2001 Fiscal Year Annual Research Report

上皮細胞におけるアミノ酸・薬物のベクトル輸送形成機構の解析

Research Project

Project/Area Number	12144201
Research Institution	The University of Tokyo
Principal Investigator	杉山雄一東京大学, 大学院・薬学系研究科, 教授 (80090471)
Co-Investigator(Kenkyū-buntansha)	金井好克杏林大学, 医学部, 助教授 (60204533)
Keywords	OATP2 / MRP2 / 極性細胞 / 経細胞輸送 / アミノ酸トランスポーター / 上皮輸送 / 膜ソーティング / 蛋白間相互作用
Research Abstract	上皮細胞における薬物のベクトル輸送について検討を加えるため、有機アニオン系化合物の肝取り込みに関与するOATP2、胆汁排泄に関与するMRP2を共発現する極性細胞(MDCK細胞)を介した化合物輸送について詳細な検討を加えた。生体内因性物質17beta estradiol-17beta-D-glucuronideや高脂血症治療薬プラバスタチンについて検討したところ、経細胞輸送の律速段階はOATP2による取り込みであることが明らかとなり、in vivoと対応したことから、本in vitro実験系がin vivoでの化合物輸送を予測する上で有用な系となることが示唆された。また、本年度はMRP2が上皮細胞頂側膜に局在する機構を明らかとするために、ヒトMRP2をプローブとして用い、yeast two hybrid法による探索を進め、候補となる蛋白を示唆しつつある。さらに、ヒト肝病態時においては、MRP2の膜局在を維持することができなくなり、細胞内へと内在化される可能性を示唆した。さらに吸収上皮におけるアミノ酸のベクトル輸送の分子機序を明らかにすることを目的として、未同定のアミノ酸トランスポーターのcDNAクローニング、及びヘテロダイマー型アミノ酸トランスポーターをモデル系とした、キメラ解析による蛋白質間相互作用と細胞膜へのsortingの機序の解析、その基盤となる会合蛋白分子のyeast two-hybrid法による探索を進行させている。平成13年度は、ヘテロ二量体型アミノ酸トランスポーターファミリーの既知のメンバーと有意の相同性を有しながら、既知の1回膜貫通型補助因子(rBAT及び4F2hc)とは連結しない新たな2種のトランスポーターを見い出した。これらとrBAT及び4F2hcとの融合タンパク質を作製することにより細胞膜へのsortingを可能とし、これらの輸送活性を実証した。すでに1回膜貫通型補助因子rBAT及び4F2hcのキメラ解析により限定したパートナー認識に関わる領域を、部位特異的変異導入により検討し、その妥当性を確認した。

Research Products

(22 results)

All Other

All Publications (22 results)

[Publications] M.Sasaki.et al.: "Transcellular Transport of Organic Anions Across a Double-transfected Madin-Darby Canine Kidney II Cell Monolayer Expressing Both Human Organic Anion-transporting Polypeptide (OATP2/SLC21A6) and Multidrug Resistance-associated Protein 2 (MRP2/ABCC2)"J. Biol. Chem.. (in press). (2002)
[Publications] J.Shoda et al.: "The expression levels of plasma membrane transporters in the cholestatic liver of patients undergoing biliary drainage and their association with the impairment of biliary secretory function"Am. J. Gastroenterol. 96. 3368-3378 (2001)
[Publications] J.Shoda et al.: "Etiologic significance of defects in cholesterol, phospholipid, and bile acid metabolisn in the liver of patients with intrahepatic calculi"Hepatology. 33. 1194-1205 (2001)
[Publications] H Tsuganezawa et al.: "A new member of the HCO3-transporter superfamily is an apical anion excanger of b-Intercalated Cells in the kidney"J. Biol. Chem.. 276. 8180-8189 (2001)
[Publications] K.Mizoguchi et al.: "Human cystinuria-related transporter : localization and functional characterization"Kidney Int. 59. 1821-1833 (2001)
[Publications] DK Kim et al.: "Expression cloning pf a Na+-independent aromatic amino acid transporter with structural similarity to H+/monocarboxylate transporters"J. Biol. Chem.. 276. 17221-17228 (2001)
[Publications] SH Cha et al.: "Identification and characterization of human organic anion transporter 3expressing predominantly in the kidney"Mol. Pharmacol.. 59. 1277-1286 (2001)
[Publications] JY Kim et al.: "Human cystine/glutamate transporter : cDNA cloning and upregulation by oxidative stress in glioma cells"Biochim. Biophys. Acta. 1512. 335-344 (2002)
[Publications] O.Yanagida et al.: "Human L-type amino acid transporter 1 (LAT1) : characterization of function and expression in tumor cell lines"Biochim. Biophys. Acta. 1514. 291-302 (2001)
[Publications] T.Nii et al.: "Molecular events involved in up-regulating human Na+-independent neutral amino acid transporter LAT1 during T-cell activation"Biochem. J.. 358. 693-704 (2001)
[Publications] A.Chairoungdua et al.: "Identification and characterization of a novel member of theheterodimeric amino acid transporter family presumed to be associated with an unknown heavy chain"J. Biol. Chem.. 276. 49390-49399 (2001)
[Publications] S.Tamai et al.: "Expression of L-type amino acid transporter 1 in a rat model of liver metastasis : positive correlation with tumor size"Cancer Detect. Prev.. 25. 439-445 (2001)
[Publications] K.Suzuki et al.: "Transientupregulation of the glial glutamate transporter GLAST in response to fibrobla growth factor, insulin-like growth factor and epidermal growth factor in cultured astrocytes"J. Cell Sci.. 114. 3717-3725 (2001)
[Publications] H.Ohkame et al.: "Expression of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in liver tumor lesionsof rat models"J. Surg. Oncol.. 78. 265-271 (2001)
[Publications] DK Kim et al.: "The human T-type amino acid transporter 1 : characterization, gene organization and chromosomallocation"Genomics. 79. 95-103 (2001)
[Publications] H.Uchino et al.: "Transport of amino acid-related compounds mediated by L-type amino acid transporter 1 (LAT1) : Insights into the mechanisms of substrate recognition"Mol. Pharmacol.. (in press). (2002)
[Publications] H.Matsuo et al.: "Idenitification of a novel Na+-independent acidic amino acid transporter with structural similarity to the member of helerodimeri amino acid transporter family associated with unknown heavy chains"Biol. Chem.. (in press). (2002)
[Publications] 金井好克: "アミノ酸トランスポーター、Annual Review腎臓2001"伊藤克己、浅野泰、遠藤仁、御手洗哲也、東原英二編中外医学社. 8 (2001)
[Publications] 金井好克: "多選択性アミノ酸トランスポーターファミリー(LATファミリー、蛋白質核酸酵素46)"共立出版. 9 (2001)
[Publications] 金井好克: "小腸および腎におけるアミノ酸吸収を担うトランスポーター(医学のあゆみ、199)"医歯薬出版. 5 (2001)
[Publications] Y.Kanai, H.Endou: "Heterodimeric amino acid transporters molecular biology and pathological and pharmacological relevance"Curr. Drug Metab. 2. 16 (2001)
[Publications] Y Kanai et al.: "The high-affinityglutamate neutral amino-acid transporter family : structure, function, and physiological relevance"In"Neurotransmitter transporters", M.E.A.Reith, Ed., Humana Press, Totowa, N.J. (in press). (2002)

2001 Fiscal Year Annual Research Report

上皮細胞におけるアミノ酸・薬物のベクトル輸送形成機構の解析

Principal Investigator

杉山 雄一 東京大学, 大学院・薬学系研究科, 教授 (80090471)

Research Products

[Publications] J.Shoda et al.: "The expression levels of plasma membrane transporters in the cholestatic liver of patients undergoing biliary drainage and their association with the impairment of biliary secretory function"Am. J. Gastroenterol. 96. 3368-3378 (2001)

[Publications] J.Shoda et al.: "Etiologic significance of defects in cholesterol, phospholipid, and bile acid metabolisn in the liver of patients with intrahepatic calculi"Hepatology. 33. 1194-1205 (2001)

[Publications] H Tsuganezawa et al.: "A new member of the HCO3-transporter superfamily is an apical anion excanger of b-Intercalated Cells in the kidney"J. Biol. Chem.. 276. 8180-8189 (2001)

[Publications] K.Mizoguchi et al.: "Human cystinuria-related transporter : localization and functional characterization"Kidney Int. 59. 1821-1833 (2001)

[Publications] DK Kim et al.: "Expression cloning pf a Na+-independent aromatic amino acid transporter with structural similarity to H+/monocarboxylate transporters"J. Biol. Chem.. 276. 17221-17228 (2001)

[Publications] SH Cha et al.: "Identification and characterization of human organic anion transporter 3expressing predominantly in the kidney"Mol. Pharmacol.. 59. 1277-1286 (2001)

[Publications] JY Kim et al.: "Human cystine/glutamate transporter : cDNA cloning and upregulation by oxidative stress in glioma cells"Biochim. Biophys. Acta. 1512. 335-344 (2002)

[Publications] O.Yanagida et al.: "Human L-type amino acid transporter 1 (LAT1) : characterization of function and expression in tumor cell lines"Biochim. Biophys. Acta. 1514. 291-302 (2001)

[Publications] T.Nii et al.: "Molecular events involved in up-regulating human Na+-independent neutral amino acid transporter LAT1 during T-cell activation"Biochem. J.. 358. 693-704 (2001)

[Publications] A.Chairoungdua et al.: "Identification and characterization of a novel member of theheterodimeric amino acid transporter family presumed to be associated with an unknown heavy chain"J. Biol. Chem.. 276. 49390-49399 (2001)

[Publications] S.Tamai et al.: "Expression of L-type amino acid transporter 1 in a rat model of liver metastasis : positive correlation with tumor size"Cancer Detect. Prev.. 25. 439-445 (2001)

[Publications] K.Suzuki et al.: "Transientupregulation of the glial glutamate transporter GLAST in response to fibrobla growth factor, insulin-like growth factor and epidermal growth factor in cultured astrocytes"J. Cell Sci.. 114. 3717-3725 (2001)

[Publications] H.Ohkame et al.: "Expression of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in liver tumor lesionsof rat models"J. Surg. Oncol.. 78. 265-271 (2001)

[Publications] DK Kim et al.: "The human T-type amino acid transporter 1 : characterization, gene organization and chromosomallocation"Genomics. 79. 95-103 (2001)

[Publications] H.Uchino et al.: "Transport of amino acid-related compounds mediated by L-type amino acid transporter 1 (LAT1) : Insights into the mechanisms of substrate recognition"Mol. Pharmacol.. (in press). (2002)

[Publications] H.Matsuo et al.: "Idenitification of a novel Na+-independent acidic amino acid transporter with structural similarity to the member of helerodimeri amino acid transporter family associated with unknown heavy chains"Biol. Chem.. (in press). (2002)

[Publications] 金井好克: "アミノ酸トランスポーター、Annual Review腎臓2001"伊藤克己、浅野泰、遠藤仁、御手洗哲也、東原英二編 中外医学社. 8 (2001)

[Publications] 金井好克: "多選択性アミノ酸トランスポーターファミリー(LATファミリー、蛋白質核酸酵素46)"共立出版. 9 (2001)

[Publications] 金井好克: "小腸および腎におけるアミノ酸吸収を担うトランスポーター(医学のあゆみ、199)"医歯薬出版. 5 (2001)

[Publications] Y.Kanai, H.Endou: "Heterodimeric amino acid transporters molecular biology and pathological and pharmacological relevance"Curr. Drug Metab. 2. 16 (2001)

[Publications] Y Kanai et al.: "The high-affinityglutamate neutral amino-acid transporter family : structure, function, and physiological relevance"In"Neurotransmitter transporters", M.E.A.Reith, Ed., Humana Press, Totowa, N.J. (in press). (2002)

杉山雄一東京大学, 大学院・薬学系研究科, 教授 (80090471)

[Publications] 金井好克: "アミノ酸トランスポーター、Annual Review腎臓2001"伊藤克己、浅野泰、遠藤仁、御手洗哲也、東原英二編中外医学社. 8 (2001)