PDZK1 and NHERF1 Regulate the Function of Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) by Modulating Its Subcellular Trafficking and Stability

Abstract

The human organic anion transporting polypeptide 1A2 (OATP1A2) is an important membrane protein that mediates the cellular influx of various substances including drugs. Previous studies have shown that PDZ-domain containing proteins, especially PDZK1 and NHERF1, regulate the function of related membrane transporters in other mammalian species. This study investigated the role of PDZK1 and NHERF1 in the regulation of OATP1A2 in an in vitro cell model. Transporter function and protein expression were assessed in OATP1A2-transfected HEK-293 cells that co-expressed PDZK1 or NHERF1. Substrate (estrone-3-sulfate) uptake by OATP1A2 was significantly increased to ∼1.6- (PDZK1) and ∼1.8- (NHERF1) fold of control; this was dependent on the putative PDZ-binding domain within the C-terminus of OATP1A2. The functional increase of OATP1A2 following PDZK1 or NHERF1 over-expression was associated with increased transporter expression at the plasma membrane and in the whole cell, and was reflected by an increase in the apparent maximal velocity of estrone-3-sulfate uptake (Vmax: 138.9±4.1 (PDZK1) and 181.4±16.7 (NHERF1) versus 55.5±3.2 pmol*(µg*4 min)−1 in control; P<0.01). Co-immunoprecipitation analysis indicated that the regulatory actions of PDZK1 and NHERF1 were mediated by direct interaction with OATP1A2 protein. In further experiments PDZK1 and NHERF1 modulated OATP1A2 expression by decreasing its internalization in a clathrin-dependent (but caveolin-independent) manner. Additionally, PDZK1 and NHERF1 enhanced the stability of OATP1A2 protein in HEK-293 cells. The present findings indicated that PDZK1 and NHERF1 regulate the transport function of OATP1A2 by modulating protein internalization via a clathrin-dependent pathway and by enhancing protein stability.

Drug Transporters and Na+/H+Exchange Regulatory Factor PSD-95/Drosophila Discs Large/ZO-1 Proteins

“…Several clinically significant SLC and ABC drug transporters harbor a carboxy-terminal PDZ ligand and therefore may bind PDZ proteins. Mounting evidence highlights the impact of PDZ proteins in regulating the membrane abundance, localization, and function of a growing number of drug transporters (Karvar et al, 2014;Park et al, 2014;Zheng et al, 2014). …”

“…Hence, NHERF proteins may have an indirect effect on drug response attributable to their influence on drug absorption and disposition in these important pharmacological barriers. Over the last decade, PDZ proteins have gained considerable attention as post-translational regulators of drug transporter function (Miyazaki et al, 2005;Kato et al, 2006;Noshiro et al, 2006;Sugiura et al, 2006Sugiura et al, , 2008Sugiura et al, , 2010Sugiura et al, , 2011Choi et al, 2011;Park et al, 2014;Wang et al, 2014;Zheng et al, 2014). To highlight these new findings, we review the current knowledge of drug transporter regulation by PDZ proteins and address the association of PDZ proteins with drug disposition and response in select disease states.…”

“…Analogous to the OCT and OAT families of drug transporters, some OATP isoforms harbor a C-terminal PDZ ligand and are binding partners with PDZ proteins (Kato et al, 2004;Wang et al, 2005;Zheng et al, 2014). Multiple PDZ proteins regulate the function of OATP1A2 (Zheng et al, 2014), which is discussed in section IV.…”

“…Individual PDZ proteins may contain multiple PDZ domains as well as other interaction modules such as ezrin-binding domains (EBDs). PDZ proteins lack intrinsic catalytic activity and therefore function primarily as scaffold proteins, where they execute a range of biologic functions that include establishing and maintaining cellular polarity, assembling multiprotein signaling complexes, and anchoring transmembrane proteins, including transporters, to the actin cytoskeleton via interactions with the ERM family of adapters (Ponting et al, 1997;Wang et al, 2007Wang et al, , 2010Wang et al, , 2012Georgescu et al, 2014;Zheng et al, 2014).…”

“…Recently, NHERF1 and NHERF3 were found to regulate OATP1A2 function by modulating protein internalization and enhancing membrane stability. The influence of NHERF1 and NHERF3 on OATP1A2 function was assessed using HEK293 cells that coexpressed NHERF1 or NHERF3 (Zheng et al, 2014). Similar to studies in OATs, estrone-3-sulfate uptake was used as a surrogate for OATP1A2 function.…”