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

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 (OATP2B1 ⌬489 -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-biotinoylaminoethylmethanethiosulfonate of mutant proteins and wild-type OATP2B1 were compared. OATP2B1 ⌬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 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.

Human Platelets Express Organic Anion-Transporting Peptide 2B1, an Uptake Transporter for Atorvastatin

“…1A). OATP2B1 glycosylation has been demonstrated (Tamai et al, 2000;Hänggi et al, 2006), and we have obtained similar results in a peptide-N Ϫ -(N-acetyl-␤-glucosaminyl)asparagine amidase assay with platelet OATP2B1 as described before for the recombinant protein (figure not shown). As a measure for the expression level of OATP2B1 in platelets compared with liver, immunoblot results were quantified by densitometric analysis (ImageQuant 5.0), and the relative optical density of the specific bands was calculated.…”

Oatp58Dc contributes to blood-brain barrier function by excluding organic anions from the Drosophila brain

“…The signature sequence spans extracellular loop 3 into transmembrane domain 6. Consistent with the Oatp family, extracellular loop 5 is large and cysteine-rich, containing 10 cysteine residues, similar to other Oatp sequences (23,24,26,38,41). Extracellular loops 2 and 5 contain putative N-linked glycosylation sites, consistent with the Oatp family (Fig.…”

Molecular Characterization of Zebrafish Oatp1d1 (Slco1d1), a Novel Organic Anion-transporting Polypeptide

“…10, A and B). Dimerization through disulfide bonds has been shown for OATP2B1 (52). Considering that cotransfection of Oatp1d1-WT and Oatp1d1-mCitrine showed plasma membrane localization (Fig.…”