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

Abstract

Previous studies identified a family of organic anion transport proteins (OATPs), many of which have C-terminal PDZ binding consensus sequences. In particular, the C-terminal four amino acids of Oatp1a1, a transporter on rat and mouse hepatocytes, comprise a consensus binding site for PDZK1. In PDZK1 knockout mice and in transfected cells where PDZK1 expression was knocked down, Oatp1a1 accumulates in intracellular vesicles. The present study tests the hypothesis that Oatp1a1 traffics to and from the cell surface in vesicles along microtubules, and that PDZK1 guides recruitment of specific motors to these vesicles. Oatp1a1-containing vesicles were prepared from wild-type and PDZK1 knockout mice. As seen by immunofluorescence, kinesin-1, a microtubule plus-end directed motor, was largely associated with vesicles from wildtype mouse liver, whereas dynein, a minus-end directed motor, was largely associated with vesicles from PDZK1 knockout mouse liver. Quantification of motility on directionally marked microtubules following addition of 50 mM ATP showed that wild-type vesicles moved equally toward the plus and minus ends whereas PDZK1 knockout vesicles moved predominantly toward the minus end, consistent with net movement toward the cell interior. These studies provide a novel mechanism by which PDZK1 regulates intracellular trafficking of Oatp1a1 by recruiting specific motors to Oatp1a1-containing vesicles. In the absence of PDZK1, Oatp1a1-containing vesicles cannot recruit kinesin-1 and associate with dynein as a predominant minus-end directed motor. Whether this is a result of direct interaction of the Oatp1a1 cytoplasmic domain with dynein or with a dynein-containing protein complex remains to be established.

Organic Anion Uptake by Hepatocytes

“…10) (23). To examine the hypothesis that subcellular trafficking of oatp1a1 required movement of oatp1a1-containing vesicles on microtubules, in vitro studies of microtubule-based motility of these vesicles was performed (191). In hepatocytes, microtubules are organized with their plus ends near the cell surface, and minus ends within the cell at a microtubule organizing center (132).…”

“…The majority of oatp1a1-containing vesicles from wild-type mice colocalized with PDZK1 (Fig. 12) (191). Vesicles were also associated with specific microtubule-based motors.…”

“…Of note is the fact that vesicles prepared from wild type and PDZK1 knockout mice differed in the distribution of microtubule-based motors (Fig. 12) (191). Kinesin-1, a microtubule-based plus end motor was highly associated with wild-type vesicles, with little associated with PDZK1 knockout vesicles.…”

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

“…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.…”

“…Evolutionarily, the OATPs are poorly conserved and orthologs in rodents may not exist in humans, making findings from studies performed in animal models difficult to translate clinically. Although multiple studies indicate that NHERF proteins play a role in regulating rodent Oatps (Wang et al, 2005(Wang et al, , 2014Sugiura et al, 2010;Choi et al, 2011), there is little information regarding how NHERF proteins affect human OATP isoforms.…”