Organic anion-transporting polypeptides are members of the solute carrier (SLC) family and key determinants for the transmembrane transport of a wide variety of compounds. OATP1B1 is predominantly expressed at the basolateral membrane of human hepatocytes and play an important role in drug clearance from the body. It has been demonstrated to be responsible for the hepatic uptake of various drugs. Computer-based hydropathy analysis predicted several putative phosphorylation sites at the amino and carboxyl termini and at intracellular loop 3 of OATP family members. Therefore, their transport functions may be regulated by phosphorylation. Previous studies have demonstrated that uptake function of OATP2B1 and OATP1A2 is regulated by protein kinase C (PKC). In the present study, we treated HEK293 cells stably expressing OATP1B1 with different PKC modulators and measured their transport activity for prototypic substrate estrone-3-sulfate. It was found that OATP1B1 uptake function was reduced upon PKC activation. Further studies indicated that PKC may affect OATP1B1 activity through regulation of the cell surface protein level. Moreover, we found out that PKC activator phorbol 12-myristate 13-acetate (PMA) not only affects the internalization of OATP1B1 but its recycling as well. Immunocytochemistry analysis revealed that internalized OATP1B1 co-localized with early and recycling endosomal markers and the co-localization of OATP1B1 with recycling endosome is dependent on PKC activation. Taken together, our present study demonstrated that PKC regulates the function of OATP1B1 by affecting internalization and recycling of the transporter protein.

Organic anion-transporting polypeptides play important roles in the uptake of various endogenous and exogenous compounds. It has been proposed that OATP family members, as membrane proteins, may form oligomers. However, oligomerization status of OATPs is still largely unclear. In the present study, HEK293 cells stably expressing OATP1B1 were generated to investigate the oligomerization status of the transporter. Chemical cross-linking and coimmunoprecipitation experiments revealed that OATP1B1 may form homo-oligomers, possibly through disulfide bonds. When wild-type OATP1B1 was coexpressed with a loss-of-function mutant W258A, cells showed reduced uptake of prototypic substrate estrone-3-sulfate (ES). Interestingly, such a coexpression did not affect OATP1B1 transport activity of high concentrations ES, implicating that oligomerization status may affect only the high affinity component of ES. OATP1B1 possesses three GXXXG motifs that have been associated with protein dimerization in other membrane proteins. When glycine residues were replaced with alanine, G219A and G393A showed drastically reduced uptake function. Further studies revealed that G219A has a similar association capability to that of the wild-type, while mutation at Gly393 may affect oligomerization status of the transporter. Kinetic analysis showed that both G219A and G393A have a dramatically reduced V for ES uptake. K of G219A was increased while that of G393A exhibited a decreased value for high affinity component of ES binding. Our studies demonstrated that OATP1B1 may function as oligomers in the high affinity site of ES while acting as monomers for the low affinity binding component of the substrate.

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ObjectiveThe family of organic anion transporting polypeptides (OATPs, gene symbol SLCO) mediates sodium‐independent transport of a wide variety of endogenous and exogenous compounds. OATPs are considered as key players in drug absorption, distribution and excretion due to their broad substrate specificity, wide tissue distribution and involvement in drug‐drug interactions. OATP1B1 is specifically located at the basolateral membrane of human hepatocytes and serves a crucial role in drug clearance from the body. Previous studies have shown that transmembrane domains (TMs) of OATP1B1 are essential structural features for proper function of the transporter.MethodsAlanine‐scanning of the putative transmembrane domain 1 and truncation of the N‐terminus of OATP1B1 were carried out, and the uptake function and protein expression of mutants were analyzed.ResultsTwo positively charged amino acid residues, K41 and K49, were identified to be important for uptake function of the transporter protein. Alanine substitution of K41 altered substrate binding at the high affinity component; while K49A exhibited increased Km value at the low affinity site. In addition, our study of the N‐terminal residues of OATP1B1 found out that residues ranging from 19–27 are essential for protein stability and uptake function of the transporter protein.ConclusionsThe N‐terminus region of OATP1B1 is important for maintaining proper function and protein expression level of the transporter.Support or Funding InformationThis work was supported by the National Natural Science Foundation of China Grants [81373473] to Mei Hong.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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