Determinants of Subcellular Trafficking of Organic Anion Transport Protein 1a1 (oatp1a1)

Abstract

Uptake of organic anionic compounds from the blood is a major function of the liver. These include endogenous compounds such as bilirubin and bile acids as well as xenobiotics such as drugs and toxins. Transporters that are expressed on the basolateral plasma membrane of hepatocytes mediate this process. Organic anion transporting polypeptides (rodents: Oatps; human: OATPs) are a family of sodium independent solute carriers that take up a broad spectrum of substrates into the liver, in exchange for intracellular bicarbonate.;Little is known regarding mechanisms regulating the subcellular distribution of oatps. Perturbation of its normal cell surface localization can obviate the function of an oatp, potentially leading to drug toxicity due to alterations in uptake and metabolism. Many Oatps, including oatp1a1, the first identified member of the oatp family, have a C-terminal PDZ consensus binding motif that interacts with PDZK1. Oatp1a1 also has serine residues upstream of the PDZ consensus binding motif (S634, S635) that can be phosphorylated and that are highly conserved among other members of the oatp family. To examine the effect of phosphorylation of these serine residues on oatp1a1 subcellular trafficking, plasmids were prepared in which these serines were mutated either to glutamic acid (E634E635, phosphomimetic) or alanine (A634A635, nonphosphorylatable). Distribution of both oatp1a1AA and oatp1a1EE was largely intracellular in transfected HEK 293T cells. Co-transfection with a plasmid encoding PDZK1 revealed that oatp1a1AA was now expressed largely on the cell surface while oatp1a1EE remained intracellular. In addition, BSP transport activity for oatp1a1AA significantly increased in the presence of PDZK1 without altering the affinity of the substrate for oatp1a1 as shown by unchanged Km. 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 oatp1a1EE internalizes quickly as compared to oatp1a1AA. To examine a physiologic role for phosphorylation on 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. As phosphorylation of oatp1a1 can occur quickly, this provides a mechanism for fast regulation of the distribution of oatp1a1 between the cell surface and intracellular vesicular pools. The proteins and motor molecules that mediate these trafficking events are unknown at present, but represent an important area for future study.