neither MRP21-319 nor MRP1323-1531 (data not shown) traffic to the plasma membrane when expressed independently also suggests that interactions between the two heterologous fragments are required for the processing of the hybrid protein through the ER and for trafficking to the apical membrane.
One striking feature of the COOH-proximal region of MRP2 CL3 is the presence of a polybasic 10 amino acid motif containing seven lysine residues (five of which are consecutive), that is not present in any other mammalian MRPs (Fig. 1). Although various point mutations of a single lysine (Lys296) in this motif did not affect the apical distribution of MRP21-319/MRP1323-1531, exchange of the entire motif for the corresponding MRP1 sequence resulted in accumulation of the protein in a subapical intracellular compartment (Fig. 6). As observed when the more extended region of MRP2 from aa 290 to 319 was exchanged for the corresponding sequence from MRP1, none of the mutant protein was detected in the basolateral membrane.
Polybasic motifs have been implicated in membrane targeting of a variety of proteins, in part, by virtue of their ability to form electrostatic interactions with phospholipids, notably phosphatidyl inositols (Heo et al., 2006). Such regions have been implicated in the membrane association of the small GTPases Rit and Rin (Fivaz and Meyer, 2003), oncoproteins such as K-ras (Yeung et al., 2008), integral membrane proteins such as CD43 and ICAM-2 (Yonemura et al., 1998), and myristoylated alanine-rich C kinase substrate and myristoylated alanine-rich C kinase substrate-related protein (Sundaram et al., 2004). Consistent with the possibility of such a role for the motif in MRP2, it is located in a predicted cytoplasmic, juxtamembrane region immediately NH2-proximal to transmembrane 6.
In some cases, regulation of the interaction of polybasic regions with the membrane involves Ser/Thr phosphorylation by PKC, which is in turn is activated by phosphatidyl-inositol hydrolysis (Sundaram et al., 2004; Yeung et al., 2008). MRP2 has been shown to be acutely regulated by PKC, activation of which results in rapid loss of the apical localization of MRP2 in liver and kidney (Kubitz et al., 2001). Analysis of CL3 of MRP2 orthologs from higher eukaryotes indicates that it contains a high-probability (>0.8), potential PKC site located in the lysine-rich motif (KKKKKSGTKK) that is conserved in all vertebrates analyzed, including: rhesus monkey (KKKKKKSGTKK), dog (KKKKKKSGTT), mouse (SKKKKKKSEAT), rat (KKKSEKTTK) and zebra fish (SKKKKKKKQK). Polybasic regions are also found associated with binding sites for PDZ proteins. Although these sites are often located in the COOH-terminal region of the protein, this is not always the case (Hung and Sheng, 2002). The sequence immediately preceding the polybasic motif in the human protein contains two matches for class III PDZ binding motifs (QDALVLEDVEKKKKKSGTKK) the NH2-proximal of which is conserved in all available sequences of MRP2 from mammals and zebra fish. Given that it is possible to eliminate the PDZ binding motif in the COOH-tail of MRP2 without affecting apical targeting of the protein, this region of CL3 may be an alternative candidate for mediating interaction with ERM proteins and PKC-regulated membrane association.
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Footnotes |
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ABBREVIATIONS: MRP, multidrug resistance protein; ABC, ATP-binding cassette; SUR, sulfonylurea receptor; GSH, glutathione; MSD, membrane spanning domain; LTC4, leukotriene C4; PDZ, postsynaptic density 95/disc-large/zona occludens; aa, amino acid(s); CL, cytoplasmic loop; E13SO4, estrone-3-sulfate; E217βG, 17β-estradiol 17-(β-D-glucuronide); mAb, monoclonal antibody; HRP, horseradish peroxidase; PCR, polymerase chain reaction; MDCK, Madin-Darby canine kidney; PAGE, polyacrylamide gel electrophoresis; ER, endoplasmic reticulum; PKC, protein kinase C.
1 Current affiliation: Department of Chemistry and Chemical Engineering, Royal Military College of Canada, Kingston, Ontario, Canada. 
2 Current affiliation: Genentech, San Francisco, California.
Address correspondence to: Dr. Roger G. Deeley, Division of Cancer Biology and Genetics, Queen's University Cancer Research Institute, Kingston, ON, Canada K7L 3N6. E-mail: deeleyr{at}post.queensu.ca
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Bakos E, Evers R, Calenda G, Tusnády GE, SzakácsG, Váradi A, and Sarkadi B. (2000) Characterization of the amino-terminal regions in the human multidrug resistance protein (MRP1). J Cell Sci 113: 4451-4461.[Abstract]
Bakos E, Evers R, Szakacs G, Tusnády GE, Welker E, Szabo K, de Haas M, van Deemter L, Borst P, Varadi A, et al. (1998) Functional multidrug resistance protein (MRP1) lacking the NH2-terminal transmembrane domain. J Biol Chem 273: 32167-32175.
Ciaccio PJ, Shen H, Kruh GD, and Tew KD (1996) Effects of chronic ethacrynic acid exposure on glutathione conjugation and MRP expression in human colon tumor cells. Biochem Biophys Res Commun 222: 111-115.[CrossRef][Medline]
Cole SPC, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Stewart AJ, Kurz EU, Duncan AM, and Deeley RG (1992) Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 258: 1650-1654.
Cui Y, Konig J, Buchholz JK, Spring H, Leier I, and Keppler D (1999) Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells. Mol Pharmacol 55: 929-937.
Deeley RG, Westlake C, and Cole SPC (2006) Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins. Physiol Rev 86: 849-899.
Fernández SB, Hollo Z, Kern A, Bakos E, Fischer PA, Borst P, and Evers R (2002) Role of the NH2-terminal transmembrane region of the multidrug resistance protein MRP2 in routing to the apical membrane in MDCKII cells. J Biol Chem 277: 31048-31055.
Fivaz M and Meyer T (2003) Specific localization and timing in neuronal signal transduction mediated by protein-lipid interactions. Neuron 40: 319-330.[CrossRef][Medline]
Gao M, Loe DW, Grant CE, Cole SPC, and Deeley RG (1996) Reconstitution of ATP-dependent leukotriene C4 transport by co-expression of both half-molecules of human multidrug resistance protein in insect Sf21 cells. J Biol Chem 271: 27782-27787.
Gao M, Yamazaki M, Loe DW, Westlake CJ, Grant CE, Cole SPC, and Deeley RG (1998) Multidrug resistance protein: identification of regions required for active transport of leukotriene C4. J Biol Chem 273: 10733-10740.
Heo WD, Inoue T, Park WS, Kim ML, Park BO, Wandless TJ, and Meyer T (2006) PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane. Science 314: 1458-1461.
Hipfner DR, Almquist KC, Leslie EM, Gerlach JH, Grant CE, Deeley RG, and Cole SPC (1997) Membrane topology of the multidrug resistance protein (MRP): a study of glycosylation-site mutants reveals an extracytosolic NH2 terminus. J Biol Chem 272: 23623-23630.
Hipfner DR, Gao M, Scheffer G, Scheper RJ, Deeley RG, and Cole SPC (1998) Epitope mapping of monoclonal antibodies specific for the 190-kDa multidrug resistance protein (MRP). Br J Cancer 78: 1134-1140.[Medline]
Hung Y and Sheng M (2002) PDZ domains: structural modules for protein complex assembly. J Biol Chem 277: 5699-5702.
Ito K, Oleschuk CJ, Westlake C, Vasa MZ, Deeley RG, and Cole SPC (2001) Mutation of Trp1254 in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity and results in loss of methotrexate transport activity. J Biol Chem 276: 38108-38114.
Ito K, Weigl KE, Deeley RG, and Cole SPC (2003) Mutation of proline residues in the NH(2)-terminal region of the multidrug resistance protein, MRP1 (ABCC1): effects on protein expression, membrane localization, and transport function. Biochim Biophys Acta 1615: 103-114.[Medline]
Keppler D, Leier I, and Jedlitschky G (1997) Transport of glutathione conjugates and glucuronides by the multidrug resistance proteins MRP1 and MRP2. Biol Chem 378: 787-791.[Medline]
Kikuchi S, Hata M, Fukumoto K, Yamane Y, Matsui T, Tamura A, Yonemura S, Yamagishi H, Keppler D, Tsukita S, et al. (2002) Radixin deficiency causes conjugated hyperbilirubinemia with loss of Mrp2 from bile canalicular membranes. Nat Genet 31: 320-325.[CrossRef][Medline]
Konno T, Ebihara T, Hisaeda K, Uchiumi T, Nakamura T, Shirakusa T, Kuwano M, and Wada M (2003) Identification of domains participating in the substrate specificity and subcellular localization of the multidrug resistance proteins MRP1 and MRP2. J Biol Chem 278: 22908-22917.
Kubitz R, Huth C, Schmitt M, Horbach A, Kullak-Ublick G, and Häussinger D (2001) Protein kinase C-dependent distribution of the multidrug resistance protein 2 from the canalicular to the basolateral membrane in human HepG2 cells. Hepatology 34: 340-350.[CrossRef][Medline]
Leslie EM, Deeley RG, and Cole SP (2008) Multidrug resistance proteins: P-glycoprotein, MRP1, MRP12, and BCRP (ABCG2) in tissue defence. Toxicol Appl Pharmacol 204: 216-237.[CrossRef]
Mason DL and Michaelis S (2002) Requirement of the NH2-terminal extension for vacuolar trafficking and transport activity of yeast Ycf1p, an ATP-binding cassette transporter. Mol Biol Cell 13: 4443-4455.
Nies AT, Konig J, Cui Y, Brom M, Spring H, and Keppler D (2002) Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2). Eur J Biochem 269: 1866-1876.[Medline]
Paulusma CC, Kool M, Bosma PJ, Scheffer GL, Borg F, Scheper RJ, Tytgat GN, Borst P, Baas F, and Oude Elferink RP (1997) A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome. Hepatology 25: 1539-1542.[CrossRef][Medline]
Paulusma CC, van Geer MA, Evers R, Heijn M, Ottenhoff R, Borst P, and Oude Elferink RP (1999) Canalicular multispecific organic anion transporter/multidrug resistance protein 2 mediates low-affinity transport of reduced glutathione. Biochem J 338: 393-401.[CrossRef][Medline]
Qian YM, Grant CE, Westlake CJ, Zhang DW, Lander PA, Shepard RL, Dantzig AH, Cole SPC, and Deeley RG (2002) Photolabeling of human and murine multidrug resistance protein 1 with the high affinity inhibitor [125I]LY475776 and azidophenacyl-[35S]glutathione. J Biol Chem 277: 35225-35231.
Qian YM, Qiu W, Gao M, Westlake CJ, Cole SPC, and Deeley RG (2001a) Characterization of binding of leukotriene C4 by human multidrug resistance protein 1: evidence of differential interactions with NH2- and COOH-proximal halves of the protein. J Biol Chem 276: 38636-38644.
Qian YM, Song WC, Cui H, Cole SPC, and Deeley RG (2001b) Glutathione stimulates sulfated estrogen transport by multidrug resistance protein 1. J Biol Chem 276: 6404-6411.
Roush DL, Gottardi CJ, Naim HY, Roth MG, and Caplan MJ (1998) Tyrosine-based membrane protein sorting signals are differentially interpreted by polarized Madin-Darby canine kidney and LLC-PK1 epithelial cells. J Biol Chem 273: 26862-26869.
Sundaram M, Cook HW, and Byers DM (2004) The MARCKS family of phospholipid binding proteins: regulation of phospholipase D and other cellular components. Biochem Cell Biol 82: 191-200.[CrossRef][Medline]
Tusnády GE, Bakos E, Varadi A, and Sarkadi B (1997) Membrane topology distinguishes a subfamily of the ATP-binding cassette (ABC) transporters. FEBS Lett 402: 1-3.[CrossRef][Medline]
Van Aubel RA, Hartog A, Bindels RJ, Van Os CH, and Russel FG (2000) Expression and immunolocalization of multidrug resistance protein 2 in rabbit small intestine. Eur J Pharmacol 400: 195-198.[CrossRef][Medline]
Westlake CJ, Cole SPC, and Deeley RG (2005) Role of the NH2-terminal membrane spanning domain of multidrug resistance protein 1/ABCC1 in protein processing and trafficking. Mol Biol Cell 16: 2483-2492.
Westlake CJ, Payen L, Gao M, Cole SPC, and Deeley RG (2004) Identification and characterization of functionally important elements in the multidrug resistance protein 1 COOH-terminal region. J Biol Chem 279: 53571-53583.
Westlake CJ, Qian YM, Gao M, Vasa M, Cole SPC, and Deeley RG (2003) Identification of the structural and functional boundaries of the multidrug resistance protein 1 cytoplasmic loop 3. Biochemistry 42: 14099-14113.[CrossRef][Medline]
Yeung T, Gilbert GE, Shi J, Silvius J, Kapus A, and Grinstein S (2008) Membrane phosphatidylserine regulates surface charge and protein localization. Science 319: 210-213.
Yonemura S, Hirao M, Doi Y, Takahashi N, Kondo T, Tsukita S, and Tsukita S (1998) Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2. J Cell Biol 140: 885-895.
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-helices are also shown (Westlake et al., 2003
) or hybrids MRP2286-319 (
) or MRP21-319 (
) at 23°C. E, LTC4 transport kinetics were determined by incubating Sf21 vesicles at 23°C for 30 s in the presence of different concentrations (25-600 nM) of [3H]LTC4.E, inset, Hanes-Woolf transformation of the data from which kinetic parameters were determined. In all cases, ATP-dependent uptake of substrates into inside-out vesicles was normalized to relative protein expression levels in B. D and E show the pooled results of two experiments with the same membrane preparations. Some error bars are contained within the limits of the symbol.