title: PDZK1 binding and serine phosphorylation regulate subcellular trafficking of organic anion transport protein 1a1.
abstract: Although perturbation of organic anion transport protein (oatp) cell surface expression can result in drug toxicity, little is known regarding mechanisms regulating its subcellular distribution. Many members of the oatp family, including oatp1a1, have a COOH-terminal PDZ consensus binding motif that interacts with PDZK1, while serines upstream of this site (S634 and S635) can be phosphorylated. Using oatp1a1 as a prototypical member of the oatp family, we prepared plasmids in which these serines were mutated to glutamic acid [E634E635 (oatp1a1(EE)), phosphomimetic] or alanine [A634A635 (oatp1a1(AA)), nonphosphorylatable]. Distribution of oatp1a1(AA) and oatp1a1(EE) was largely intracellular in transfected human embryonic kidney (HEK) 293T cells. Cotransfection with a plasmid encoding PDZK1 revealed that oatp1a1(AA) was now expressed largely on the cell surface, while oatp1a1(EE) remained intracellular. To quantify these changes, studies were performed in HuH7 cells stably transfected with these oatp1a1 plasmids. These cells endogenously express PDZK1. Surface biotinylation at 4°C followed by shift to 37°C showed that oatp1a1(EE) internalizes quickly compared with oatp1a1(AA). To examine a physiological role for phosphorylation in oatp1a1 subcellular distribution, studies were performed in rat hepatocytes exposed to extracellular ATP, a condition that stimulates serine phosphorylation of oatp1a1 via activity of a purinergic receptor. Internalization of oatp1a1 under these conditions was rapid. Thus, although PDZK1 binding is required for optimal cell surface expression of oatp1a1, phosphorylation provides a mechanism for fast regulation of the distribution of oatp1a1 between the cell surface and intracellular vesicular pools. Identification of the proteins and motor molecules that mediate these trafficking events represents an important area for future study.
authors.affiliation: Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Jo H
authors.initials: JH
authors.lastname: Choi
authors.forename: John W
authors.initials: JW
authors.lastname: Murray
authors.forename: Allan W
authors.initials: AW
authors.lastname: Wolkoff
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D018698:Glutamic Acid; D057809:HEK293 Cells; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D027361:Organic Anion Transporters / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine; D013997:Time Factors; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D027361:Organic Anion Transporters; C484659:PDZK1 protein, human; C514896:PDZK1 protein, mouse; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D012694:Serine; D000255:Adenosine Triphosphate; D000409:Alanine