Oligomerization Study of Human Organic Anion Transporting

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Oligomerization study of human organic anion transporting polypeptide 1B1 Chunxu Ni, Xuan Yu, Zihui Fang, Jiujiu Huang, and Mei Hong Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00649 • Publication Date (Web): 09 Dec 2016 Downloaded from http://pubs.acs.org on December 14, 2016

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Molecular Pharmaceutics

Oligomerization study of human organic anion transporting polypeptide 1B1 Chunxu Ni1, #, Xuan Yu1, #, Zihui Fang1, Jiujiu Huang1, 2, Mei Hong1, 2, * 1

College of Life Sciences, South China Agricultural University, Guangzhou, China

2

Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural

Organisms

#

These two authors contributed equally.

*To whom correspondence should be addressed: Mei Hong, College of Life Sciences, South China Agricultural University, Guangzhou, China, Tel: (8620)8528-0901; Fax: (8620)8528-2180; Email: [email protected]

Abbreviations: ABC, ATP binding cassette; ES, estrone-3-sulfate; OAT, organic anion transporter; OATP, organic anion transporting polypeptide; OCT, organic cation transporter; TM, transmembrane domain; URAT, urate transporter

1

ACS Paragon Plus Environment


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Abstract Organic anion-transporting polypeptides play important roles in the uptake of various endogenous and exogenous compounds. It has been proposed that OATP family members, as membrane proteins, may form oligomers. However, oligomerization status of OATPs is still largely unclear. In the present study, HEK293 cells stably expressing OATP1B1 were generated to investigate the oligomerization status of the transporter. Chemical cross-linking and co-immunoprecipitation experiments revealed that OATP1B1 may form homo-oligomers, possibly through disulfide bonds. When wild-type OATP1B1 was co-expressed with a loss-of-function mutant W258A, cells showed reduced uptake of prototypic substrate estrone-3-sulfate (ES). Interestingly, such a co-expression did not affect OATP1B1 transport activity of high concentrations ES, implicating that oligomerization status may affect only the high affinity component of ES. OATP1B1 possesses three GXXXG motifs that have been associated with protein dimerization in other membrane proteins. When glycine residues were replaced with alanine, G219A and G393A showed drastically reduced uptake function. Further studies revealed that G219A has a similar association capability to that of the wild-type, while mutation at Gly393 may affect oligomerization status of the transporter. Kinetic analysis showed that both G219A and G393A have a dramatically reduced Vmax for ES uptake. Km of G219A was increased while that of G393A exhibited a decreased value for high affinity component of ES binding. Our studies demonstrated that OATP1B1 may function as oligomers in the high affinity site of ES, while act as monomers for the low affinity binding component of the substrate.

Key words Drug transporter; GXXXG motif; oligomerization; organic anion transporting polypeptide; uptake function

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Introduction Organic anion-transporting polypeptides (human OATPs; other species Oatps; gene symbol SLC21/SLCO) are members of the solute carrier (SLC) family and are responsible for the transport of various structurally independent compounds1. It has been demonstrated that endogenous compounds such as bile acids, hormones and their conjugates as well as pharmaceuticals including antibiotics, antidiabetic drugs, anti-inflammatory drugs, antifungals, antivirals, antihistamines, antihypertensives, fibrates, statins, cardiac glycosides, immunosuppressants, and anticancer drugs are transported by OATPs2. So far 12 members of the human OATP family, including OATP1A2, 1B1, 1B3, 1B7, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1, have been cloned3-6, though SLCO1B7 was proposed as a pseudogene because OATP1B7 is considered non-functional7. Some OATPs are expressed ubiquitously; while others, such as OATP1B1 and OATP1B3, are predominantly found in certain organs or tissues. OATP1B1 is the major OATP located at the basolateral membrane of human hepatocytes and play a crucial role in drug clearance from the body8. In recent years, more and more drugs were shown to be transported by OATP1B19,10. Oligomerization often plays essential roles in different aspects of membrane protein function11. Recent studies have shown that transporter proteins often exist as oligomers, either as homo-oligomers or hetero-oligomers. An example of homo-oligomerization of drug transporters is the human organic anion transporter OAT1. Chemical cross-linking as well as gel filtration chromatography of membrane proteins indicated that OAT1 is present as multimeric complexes in cell membranes12. Further study revealed that transmembrane domain 6 of OAT1 contains a contact region for oligomerization and that cell surface expression of OAT1 was reduced when its oligomerization status was disrupted13. It was also demonstrated that rOat1, rOct1 and rOct2 would form homo-oligomers in detergent solution14. The integrity of the large extracellular loop of rOct1, which is critically dependent on disulfide bond formation, is important for homo-oligomerization of rOct115. A typical example for the functional oligomerization of drug transporters is the ATP-binding cassette (ABC) transporter ABCG2, which is widely accepted to form homo-dimers to exert its function16. Many drug transporters were also found to be associated with other proteins to

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form hetero-oligomers. For instance, interaction of PDZ proteins with urate-anion exchanger URAT1, human OAT4, OCT family members OCTN1 and OCTN2 have been demonstrated17,18. Wang et al. identified PDZK1 as the major interacting protein with rat Oatp1a1. Disruption of the association can affect proper targeting and sorting of Oatp1a1 to the basolateral plasma membrane, suggesting oligomerization between Oatp1a1 and PDZK1 plays an important role in protein subcellular localization and function19. Although a study revealed that several OATPs including OATP1A2, OATP2B1, OATP3A1, OATP4A1 and OATP1C1 contain a Class I PDZ binding domain at their carboxyl termini and the C-termini of OATP1A2, OATP3A1 and OATP1C1 interact with different PDZ proteins20, OATP1B1 and 1B3 do not contain such a domain at their C-termini, implicating that they may utilize other manners to regulate protein sorting. Wang et al. demonstrated that in HeLa cells stably expressing OATP2 (former name of OATP1B1) under regulation of a zinc-inducible promoter, a major band of the transporter protein was shown at around 188 kD in the absence of dithiothretol (DTT) reduction, while addition of DTT resulted in a band with the size of around 75 kDa, suggesting the transporter may exist as oligomers and that disulfide bonds may play a role in oligomerization21. In the current study, we investigated the oligomerization status of OATP1B1 and found out that OATP1B1 may form homo-oligomers, possibly through disulfide bonds. Mutagenesis studies of the conserved GXXXG motif within transmembrane domains, a motif that has been related with protein processing and oligomerization of membrane proteins22-24, revealed two critical glycine residues for ES transport of OATP1B1. Experimental Section Materials -[3H]estrone-3-sulfate (ES) was purchased from Perkin-Elmer Life Sciences (Waltham,

MA).

Sulfosuccinimidyl

2-(biotinamido)-ethyl-1,

3-dithiopropionate

(NHS-SS-biotin), streptavidin-agarose beads, cross-linking reagents bis[sulfosuccinimidyl] suberate (BS3) and 3,3´-dithiobis [sulfosuccinimidylpropionate] (DTSSP) were from Thermo Fisher Scientific Inc. (Waltham, MA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Cell culture and transfection of plasmid constructs into cells -HEK293 cells (ATCC, 4


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Manassas, VA) were grown at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. For transient transfection, confluent cells in 48-well or 6-well plate were transfected with DNA plasmid using LipofectAMINE 2000 reagent (Invitrogen) following manufacturer’s instructions. Transfected cells were incubated for 48 hrs at 37 °C and then used for transport assay and cell surface biotinylation. Establishment of HEK293 cells stably expressing OATP1B1 -Confluent cells in 6-well plate were transfected with pReceiverM07 vector (M07)/pReceiver M07 vector containing the SLCO1B1 cDNA with 3-HA tags (OATP1B1-HA) or pReceiver M03 (M03) vector/pReceiver M03 vector containing the SLCO1B1 cDNA with green fluorescence protein (GFP) tag (OATP1B1-GFP) at the C-terminus using LipofectAMINE 2000 reagent. All of these constructs are OmicsLinkTM Expression-Ready ORF cDNA clones purchased from Genecopoeia (Rockville, MD). They contain a cytomegalovirus (CMV) promoter for expression of exogenous genes in mammalian systems. Seventy-two hours after transfection, transfection medium was replaced with DMEM containing 0.8mg/ml of G418 for selection. Ten days after antibiotic selection, single colonies were picked up and transferred to 60mm cell culture dishes for further propagation. Uptake assay, cell surface biotinylation and western blotting -Uptake function and cell surface expression of the transporter protein were measured as described previously25,26. Briefly, cells in 48-well plate were incubated with uptake solution containing [3H]ES at 37°C for 2 min (1min for kinetic analysis, and transport kinetic values were calculated using the Eadie-Hofstee transformation.) and uptake was stopped by addition of ice-cold phosphate-buffered saline (PBS) solution. Cells were then washed with cold PBS and solubilized in 0.2N NaOH. The radioactivity of the cell lysate was measured with a liquid scintillation counter Triathler-Hidex (Hidex, Turku, Finland). Cell

surface

level

of

OATP1B1

and

its

mutants

was

examined

using

the

membrane-impermeable biotinylation reagent NHS-SS-biotin25,26. In brief, cells in 6-well plate were labelled with NHS-SS-biotin, lysed with RIPA lysis buffer25 and cell debris was removed by centrifugation. Streptavidin-agarose beads were added to the supernatant and

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bound proteins were released in Laemmli sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 2.5% β-mercaptoethanol and 0.02 % bromophenol blue), loaded onto a 7.5% SDS-polyacrylamide gel, separated by electrophoresis, then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). OATP1B1 was detected with anti-HA antibody (1:1000 dilution, Cell Signaling Technology, Danvers, MA) or anti-GFP antibody (1:1000, Beyotime Biotechnology, Inc., Jiangsu, China). Immunofluorescence analysis – Immunofluorescence analysis of the OATP1B1-expressing cells was performed as described previously27. Briefly, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and incubated with anti-HA antibody (1:400 dilution) overnight at 4°C. Bound primary antibodies were detected by reaction with Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen) diluted 1:1000 for 1 h. Cells expressing OATP1B1-GFP were fixed with paraformaldehyde, mounted in Fluoromount mounting medium and observed with a Zeiss Axio Observer D1 fluorescence microscope (Carl Zeiss, Oberkochen, Germany) Cross-linking of membrane proteins -Chemical cross-linking was carried out by incubating confluent intact cells in 6-well plate (2.0×106 cells) with cross-linking reagents (BS3 diluted in 20 mM sodium phosphate and 0.15 M NaCl, pH 8.0 or DTSSP in 0.1 M phosphate, 0.15 M NaCl, pH 7.2) for 1h at room temperature. Both cross-linkers are water-soluble and suitable for cell-surface protein cross-linking. These N-hydroxysuccinimide esters readily react with the primary amines in the side chain of lysine residues. Since DTSSP contains a disulfide bond in its structure, it is thiol-cleavable. The reaction was stopped by addition of 0.1 M Tris-HCl, pH 7.5, for 15 min at room temperature. Cells were then scraped down, re-suspended in 300 µl isolation buffer (250 mM sucrose, 20 mM HEPES, 1.5 mM MgCl2, 1 mM EDTA, pH 7.4, protease inhibitors phenylmethylsulfonyl fluoride, 200 µg/ml, leupeptin, 3 µg/ml), placed on ice and subjected to sonication with a Branson S450-D digital sonifier (Branson Ultrasonic, Danbury, CT). The sonication procedure was as follows: 4 rounds of 8-second sonication on ice, with 5 seconds intervals in between at 20 amplitude microns power. Cell debris was removed by centrifugation at 1,000×g, the supernatant was then centrifugated at 17,000×g for 30 min at 4°C to collect the membrane fractions, which were

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re-dissolved in appropriate volume of isolation buffer. Proteins were then denatured, separated by electrophoresis and subjected to immunoblotting. Co-immunoprecipitation -HEK293 cells co-expressing OATP1B1-GFP and OATP1B1-HA were washed three times with cold PBS and lysed in immunoprecipitation buffer (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 2 mM EDTA, 3% glycerol, protease inhibitors phenylmethylsulfonyl fluoride, 200 µg/ml, leupeptin, 3 µg/ml). Cell debris was removed by centrifugation at 12,000×g for 20 min at 4°C. Supernatants were transferred to new Eppendorf tubes and pre-cleaned with 60 µl of protein G agarose beads (Thermo Scientific). Agarose beads were then removed by centrifugation at 1,000×g for 1 min at 4°C and supernatants were incubated with anti-HA or anti-GFP antibody at 4°C overnight. Fifty microliters of protein G agarose beads were then added and mixed with end-over-end rotating at 4 °C for 1.5 h. Agarose beads-bound proteins were eluted with Laemmli sample buffer containing β-mecaptoethanol and analyzed by immunoblotting with anti-GFP or anti-HA antibody. Site-directed Mutagenesis -Mutant transporters were generated using QuickChange Lightning Site-Directed Mutagenesis Kit from Agilent (Santa Clara, CA). OATP1B1-HA or OATP1B1-GFP was used as template for the mutagenesis. All mutant sequences were confirmed by the dideoxy chain termination method. Statistical analysis -Data statistical analysis was performed using one-way analysis of variance with Bonferroni's post hoc test.. Differences between means are regarded as significant if p50% of wild-type OATP1B1), while two mutants, i.e. G219A and G393A, demonstrated markedly reduced transport activity (≤20% of OATP1B1) (Fig. 5A). In addition to the single mutants, we also generated double mutants of which both glycine residues of the same GXXXG motif were

10


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mutated to alanine. However, no further functional reduction was observed in these double mutants compared with G219A or G393A (Fig. 5B). Cell surface expression of G219A was comparable with that of wild-type OATP1B1, while that of G393A showed a reduced protein level (~60% of the wild-type transporter) (Fig. 5C). We also investigated total protein expression of these two mutants and obtained similar results as the cell surface expression analysis (data not shown). These results suggested that mutation of Gly393 may affect protein stability of OATP1B1. However, the decreased level of G393A on plasma membrane could not account for its dramatically reduced uptake, which was ~20% of that of wild-type OATP1B1. Oligomerization study of G219A and G393A -Since GXXXG motif was considered an important dimerization motif16 and that mutation of either of the glycine residues would result in destabilizing the dimer31, we then used chemical cross-linking with BS3 to see whether mutations at Gly219 and Gly393 led to dissociation of the oligomers. As shown in Figure 6A, the amount of oligomers in G219A was comparable to wild-type OATP1B1, while that of G393A decreased significantly. Co-immunoprecipitation study also revealed a decreased oligomerization status of G393A (Fig. 6B). These results implicated that G393A may be involved in oligomerization of OATP1B1. Furthermore, co-expression of G219A or G393A with wild-type OATP1B1 led to reduction of low concentration ES uptake but increased transport function for high concentration of ES by wild-type OATP1B1 (Fig. 6C&D), a phenomenon similar to that of W258A. Kinetic analysis of G219A and G393A -Since G219A and G393A both showed dramatically reduced ES uptake function, we performed kinetic analysis and found out that both mutants exhibited a significantly reduced Vmax compared with OATP1B1 wild-type (Fig. 7), suggesting a decreased turn-over number of the transporter protein. Km of G219A was significantly higher than that of wild-type OATP1B1, while that of G393A was decreased for the high affinity component of ES uptake (Fig. 7A and Table 1). As for the ES low affinity binding site, no change was observed for Km value of G219A and G393A (Fig. 7B, Table 1).

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Discussion In a previous study for hepatocyte bilirubin transport of OATP2 (former name of OATP1B1), Wang et al. found HeLa cells stably expressing OATP2 exhibited a major band of around 188 kD in the absence of DTT reduction, while addition of DTT resulted in a band with the size of around 75 kDa. Such an observation suggested that OATP2 may exist as oligomers21. In the present study, a higher size band with the molecular weight of around 190kD was observed in samples denatured in the absence of β-mercaptoethanol, indicating that disulfide bonds may be involved in the formation of the protein complexes (Fig. 2B). Treatment of intact cells with membrane impermeable cross-linkers BS3 and DTSSP both resulted in a signal at the position of ~190kD, implicating the presence of oligomers, possibly dimers, on the cell surface. In addition to the 190 kD signal, higher size band of OATP1B1 was observed after cross-linking, suggesting that other forms of oligomeric complexes may exist (Fig. 2A&B). Upon cross-linking, the intensity of oligomers increased while that of OATP1B1 monomer (~95kDa) reduced. But even with a high concentration of BS3 (5 mM), monomer bands were still present. We speculated that this may be due to the efficiency of the cross-linking reagents. We had applied higher concentrations of BS3 (>5 mM) and found out that the higher the concentration of BS3 applied, the greater reduction of monomeric OATP1B1 level was observed. However, at such high BS3 concentrations, only aggregrated signals but not distinctive bands of OATP1B1 at high molecular weights were observed (data now shown). In addition, we could not rule out the possibility that some monomeric form of OATP1B1 may also exit on the plasma membrane. The association of OATP1B1 into oligomeric units was further confirmed by the capability of differently tagged OATP1B1 to co-precipitate, which indicated that the higher size bands observed after cross-linking are homo-oligomers (Fig. 3). If the formation of oligomers is required for uptake function of the transporter, the introduction of abnormal mutants would disrupt at least a portion of the functional oligomers and thus resulted in reduction of the transport activity compared with WT-OATP1B1-expressing

cells.

Indeed,

cells

co-expressing

OATP1B1

and

its

12


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loss-of-function mutant W258A showed marginal but significant decreased uptake function compared with cells transfected with the same amount of OATP1B1 (Fig. 4A&B).. W258A had dramatically decreased ES uptake function but the cell surface expression of this mutant was comparable to wild-type OATP1B126, and co-expression of this mutant did not reduce cell surface expression of the transporter protein (data not shown). In addition, when more loss-of-function mutant was introduced in the co-expression system, the uptake function of OATP1B1 was affected more dramatically (Fig. 4A). These results thus suggested that the formation of oligomers may be involved in maintaining proper function of the transporter. However, the suppressive effect caused by the mutant was relatively small. We speculated that may be due to several reasons. Firstly, W258A needs to form an oligomer with OATP1B1 to exert its effect. Therefore, even if OATP1B1 forms a dimer, the chance of W258A affecting OATP1B1 may only be 1/3 (considering there are half wild-type OATP1B1 and half W258A in the expression system). Secondly, though transport function of W258A is significantly reduced, the mutant still shows around 15% activity compared with wild-type OATP1B1. Thus it may still contribute to the transport activity of the co-expression system. Thirdly, we still need to clarify how many units are present in the oligomeric structure of OATP1B1. If more than two monomers are present in one multimeric complex, the presence of one or more mutated monomer(s) may cause different effects on the uptake function. An interesting finding of the current study was that the dominant-negative effect was only observed in uptake of low concentration ES (50 nM). When transport activity was measured for high concentration of ES (20 µM), cells co-expressing W258A and OATP1B1 wild-type showed increased uptake function compared with cells that transfected with equal amount of OATP1B1(Fig. 4D), indicating that oligomerization status may not be required for the low affinity binding site of ES. The GXXXG motif within transmembrane domains has been related to protein processing and oligomerization in many proteins22-24. There are three GXXXG motifs within the structure of OATP1B1 and single mutation of most of these glycine residues resulted in comparable uptake function to the wild-type OATP1B1. However, two mutants, G219A and G393A, showed dramatically reduced transport activity (Fig. 5A). Both Gly219 and

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Gly393 are highly conserved among human OATPs, with all members having the same amino acid residue at these positions. Despite impaired function, cell surface expression of G219A was similar to that of wild-type OATP1B1, while G393A exhibited around 40% reduction of cell surface protein level. Further, chemical cross-linking (Fig. 6A) and co-immunoprecipitation (Fig. 6B) of G393A exhibited reduced association of the transporter (~60% of that of wild-type OATP1B1), suggesting that this glycine residue may be involved in oligomer formation. When we analyzed the kinetic parameters of these two mutants, we found out that both G219A and G393A exhibited a dramatic reduction of Vmax compared with wild-type OATP1B1 (Fig. 7), suggesting mutation at these glycine residues may alter turn-over rate of the protein, thus reduced its transport function. Co-expression of G219A or G393A with wild-type OATP1B1 also resulted in a reduction of low concentration ES uptake by OATP1B1 (Fig. 6C). Interestingly, we found out that G393A affected wild-type OATP1B1 function more dramatically than G219A, though ES uptake by G393A per se was higher than G219A (Fig. 5A). We speculated that may be due to the reduced cell surface expression of G393A (Fig. 5C) and its decreased association with OATP1B1 wild-type, which further impaired ES uptake by the oligomers. Co-expression of G219A or G393A with OATP1B1 also resulted in increased uptake at the low affinity binding site of ES (Fig. 6D), further proved that OATP1B1 monomers may function independently for ES uptake at high concentration ranges. In summary, our current study demonstrated that OATP1B1 may form homo-oligomers in intact cells, possibly through disulfide bonds. Such an oligomerization status affects its transport function at the ES high affinity binding site, while at the low affinity component for the substrate, monomers of the multimeric complexes may function independently. Since OATP1B1 is a major site for drug absorption and/or drug-drug interaction, disruption of its oligomerization status may affect bioavailability of multiple clinically important drugs, especially those that were found to be transported by the high affinity binding site of estrone-3-sulfate. It was also found that Gly393 at the conserved GXXXG motif may play a role in OATP1B1 oligomerization. Further studies are needed to reveal other critical domains that are involved in oligomer formation of OATP1B1. Additionally, it should be noted that 14


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our current studies were based on cell system, of which may not completely reflect the native membrane environment of the liver. Additional protein- protein interactions in the liver, where other OATP members such as OATP1B3 and OATP2B1 co-exist, may occur. Under such conditions, different OATP isoforms may be associated with each other to form hetero-oligomers, which may also be essential for the function of these transporters. Acknowledgements This work was supported by the National Natural Science Foundation of China Grant [81373473]

and

Natural

Science

Foundation

of

Guangdong

Province

Grant

[2015A030312005] to Mei Hong. References 1.

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Mikkaichi T, Suzuki T, Tanemoto M, Ito S, Abe T (2004) The organic anion transporter (OATP) family. Drug Metab Pharmacokinet 19: 171–179.

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Nakanishi, T.; Tamai, I. Genetic polymorphisms of OATP transporters andtheir impact on intestinal absorption and hepatic disposition of drugs. Drug Metab. Pharmacokinet. 2012, 27, 106-121.

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Noé, J.; Portmann, R.; Brun, M. E.; Funk, C. Substrate-dependent drug-drug interactions between gemfibrozil, fluvastatin and other organic anion-transporting peptide (OATP) substrates on OATP1B1, OATP2B1, and OATP1B3. Drug Metab. Dispos. 2007, 35, 1308-1314.

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Figure Captions Figure 1 Characterization of HEK293 cells stably expressing OATP1B1-HA and OATP1B1-GFP. A. Representative blot for plasma membrane protein expression of OATP1B1-HA (left) and OATP1B1-GFP (right). Cells were biotinylated with NHS-SS-biotin and precipitated with streptavidin beads, separated by SDS-PAGE, followed by western blotting with anti-HA or anti-GFP antibody. Same blot was probed with anti-integrin antibody and shown as loading control. Three independent experiments were performed to analyze cell surface expression of OATP1B1 in the stable clones. B. Immunocytochemical analysis of OATP1B1-HA (upper panel) and OATB1B1-GFP (lower panel). C. Uptake of estrone-3-sulfate by OATP1B1. Transport of 50 nM estrone-3-sulfate in HEK293 cells expressing OATP1B1 was measured at 37°C at a 2 min interval. The results represent data from three experiments, with duplicate measurements for each sample. The results shown are means ± S.D. (n = 3). Asterisks indicate values are significantly different (p

Oligomerization Study of Human Organic Anion Transporting

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