Research Article: Organic anion transporting polypeptide 1B3 can form homo- and hetero-oligomers

Date Published: June 23, 2017

Publisher: Public Library of Science

Author(s): Yuchen Zhang, Kelli H. Boxberger, Bruno Hagenbuch, Hendrik W. van Veen.


OATP1B3 is a 12 transmembrane domain protein expressed at the basolateral membrane of human hepatocytes where it mediates the uptake of numerous drugs and endogenous compounds. Previous western blot results suggest the formation of OATP1B3 multimers. In order to better understand the function of OATP1B3 under normal physiological conditions, we investigated its oligomerization status. We transiently transfected OATP1B3 with a C-terminal His-, FLAG- or HA-tag in HEK293 cells and used co-immunoprecipitation and a Proximity Ligation Assay to detect interactions between the different constructs. All three constructs retained similar transport rates as wild-type OATP1B3. Immunofluorescence experiments indicated 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, anti-FLAG antibodies were able to pull down FLAG-tagged OATP1B3 (positive control) and co-transfected His- or HA-tagged OATP1B3, demonstrating the formation of homo-oligomers and suggesting that the C-terminal part is not involved in oligomer formation. We confirmed co-localization of His- and FLAG-tagged OATP1B3 in transfected HEK293 cells with the Proximity Ligation Assay. Transport studies with a non-functional OATP1B3 mutant suggest that the individual subunits and not the whole oligomer are the functional units in the homo-oligomers. In addition, we also detected OATP1B3-FLAG co-localization with OATP1B1-His or NTCP-His, suggesting that OATP1B3 also hetero-oligomerizes with other transport proteins. Using the Proximity Ligation Assay with transporter specific antibodies, we demonstrate close association of OATP1B3 with NTCP in frozen human liver tissue. These findings demonstrate that OATP1B3 can form homo- and hetero-oligomers and suggest a potential co-regulation of the involved transporters.

Partial Text

Organic anion transporting polypeptide (OATP) 1B3 is a multispecific drug uptake transporter belonging to the SLCO superfamily [1]. Under normal physiological conditions, OATP1B3 and the closely related OATP1B1 are selectively expressed at the sinusoidal membrane of human hepatocytes [2]. The two drug transporters are responsible for the uptake of numerous endogenous compounds, such as bile acids and hormones, and many xenobiotics, including various drugs, into hepatocytes [3]. In addition, conjugated bile acids, including taurocholate or glycocholate, are transported into human hepatocytes via Na+/Taurocholate Cotransporting Polypeptide (NTCP) [4] which has recently also been identified as the receptor for human hepatitis B and D virus [5].

In this study, we demonstrated that the important hepatocellular drug transporter OATP1B3 can form homo-dimers or -oligomers. Within these dimers/oligomers, an individual OATP1B3 seems to be the functional unit because co-transfections with a nonfunctional OATP1B3-K41C mutant did not affect uptake of E3S and E17βG. Furthermore, we also established that OATP1B3 can form hetero-dimers/oligomers in HEK293 cells with other hepatocellular transporters like OATP1B1 and NTCP, and we verified the interaction with NTCP using frozen human liver sections.




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