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Transmembrane Domain 1 of Human Organic Anion Transporting Polypeptide 2B1 Is Essential for Transporter Function and Stability S

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Transmembrane Domain 1 of Human Organic Anion Transporting Polypeptide 2B1 Is Essential for Transporter Function and Stability S Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2018/06/05/mol.118.111914.DC1 1521-0111/94/2/842–849$35.00 https://doi.org/10.1124/mol.118.111914 MOLECULAR PHARMACOLOGY Mol Pharmacol 94:842–849, August 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics Transmembrane Domain 1 of Human Organic Anion Transporting Polypeptide 2B1 Is Essential for Transporter Function and Stability s Zihui Fang,1 Jiujiu Huang,1 Jie Chen, Shaopeng Xu, Zhaojian Xiang, and Mei Hong College of Life Sciences (Z.F., J.H., J.C., S.X., Z.X., M.H.) and Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms (J.H., M.H.), South China Agricultural University, Guangzhou, China Received January 29, 2018; accepted May 24, 2018 Downloaded from ABSTRACT Organic anion transporting polypeptides (OATPs, gene symbol OATP2B1 revealed that substitution of L58 with alanine SLCO) are important membrane transporter proteins that dramatically altered the Km value, and mutation of V52, H55, mediate the uptake of wide ranges of endogenous and Q59, and L69 resulted in significantly reduced substrate exogenous compounds. OATP2B1 has been found in multiple turnover number, whereas A61V, Q62A, and S66A exhibited molpharm.aspetjournals.org organs and tissues, including the liver, small intestine, kidney, significantchangeinbothKm and Vmax values. In addition, brain, placenta, heart, skin, as well as skeletal muscle, and is phenylalanine at position 51 seems to play an important role in proposed to be involved in the uptake of orally administered maintaining proper folding of OATP2B1 because alanine re- drugs. Quite a few reports have demonstrated that trans- placement of F51 caused accelerated degradation of the membrane domains (TMs) are crucial for proper functions of transporter protein. Although proteasome and lysosome inhib- OATP family members. Comparative modeling proposed that itors could partially recover protein level, the mutant transporter TM1, along with TM2, 4, and 5 of the N-terminal half of remained nonfunctional. Taken together, the identification of OATP2B1, may be localized within the substrate interaction nine essential amino acid residues within TM1 of OATP2B1 pocket and are important for uptake function of the transporter. suggested that the transmembrane domain is important for Alanine scanning of the putative transmembrane domain 1 of maintaining proper function of the transporter. at ASPET Journals on September 28, 2021 Introduction interactions, OATPs have been extensively recognized as key determinants for drug absorption, distribution, and excretion Organic anion transporting polypeptides (OATPs, gene (Shitara et al., 2005; Poirier et al., 2007). symbol SLCO) belong to the solute carrier family and mediate OATP2B1 has been found in multiple organs and tissues, sodium-independent transport of various endogenous and including the liver, small intestine, kidney, brain, placenta, exogenous compounds (Hagenbuch and Gui, 2008). Bile salts, heart, skin, as well as skeletal muscle (Tamai et al., 2000; hormones and their conjugates, toxins, and a wide range of Kullak-Ublick et al., 2001; Hagenbuch and Meier, 2004; drugs have been found to be transported by OATPs. Twelve Niessen et al., 2009; Knauer et al., 2010, 2013). In the liver, human OATP family members have been found so far: OATP1A2, 1B1, 1B3, 1B7, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, OATP2B1 is involved in hepatic uptake of a wide spectrum of 5A1, and 6A1 (Hagenbuch and Meier, 2003; Nakanishi and xenobiotics, and many organic anions, mono- and dicarboxylic Tamai, 2012). However, SLCO1B7 was proposed as a pseu- acids (Kobayashi et al., 2003; Sai et al., 2006), steroid dogene because OATP1B7 is considered as nonfunctional hormones and their derivatives (Grube et al., 2006), as well (Stieger and Hagenbuch, 2014). Some OATP family members as drugs such as rifamycin SV (Vavricka et al., 2002), prav- are predominantly expressed in certain organs or tissues. For astatin (Kobayashi et al., 2003), cyclosporine, and gemfibrozil example, OATP1B1 and OATP1B3 are found only in the liver, (Tamai et al., 1997) are inhibitors of the transporter. whereas others were reported as being expressed ubiquitously OATP2B1 exhibits high expression in the apical membrane (König et al., 2006). Due to their broad substrate specificity, of human intestinal epithelial cells and may function as a wide tissue distribution, and the involvement of drug-drug pH-dependent organic anion transporter (Kis et al., 2010). Since the physiologic microclimate pH in the intestinal lumen This work was supported by the National Natural Science Foundation of is weakly acidic, OATP2B1 that shows higher activity at acidic China [Grant 81373473] and National Natural Science Foundation of Guangdong Province [Grant 2015A030312005] to M.H. pH is believed to be involved in the uptake of drugs adminis- 1Z.F. and J.H. contributed equally to this work. tered orally (Kobayashi et al., 2003; Tamai, 2012). Although https://doi.org/10.1124/mol.118.111914. s This article has supplemental material available at molpharm. extensive studies have been carried out to identify substrates aspetjournals.org. of OATPs, the underlying mechanisms of substrate binding ABBREVIATIONS: ES, estrone-3-sulfate; MG132, carbobenzoxy-Leu-Leu-leucinal; NHS-SS-biotin, sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3- dithiopropionate; OATP, organic anion transporting polypeptide; TM, transmembrane domain. 842 Transmembrane Domain 1 Is Important for OATP2B1 Function 843 and/or recognition remain largely unclear because high- cells expressing OATP2B1 or mutants were labeled on ice with NHS- resolution crystal structures of mammalian drug transporters SS-biotin in two successive 20-minute incubations and lysed with are still not available (Niessen et al., 2009). radioimmunoprecipitation assay buffer [50 mM Tris, 150 mM NaCl, Transmembrane domains (TMs) have been demonstrated to 0.1% SDS, 1% NP40, and protease inhibitors phenylmethylsulfonyl m m be important for proper functions of various transporters. fluoride (200 g/ml) and leupeptin (3 g/ml), pH 7.4]. The labeled proteins in supernatant were then pull downed by streptavidin- Previous studies have identified quite a few essential amino agarose beads, released in 4Â Laemmli buffer and loaded onto a acids located within transmembrane domains of OATP mem- 7.5% SDS–polyacrylamide electrophoresis gel, transferred electropho- bers. For example, amino acid residues within TM2 (Li et al., retically to a polyvinylidene difluoride membrane (Millipore, Billerica, 2012), TM6 (Huang et al., 2013), TM10 (Gui and Hagenbuch, MA), and detected with anti-hemagglutinin antibody (Cell Signaling 2009; Ohnishi et al., 2014), and TM11 (Weaver and Hagen- Technology, Danvers, MA). buch, 2010; Hong et al., 2015) were shown to be crucial for Uptake Assay. HEK293 cells in a 48-well plate were used for proper function of OATP1B1, and TM8 (Miyagawa et al., 2009) transport measurement as described before (Li et al., 2012; Huang is believed to be important for substrate recognition of the et al., 2013) with minor modification. In brief, cells were incubated 3 3 transporter. As for OATP1B3, K41 in TM1 and R580 in TM11 with uptake solution that contained [ H]ES (pH 7.4) or [ H]taurocholic are believed to be pivotal to the transporter function (Glaeser acid (pH 5.0) at 37°C for 2 minutes (1 minute for kinetic analysis), and the reaction was stopped by ice-cold phosphate-buffered saline solu- et al., 2010). In addition, G45 within TM1 along with Y537, tion. Cells were then washed twice with cold phosphate-buffered Downloaded from S545, and T550 within TM10 (Gui and Hagenbuch, 2008; solution, solubilized in 0.2 N NaOH followed by neutralization with DeGorter et al., 2012) were demonstrated to be critical for the 0.2 N HCl, and radioactivity of the cell lysate was measured using a transport of OATP1B3-specific substrate CCK8. TM6 of Triathler-Hidex (Hidex, Turku, Finland) liquid scintillation counter. OATP1A2 seems to be an important region for substrate The uptake count was standardized by the amount of protein in each binding, protein stability, and trafficking (Chan et al., 2015). well. However, little information is available for the role of TMs Statistical Analysis. Data statistical analysis was carried out within OATP2B1. using one-way analysis of variance with Bonferroni’s post hoc test. molpharm.aspetjournals.org , Comparative modeling proposed that transmembrane helix Differences between means are regarded as significant if P 0.05. 1 along with TM2, 3, and 5 of the N-terminal half of OATP2B1 may be involved in formation of the substrate interaction pore Results (Meier-Abt et al., 2005). In the present study, alanine scanning of the putative TM1 of OATP2B1 identified nine Characterization of OATP2B1 Function in HEK293 amino acid residues that are essential for the transporter Cells. To see whether OATP2B1 could be properly expressed function. Kinetic analysis of the functionally impaired mu- and function in HEK293 cells, we first analyzed cell surface tants showed that Km and/or Vmax values of most of the expression and kinetic parameters of OATP2B1. As shown in mutants were affected, and that F51A exhibited dramatically Fig. 1, OATP2B1 was expressed and correctly targeted to the at ASPET Journals on September 28, 2021 reduced protein expression. The identification of nine critical plasma membrane and exhibited a Km value of 6.76 6 0.71 amino acid residues within TM1 of OATP2B1 suggested that mM, which is consistent with the previously reported Km of the transmembrane domain is important for maintaining 8.09 6 1.67 mM (Nozawa et al., 2004) for OATP2B1 expressed proper function of the transporter protein. in HEK293 cells. These results indicated that OATP2B1 was properly expressed and functioned within HEK293 cells. Effect of TM1 Mutants on OATP2B1 Uptake Function Materials and Methods and Protein Expression. To analyze critical amino acid Materials. [3H]Estrone-3-sulfate (ES) and [3H]taurocholic acid residues within TM1, we performed alanine scanning. Each of were obtained from PerkinElmer Life Sciences (Waltham, MA).
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