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.
Highlights
Organic anion transporting polypeptides (OATPs) encoded by solute carrier transporter (SLC) genes are a family of membrane transporter proteins that are widely expressed in human tissues
The interaction between PDZ proteins and human organic anion transporting polypeptide 1A2 (OATP1A2) was identified by yeast two-hybrid library screening [34], but the functional significance has not been established
N-Flag tagged OATP1A2 and N-myc tagged PDZK1 and NHERF1 constructs for use in this study. This enabled the use of the highly specific anti-flag and anti-myc antibodies in the analysis of OATP1A2/PDZ protein interactions
Summary
Organic anion transporting polypeptides (OATPs) encoded by solute carrier transporter (SLC) genes are a family of membrane transporter proteins that are widely expressed in human tissues. OATPs are regulated by interactions with chaperones that include the PDZ (PSD95, D1g and ZO1) domaincontaining proteins [26,27,28]. These proteins interact with small canonical sequences of 3–4 amino acid residues within C-termini of target proteins to direct and anchor the latter to specific regions of the cell [29]. The PDZ proteins PDZK1 and NHERF1 have been shown to regulate the subcellular trafficking of certain transporters and to be essential for optimal transport function [30,31,32]. A recent study implicated PDZK1 in the selective recruitment of microtubule-based motor proteins that mediate murine Oatp1a1 trafficking to the plasma membrane [33]
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