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.

Oatp1a1 Requires PDZK1 to Traffic to the Plasma Membrane by Selective Recruitment of Microtubule-Based Motor Proteins

“…Further studies were performed in 293T cells expressing oatp1a1 in the presence or absence of PDZK1 (Choi et al, 2011). Similar to results in mice, in the absence of PDZK1, distribution of oatp1a1 was largely intracellular.…”

“…Many of these compounds are poorly soluble in aqueous solution, and circulate bound to proteins such as albumin (Wolkoff et al, 1987;Choi et al, 2009). Uptake involves extraction from the protein carrier and is mediated by a specific transporter(s) on the basolateral (sinusoidal) surface of the cell (Meier et al, 1997;Wolkoff, 2012).…”

“…These proteins have been shown to play a role in clearance of anionic drugs from the circulation, and mutations have been associated with several reports of toxicity in patients due to reduced clearance of drug by the liver (Link et al, 2008;Clarke and Cherrington, 2012;Nakanishi and Tamai, 2012). Many of the members of the OATP family have been found to have PDZ binding consensus sequences at their C termini (Choi et al, 2009). Previous studies of the rat oatp1a1 transporter showed that its C-terminal four amino acids (KTKL) comprise a consensus site that binds PDZK1 (Wang et al, 2005;Choi et al, 2011).…”

“…Many of the members of the OATP family have been found to have PDZ binding consensus sequences at their C termini (Choi et al, 2009). Previous studies of the rat oatp1a1 transporter showed that its C-terminal four amino acids (KTKL) comprise a consensus site that binds PDZK1 (Wang et al, 2005;Choi et al, 2011). The mouse homolog has the same C-terminal sequence, and traffics poorly to the hepatocyte surface in PDZK1 knockout mice, accumulating in vesicular structures within cells (Wang et al, 2005).…”

Hypoxia/Reoxygenation Stress Signals an Increase in Organic Anion Transporting polypeptide 1a4 (Oatp1a4) at the Blood–Brain Barrier: Relevance to CNS Drug Delivery

“…41 Furthermore, Oatp transporters that possess a consensus PDZ motif at their C-terminus (i.e., Oatp1a1) have been shown to exhibit subcellular trafficking properties in human embryonic kidney cells that were transfected with the transporter of interest. 42 While Oatp1a4 lacks a consensus PDZ domain, we believe that protein trafficking is likely involved in regulating Oatp1a4 expression at the brain microvascular endothelium after an H/R insult. Studies to assess 'pools' of 'relocating Oatp1a4 transporter' in the context of H/R stress are currently being undertaken in our laboratory.…”