Interaction with PDZK1 Is Required for Expression of Organic Anion Transporting Protein 1A1 on the Hepatocyte Surface

Abstract

Although many organic anion transport protein (Oatp) family members have PDZ consensus binding sites at their C termini, the functional significance is unknown. In the present study, we utilized rat Oatp1a1 (NM_017111) as a prototypical member of this family to examine the mechanism governing its subcellular trafficking. A peptide corresponding to the C-terminal 16 amino acids of rat Oatp1a1 was used to affinity-isolate interacting proteins from rat liver cytosol. Protein mass fingerprinting identified PDZK1 as the major interacting protein. This was confirmed by immunoprecipitation of an Oatp1a1-PDZK1 complex from cotransfected 293T cells as well as from native rat liver membrane extracts. Oatp1a1 bound predominantly to the first and third PDZ binding domains of PDZK1, whereas the high density lipoprotein receptor, scavenger receptor B type I binds to the first domain. Although it is possible that PDZK1 forms a complex with these two integral membrane proteins, this did not occur, suggesting that as yet undescribed factors lead to selectivity in the interaction of these protein ligands with PDZK1. Oatp1a1 protein expression was near normal in PDZK1 knock-out mouse liver. However, it was located predominantly in intracellular structures, in contrast to its normal basolateral plasma membrane distribution. Plasma disappearance of the Oatp1a1 ligand [35S]sulfobromophthalein was correspondingly delayed in knock-out mice. These studies show a critical role for oligomerization of Oatp1a1 with PDZK1 for its proper subcellular localization and function. Because its ability to transport substances into the cell requires surface expression, this must be considered in any assessment of physiologic function.

Pharmacokinetics and Hepatic Uptake of Eltrombopag, a Novel Platelet-Increasing Agent

“…The CL tot of ELT in pdzk1(Ϫ/Ϫ) mice was not completely reduced but was significantly lower than that in wild-type mice (77.3 Ϯ 4.9 and 55.2 Ϯ 3.9 in wild-type and pdzk1(Ϫ/Ϫ) mice, respectively; n ϭ 4 for each). PDZK1 regulates expression and/or localization of various transporters in vivo (Wang et al, 2005;Sugiura et al, 2008). In particular, Oatp1a1 is down-regulated in pdzk1(Ϫ/Ϫ) mice (Wang et al, 2005).…”

“…Plasmid DNA encoding human OATP1B1, OATP1B3, OATP2B1, organic cation transporter (OCT) 1, or OCTN2, all of which had been subcloned in pcDNA3 vector, was transiently transfected into HEK293/PDZK1 cells according to the calcium phosphate precipitation method. This HEK293/ PDZK1 cell line was used because the PDZ adaptor protein PDZK1 increases the transport activity of various xenobiotic transporters in vitro (Sugiura et al, 2006) and regulates expression of basolateral membrane transporter in hepatocytes in vivo (Wang et al, 2005), although a possible interaction of PDZK1 with OATP1B1, OATP1B3, OATP2B1, or OCT1 has not yet been examined. At 48 h after transfection, the cells were harvested and suspended in transport buffer, and uptake studies were performed by the silicon oil layer method as described above using isolated hepatocytes.…”

Hepatobiliary transporters and drug‐induced cholestasis†

“…[21][22][23] Furthermore, PDZK1 was demonstrated to be a critical determinant for the proper subcellular localization and function of rat Oatp1a1. 24 The transcriptional regulation of basolaterally expressed MRPs is not fully elucidated. Studies in mice support the notion that Mrp3 and Mrp4 are induced through a pregnane X receptor-mediated pathway.…”

PDZK1 Regulates Two Intestinal Solute Carriers (Slc15a1 and Slc22a5) in Mice

“…However, most of the data suggesting such functional regulation by PDZK1 were obtained in in vitro double-transfection studies with cultured cell lines. Although Wang et al (2005) recently showed an essential role of PDZK1 in basolateral expression of organic anion transporting peptide 1a1 in the liver, the in vivo evidence for a fundamental role of PDZK1 as a regulatory mechanism for xenobiotic transporters is quite limited in small intestine.…”