The Eighth and Ninth Transmembrane Domains in Organic Anion Transporting Polypeptide 1B1 Affect the Transport Kinetics of Estrone-3-Sulfate and Estradiol-17 -D-glucuronide

Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are responsible for the hepatic uptake of organic anions. They share similar sequences and structures with 12 putative transmembrane domains (TMs). Their substrate specificities are very broad and overlap each other, whereas each transporter specifically recognizes certain substrates. Because the homology of the amino acid sequence in the latter part of OATP1B1 and OATP1B3 is relatively low, to determine which TMs in the latter part of OATP1B1 are important for its substrate recognition, we constructed several cell lines expressing chimeric transporters in which some TMs of OATP1B1 were substituted with those of OATP1B3, and we investigated the transport kinetics of estrone-3-sulfate (E-sul; a substrate preferentially accepted by OATP1B1) and estradiol-17 -D-glucuronide (EG; a substrate accepted by both transporters). As the number of substituted TMs at the N terminus with those of OATP1B3 increased, the Km value of E-sul greatly increased and its uptake clearance decreased. The substitution of TM7 or TM9 of OATP1B1 with that of OATP1B3 (named 1B1-TM7 or 1B1-TM9) did not change the transport kinetics of EG, whereas the Km value of E-sul in 1B1-TM9 increased 7.4-fold. Conversely, the substitution of TM8 resulted in an 18-fold increase in the Km value of E-sul and abolished the transporter-mediated uptake of EG. These results suggest that TM8 in OATP1B1 is critical for the substrate recognition of both E-sul and EG and that TM9 is important for the recognition of E-sul, whereas it is interchangeable with that of OATP1B3 for EG transport. To protect the body from exposure to various endogenous and exogenous compounds, many kinds of transporters and metabolic enzymes are expressed, and substrate specificity of each protein is generally very broad. However, the underlying mechanisms to explain why each drug transporter can accept a variety of compounds as substrates so far remain to be elucidated because the crystal structures of mammalian drug transporters have not yet been solved. Recently, some reports have created in silico models to explain the substrate specificity of drug transporters by using ligand-based drug design approach (Chang et al., 2005). However, there are few studies identifying the exact binding sites of multiple substrates in drug transporters from in silico analysis. Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are predominantly expressed on the basolateral membrane of human hepatocytes (Abe et al., 1999, 2001; Hsiang et al., 1999; König et al., 2000a,b). The genomic sequence of OATP1B1 and OATP1B3 is arranged in tandem on the same locus, and their genomic exon-intron coordination is very similar (König et al., 2000a). The homology of their amino acid sequences is 80%, and they share the same structure with 12 putative transmembrane domains (TMs). The substrate specificity of OATP1B1 and OATP1B3 is very broad, and various structurally unrelated compounds can be accepted by these transporters as substrates. Moreover, the substrate specificity of OATP1B1 often overlaps with that of OATP1B3, such as that for estradiol 17 -D-glucuronide (EG), taurocholate, thyroid hormones, sulfobromophthalein, and clinically used drugs (including HMG-CoA reductase inhibitors and angiotensin II receptor antagonists) (Tirona and Kim, 2007). Several lines of clinical evidence have suggested that the change in the transport function of OATP1B1 caused by genetic polymorphisms and drug-drug interactions affects the pharmacokinetics and subsequent pharmacological/toxicological effect of multiple anionic drugs (Shitara et al., 2005; Maeda and Sugiyama, 2008). Hirano et al. (2004) have esThis study was supported by a grant-in-aid for scientific research (A) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan [Grant KAKENHI: 20249008]. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.148411. ABBREVIATIONS: OATP, organic anion transporting polypeptide; TM, transmembrane domain; EG, estradiol-17 -D-glucuronide; E-sul, estrone3-sulfate; SN-38, 7-ethyl-10-hydroxycamptothecin; CCK-8, cholecystokinin octapeptide; HEK, human embryonic kidney; PBS, phosphatebuffered saline; TTBS, Tris-buffered saline containing 0.05% Tween 20. 0022-3565/09/3292-551–557$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 329, No. 2 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 148411/3466557 JPET 329:551–557, 2009 Printed in U.S.A. 551 at A PE T Jornals on A ril 2, 2017 jpet.asjournals.org D ow nladed from tablished several methodologies for estimating the contribution of OATP1B1 and OATP1B3 to the overall hepatic uptake of compounds. These approaches have revealed that HMGCoA reductase inhibitors (pitavastatin and rosuvastatin) are generally taken up into human hepatocytes predominantly via OATP1B1 (Hirano et al., 2004; Kitamura et al., 2008), whereas angiotensin II receptor antagonists (valsartan and olmesartan) are taken up via both OATP1B1 and OATP1B3 to the same extent (Yamashiro et al., 2006; Yamada et al., 2007). Thus, OATP1B1 and OATP1B3 are responsible for the hepatic uptake of organic anions in humans. Although these transporters show overlapping substrate specificities, some substrates are highly specific to each transporter. For example, E-sul, caspofungin, and SN-38 (active metabolite of irinotecan) are reported to be specifically transported by OATP1B1 but not OATP1B3 (Hirano et al., 2004; Nozawa et al., 2005; Sandhu et al., 2005). Alternatively, cholecystokinin octapeptide (CCK-8), telmisartan, and docetaxel are transported only by OATP1B3 (Ismair et al., 2001; Smith et al., 2005; Ishiguro et al., 2006). This suggests that even the substrate recognition properties of highly homologous transporters, OATP1B1 and OATP1B3 are somewhat different and may be the result of small differences in their amino acid sequences. To explore the amino acids important for discriminating the substrate specificity of OATP1B1 and OATP1B3, the creation of several chimeric proteins using parts of OATP1B1 and OATP1B3 is a powerful approach for determining which TMs are interchangeable and for narrowing the candidate binding sites of transporter substrates. During the preparation of our article, Gui and Hagenbuch (2008) showed that three amino acids in TM10 of OATP1B3 are critical for the transport of CCK-8 by using several kinds of chimeric proteins in which TMs of OATP1B3 are substituted with those from OATP1B1. However, little is known about the molecular mechanism for the broad but strict substrate specificity of OATP1B1, and the important binding sites for its substrates have not been clarified. Especially in the latter part of OATP1B1 and OATP1B3 sequences, the homology of amino acid sequence is relatively lower, which implies that this region might be important for substrate recognition by OATP1B1. Therefore, the purpose of this study was to clarify the difference and similarity of the substrate recognition properties of OATP1B1 and OATP1B3. To achieve this goal, we especially focused on the latter half of TMs and observed the effect of the exchange of TMs in OATP1B1 with those of OATP1B3 on the transport properties of their common substrate, EG and the OATP1B1-selective substrate, E-sul, by using a series of chimeric OATP1B1 transporters. Materials and Methods Materials. [H]EG (45 Ci/nmol) and [H]E-sul (46 Ci/nmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). [H]CCK-8 (77 Ci/mmol) was purchased from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK). All other chemicals were commercially available and of reagent grade. Definition of the Names for Chimeric OATP1B1 Proteins. As shown in Fig. 1, a chimeric OATP1B1 protein in which the first to Nth TMs of OATP1B1 were substituted with those of OATP1B3 is named 1B1-TM1-N. A mutated protein, in which the Nth TM of OATP1B1 was replaced with that of OATP1B3 is named 1B1-TMN. Construction of the Plasmid Vectors for Chimeric Proteins. Using previously constructed pcDNA3.1( )/Neo vectors carrying human OATP1B1 and OATP1B3 cDNA (Hirano et al., 2004) as templates, mammalian expression vectors for chimeric proteins including 1B1-TM1-3, 1B1-TM1-6, 1B1-TM1-7, 1B1-TM1-8, and 1B1TM1-9 were amplified by overlap extension polymerase chain reaction. The pcDNA3.1( )/Neo expression vectors for mutated proteins, whose single TM of OATP1B1 was substituted with that from OATP1B3 including 1B1-TM7, 1B1-TM8, and 1B1-TM9 were constructed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) as described previously (Geiser et al., 2001). The sequences of all the constructs were verified using a DNA sequencer (3130xl Genetic Analyzer; Applied Biosystems, Foster City, CA). Cell Culture and Transfection of Plasmid Vectors into Cells. Each pcDNA3.1( )/Neo expression vector carrying cDNA of chimeric transporter was transfected into HEK293 cells using FuGENE 6 reagent (Roche Diagnostics, Indianapolis, IN) as described previously (Hirano et al., 2004). The transfected HEK293 cells were selected with Geneticin (G418, 800 g/ml; Invitrogen, Carlsbad, CA). HEK293 cells stably expressing each type of chimeric transporter were cultured in low-glucose Dulbecco’s modified Eagle’s medium (Invitrogen) with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and 1% antibiotic-antimycotic solution (Invitrogen) at 37°C under 5% CO2 and 95% humidity. Western Blot Analysis. Crude membrane was prepared from HEK293 cells as reported previously (Sasaki et al., 2002). After the crude membrane fraction was suspended in PBS, it was immediately frozen in liquid N2 and stored at 80°C until used. The protein concentration of the crude membrane fractions prepared from