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First published on June 5, 2006; DOI: 10.1124/mol.105.019547

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Received for publication November 3, 2005.
Revised March 22, 2006.
Accepted for publication May 22, 2006.

FUNCTIONAL ANALYSIS OF THE EXTRACELLULAR CYSTEINE RESIDUES IN THE HUMAN ORGANIC ANION TRANSPORTING POLYPEPTIDE, OATP2B1

Emanuel Hanggi 1, Anne Freimoser Grundschober 1, Simone Leuthold 1, Peter J Meier 1, Marie Vivien St-Pierre 1*

1 Division of Clinical Pharmacology and Toxicology, University Hospital Zurich

* Address correspondence to: E-mail: stpierre{at}kpt.unizh.ch

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 ten conserved cysteines. To elucidate the structure-function relationship of this cysteine rich ectodomain, a truncated OATP2B1 lacking ten extracellular cysteines (OATP2B1{Delta}489-557) and ten 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-biotinoylaminoethylmethanethiosulfonate (MTSEA) of mutant proteins and wild-type OATP2B1 were compared. OATP2B1{Delta}489-557 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 to OATP2B1, C493A and C557A transported estrone-3-sulfate and dehydroepiandrosterone sulfate less efficiently, whereas all others mutants were functionally impaired. MTSEA labeled free cysteines in all Cys to Ala mutants but not in OATP2B1, suggesting that all ten 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.


Key words: Organic anion, Structure-activity relationships and modeling, Mutagenesis/Chimeric approaches


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