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The N‐terminal Region of Organic Anion Transporting Polypeptide 1B3 (OATP1B3) Plays an Essential Role in Regulating its Plasma Membrane Trafficking
Author(s) -
Chun SeEun,
Thakkar Nilay,
Park Ji Eun,
Han Songhee,
Maeng Sang Hyun,
Lim YoungRan,
Machado Daniel,
Lee Wooin
Publication year - 2016
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.30.1_supplement.935.10
Subject(s) - hek 293 cells , amino acid , organic anion transporting polypeptide , organic anion transporter 1 , chemistry , transfection , point mutation , gene , biochemistry , microbiology and biotechnology , mutation , biology , transporter
Organic anion transporting polypeptide 1B3 (OATP1B3) mediates the hepatic uptake of various endogenous and xenobiotic compounds and it is shown to influence the disposition of clinically important drugs including statins and paclitaxel. Recently, we and others reported the expression of cancer‐type OATP1B3 which arises from the use of an alternative transcription initiation site and lacks the N‐terminal 28 amino acids compared to liver‐type OATP1B3. We also noted that cancer‐type OATP1B3 displays mainly a cytoplasmic expression pattern clearly different from a membranous expression pattern of liver‐type OATP1B3, in both clinical cancer tissue specimens and cancer cell lines. In this study, we set out to verify the importance of the N‐terminal region of OATP1B3 in its membrane trafficking and to identify responsible sequence motif(s) in that region. We first confirmed our previous findings by transfecting the expression plasmid for cancer‐type or liver‐type OATP1B3 fused with the C‐terminal myc tag using HCT8 and HEK293T cells. Subsequently, we prepared a number of expression plasmids harboring point mutations at various amino acid positions (K or R with a positive charge, S with potential for phosphorylation, ASSE tetrapeptide with potential for forming a beta‐turn) within the N‐terminal 28 amino acids of OATP1B3 via site‐directed mutagenesis. These constructs were transiently transfected in HEK293T cells and compared for their cellular localization by immunoblotting analyses of fractionated samples. Our results showed that various point mutations within the N‐terminal 28 amino acids do not alter the extent of membrane trafficking of OATP1B3. As an alternative approach, we prepared a construct for the N‐terminal 50 amino acid sequence of OATP1B3 fused with the C‐terminal myc tag and we found that the resulting fusion protein is effectively localized to the plasma membrane. Similar experiments were carried out for OATP1B1, a closely related member to OATP1B3 and the results showed that the importance of the N‐terminal region in regulating membrane trafficking is also applicable to OATP1B1. Further investigations are ongoing to identify essential amino acid residue(s)/motif(s) for membrane trafficking within the N‐terminal 50 amino acids of OATP1B3 and OATP1B1. Our current findings may provide important mechanistic insights into the regulation of OATP1B3 and OATP1B1 expression and membrane localization in physiological and pathological conditions.