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Organic Anion Transporting Polypeptide 1B3 can Form Homo‐ and Heterodimers
Author(s) -
Zhang Yuchen,
Hagenbuch Bruno
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.939.5
Subject(s) - hek 293 cells , chemistry , immunoprecipitation , organic anion transporter 1 , transfection , transmembrane domain , cytoplasm , transporter , extracellular , organic anion transporting polypeptide , microbiology and biotechnology , biochemistry , amino acid , biology , receptor , gene
OATP1B3 is a 12 transmembrane domain protein expressed at the basolateral membrane of human hepatocytes where it mediates the uptake of numerous drugs and many endogenous compounds. Previous western blot results suggest the formation of OATP1B3 multimers. In order to better understand post‐translational regulation and its effects on the mechanism of OATP1B3‐mediated transport, we investigated the oligomerization status of OATP1B3. We used transient transfection of C‐terminally His‐, FLAG‐ or HA‐tagged OATP1B3 and OATP1B1 in HEK293 cells, co‐immunoprecipitation with and without cross‐linking and a Proximity Ligation Assay (Duolink PLA) to detect interactions between the different constructs. Despite similar transport rates for estradiol‐17β‐glucuronide and estrone‐3‐sulfate, immunofluorescence experiments demonstrated that in contrast to wild‐type, His‐ and FLAG‐tagged OATP1B3 where the C‐terminal end is on the cytoplasmic side of the membrane, the C‐terminal end of HA‐tagged OATP1B3 is extracellular. After cross‐linking with DTSSP anti‐FLAG antibodies were able to pull down FLAG‐tagged OATP1B3 (positive control) but also co‐transfected His‐ or HA‐tagged OATP1B3, demonstrating that OATP1B3 can form homodimer and suggesting that the C‐terminal part of the protein is not involved in the dimerization. Using the Duolink PLA technique, we could confirm co‐localization of His‐ and FLAG‐tagged OATP1B3 in transfected HEK293 cells. In addition, we were also able to detect OATP1B3‐FLAG OATP1B1‐His co‐localization suggesting that OATP1B3 not only homo‐dimerizes but also hetero‐dimerizes with OATP1B1. These findings suggest that OATP1B3 and OATP1B1 can form dimers and that the two OATPs might be co‐regulated at the post‐translational level.