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Research ArticleArticle

Functional Analysis of the Extracellular Cysteine Residues in the Human Organic Anion Transporting Polypeptide, OATP2B1

Emanuel Hänggi, Anne Freimoser Grundschober, Simone Leuthold, Peter J. Meier and Marie V. St-Pierre
Molecular Pharmacology September 2006, 70 (3) 806-817; DOI: https://doi.org/10.1124/mol.105.019547
Emanuel Hänggi
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Anne Freimoser Grundschober
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Simone Leuthold
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Peter J. Meier
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Marie V. St-Pierre
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Abstract

Organic anion transporting polypeptide (OATP) superfamily member 2B1 (OATP2B1) mediates the uptake of steroid hormone precursors and selected drugs in the placenta, liver, mammary gland, brain, and intestine. This action is modulated by sulfhydryl reagents. Common to all OATPs is a large extracellular loop between transmembrane domains IX and X with 10 conserved cysteines. To elucidate the structure-function relationship of this cysteine rich ectodomain, a truncated OATP2B1 lacking 10 extracellular cysteines (OATP2B1Δ489-557) and 10 OATP2B1 mutants containing individual Cys-to-Ala substitutions were generated and expressed in CHO-K1 cells. The immunolocalization, cell-surface expression, transport activity, and free cysteine labeling with N-biotinoylaminoethylmethane-thiosulfonate of mutant proteins and wild-type OATP2B1 were compared. OATP2B1Δ489-557 accumulated intracellularly. Nine Cys-to-Ala substitutions, C489A, C495A, C504A, C516A, C520A, C539A, C541A, C553A, and C557A, were misprocessed, appearing predominantly as core-glycosylated, 60-kDa proteins and as 180-kDa complexes. Only C493A was a fully glycosylated 75-kDa protein expressed at the cell surface. Thapsigargin partially corrected the misprocessing of mutants. Compared with OATP2B1, C493A and C557A transported estrone-3-sulfate and dehydroepiandrosterone sulfate less efficiently, whereas all other mutants were functionally impaired. MTSEA labeled free cysteines in all Cys-to-Ala mutants but not in OATP2B1, suggesting that all 10 extracellular cysteines are normally disulfide-bonded. Our findings show that the trafficking and function of OATP2B1 is vulnerable to changes in the cysteine residues of extracellular loop IX-X.

Footnotes

  • This work was supported by a grant from the University of Zürich Forschungskommission 2003 (to M.V.S.) and grants from the National Science Foundation, Switzerland (3100-67173 to M.V.S. and 31-64140 to P.J.M.).

  • ABBREVIATIONS: OATP, organic anion transporting polypeptide; DHEAS, dehydroepiandrosterone sulfate; PCR, polymerase chain reaction; HA, hemagglutinin A; CHO, Chinese hamster ovary; sulfo-NHS-SS-biotin, sulfonated N-hydroxysuccinimide biotin; MTSEA-biotin, N-biotinoylaminoethyl methanethiosulfonate; PBS, phosphate-buffered saline; PNGase F, peptide N-glycosidase F; DTSSP, 3,3′-dithiobis-[sulfosuccinimidyl-propionate]; BS3, bis[sulfosuccinimidyl]suberate; PAGE, polyacrylamide gel electrophoresis; Ni-NTA, nickel-nitrilotriacetic acid; ALLN, N-acetyl-l-leucyl-l-leucyl-l-norleucinal; ER, endoplasmic reticulum.

  • ↵1 E.H. and A.F.G. contributed equally to this work

    • Received November 2, 2005.
    • Accepted May 22, 2006.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 70 (3)
Molecular Pharmacology
Vol. 70, Issue 3
1 Sep 2006
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Research ArticleArticle

Functional Analysis of the Extracellular Cysteine Residues in the Human Organic Anion Transporting Polypeptide, OATP2B1

Emanuel Hänggi, Anne Freimoser Grundschober, Simone Leuthold, Peter J. Meier and Marie V. St-Pierre
Molecular Pharmacology September 1, 2006, 70 (3) 806-817; DOI: https://doi.org/10.1124/mol.105.019547

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Research ArticleArticle

Functional Analysis of the Extracellular Cysteine Residues in the Human Organic Anion Transporting Polypeptide, OATP2B1

Emanuel Hänggi, Anne Freimoser Grundschober, Simone Leuthold, Peter J. Meier and Marie V. St-Pierre
Molecular Pharmacology September 1, 2006, 70 (3) 806-817; DOI: https://doi.org/10.1124/mol.105.019547
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