title: The N-terminal region of organic anion transporting polypeptide 1B3 (OATP1B3) plays an essential role in regulating its plasma membrane trafficking.
abstract: Organic anion transporting polypeptide 1B3 (OATP1B3) is a major influx transporter mediating the hepatic uptake of various endogenous substrates as well as clinically important drugs such as statins and anticancer drugs. However, molecular mechanisms controlling the membrane trafficking of OATP1B3 have been largely unknown. Several reports recently indicated the presence of a distinct, cancer-type OATP1B3 variant lacking the N-terminal 28 amino acids compared to OATP1B3 expressed in non-malignant hepatocytes. Interestingly, the cancer-type OATP1B3 variant is located predominantly in the cytoplasm, implicating the involvement of the N-terminal region of OATP1B3 in its membrane trafficking. In the current study, we set out to experimentally validate the importance of the N-terminal region of OATP1B3 and to identify responsible sequence motif(s) in that region. A number of truncation or point mutants of OATP1B3 were transiently expressed in HEK293T, HCT-8 or MDCK II cells and their expression in cytoplasmic and surface membrane fractions were analyzed by immunoblotting. Our results indicated that the N-terminal sequence of OATP1B3, in particular, at the amino acid positions between 12 and 28, may be indispensable in its membrane trafficking. Moreover, our results using a fusion construct indicated that the first 50 amino acids of OATP1B3 are sufficient for its membrane localization. The importance of the N-terminal region in membranous localization was shared among the other OATP1B subfamily members, OATP1B1 and rat Oatp1b2. Our efforts to identify the responsible amino acid(s) or structure motif(s) in the N-terminal region did not pinpoint individual amino acids or motifs with putative secondary structures. Our current findings however demonstrate that the N-terminal region is important for the membrane localization of the OATP1B subfamily members and should facilitate future investigations of the mechanisms involved in the regulation and membrane trafficking of these important transporter proteins.
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-Gu, Seoul, Republic of Korea.
authors.forename: Se-Eun
authors.initials: SE
authors.lastname: Chun
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY, USA.
authors.forename: Nilay
authors.initials: N
authors.lastname: Thakkar
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-Gu, Seoul, Republic of Korea.
authors.forename: Yunseok
authors.initials: Y
authors.lastname: Oh
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-Gu, Seoul, Republic of Korea.
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Park
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-Gu, Seoul, Republic of Korea.
authors.forename: Songhee
authors.initials: S
authors.lastname: Han
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-Gu, Seoul, Republic of Korea.
authors.forename: Gongmi
authors.initials: G
authors.lastname: Ryoo
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-Gu, Seoul, Republic of Korea.
authors.forename: Hyunggu
authors.initials: H
authors.lastname: Hahn
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-Gu, Seoul, Republic of Korea.
authors.forename: Sang Hyun
authors.initials: SH
authors.lastname: Maeng
authors.affiliation: Department of Biological Sciences, Konkuk University, Seoul, Republic of Korea.
authors.forename: Young-Ran
authors.initials: YR
authors.lastname: Lim
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-Gu, Seoul, Republic of Korea.
authors.forename: Byung Woo
authors.initials: BW
authors.lastname: Han
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-Gu, Seoul, Republic of Korea. Electronic address: wooin.lee@snu.ac.kr.
authors.forename: Wooin
authors.initials: W
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D051379:Mice; D029362:Organic Anion Transporters, Sodium-Independent / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D017354:Point Mutation; D021381:Protein Transport; D051381:Rats; D017386:Sequence Homology, Amino Acid; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D029362:Organic Anion Transporters, Sodium-Independent; C421209:SLCO1B3 protein, human; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3
keywords: Membrane trafficking; N-terminal region; OATP; OATP1B3; Transporter