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.

Post-translational regulation of the major drug transporters in the families of organic anion transporters and organic anion–transporting polypeptides

“…It is now known that the positive cytoplasmic immunostaining was from the cancer-type OATP1B3 (674 aa, lacking the Nterminal 28 amino acids compared to the liver-type OATP1B3 protein of 702 aa) (40). Using the Nterminal truncation mutants, a follow-up study informed that the N-terminal sequence of OATP1B3 (in particular, the amino acid positions between 12 and 18 within the region lacking in the cancer-type OATP1B3) is important for membrane trafficking of OATP1B3 (128). The structural motifs or individual amino acids responsible for the trafficking of OATP1B3 were not, however, identified in that region.…”

Regulation of Organic Anion Transporting Polypeptides (OATP) 1B1- and OATP1B3-Mediated Transport: An Updated Review in the Context of OATP-Mediated Drug-Drug Interactions

“…Compared to wild-type OATP1B3 that is highly expressed in the liver, the cancer-type OATP1B3 lacks an N-terminus encoding region. The putative protein expression of the cancer-type OATP1B3 is primarily expressed in the cytosol and has minimal transport function when expressed exogenously in cancer cell lines [ 109 , 110 ].…”

Characterization of Liver- and Cancer-type-Organic Anion Transporting Polypeptide (OATP) 1B3 Messenger RNA Expression in Normal and Cancerous Human Tissues

“…Lt-OATP1B3 is expressed on the plasma membrane in human liver tissue [8], in cultured human hepatocytes [22], and in the HCT8 colon cancer cell line, when expressed exogenously [17]. However, Ct-OATP1B3 has been shown to be predominantly expressed in the cytosol when transfected into HCT8, MDCK II and HEK293T cells [17, 23].…”