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

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: 138.964.1 (PDZK1) and 181.4616.7 (NHERF1) versus 55.563.2 pmol*(mg*4 min)21 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.This work was supported in part by The National Health and Medical Research Council of Australia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Single nucleotide differences (SNDs) continue to contaminate the dbSNP database with consequences for human genomics and health

10.1002/humu.22735Human Mutation362196-19

Clathrin-dependent internalization of OATP1A2 by PDZK1 and NHERF1.

<p>(A) Disruption of the clathrin-dependent pathway impairs OATP1A2-N-flag internalization. HEK-293 cells over-expressing OATP1A2-N-flag were treated with K⁺ depletion buffer or 10 mM acetic acid for 30 mins, relative to PBS-treated control. Internalization continued at 37°C for 5 or 10 mins as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094712#s2" target="_blank">Materials and Methods</a>,” followed by immunoblotting for OATP1A2-N-flag. (B) Internalized OATP1A2-N-flag as a percentage of the total initial pool of biotinylated OATP1A2-N-flag at the cell surface (means±S.E. of 3 individual experiments). *: Different from control: <i>P</i><0.05. (C) <i>Top panel</i>: immunoblot analysis of OATP1A2-N-flag in HEK-293 cells containing co-expressed PDZK1-N-myc or NHERF1-N-myc after treatment with 10 mM acetic acid for 30 mins, followed by internalization at 37°C for 5 or 10 mins. <i>Bottom panel</i>: Internalized OATP1A2-N-flag as a percentage of the total initial pool of biotinylated OATP1A2-N-flag at the cell surface (means±S.E. of 3 individual experiments). (D) <i>Top panel</i>: immunoblot analysis of OATP1A2-N-flag in HEK-293 cells containing co-expressed PDZK1-N-myc or NHERF1-N-myc after treatment with K⁺ depletion buffer for 30 mins, followed by internalization at 37°C for 5 or 10 mins. <i>Bottom panel</i>: Internalized OATP1A2-N-flag as a percentage of the total initial pool of biotinylated OATP1A2-N-flag at the cell surface (means±S.E. of 3 individual experiments). Blotting for β-actin in separate aliquots of total lysates was used to confirm uniform protein loading.</p

Michaelis-Menten plot of E3S transport kinetics by OATP1A2 with or without co-transfected PDZK1 or NHERF1.

<p>E3S uptake was conducted over a 4-specific uptake by vector transfected cells and was also standardized to the amount of protein in each well. Kinetic parameters were calculated by nonlinear regression. Values are means ± S.E. (n = 3). **: Different from control: <i>P</i><0.01.</p

Association of OATP1A2 with PDZK1 and NHERF1.

<p>(A) co-immunoprecipitation of OATP1A2-N-flag and PDZK1-N-myc or NHERF1-N-myc. HEK-293 cells that co-expressed OATP1A2-N-flag and either PDZK1-N-myc or NHERF1-N-myc were lysed and subjected to immunoprecipitation with anti-flag antibody, followed by immunoblotting with anti-myc antibody. (B) Direct immunoblotting for N-Flag and N-myc tags, respectively, in the lysates of HEK-293 cells co-expressed with OATP1A2-N-flag and either PDZK1-N-myc or NHERF1-N-myc prior to immunoprecipitation.</p

Altered transport function and expression of OATP1A2 in HEK-293 cells in the presence and absence of co-transfected PDZK1 and NHERF1.

<p>(A) Transport of 300 nM [³H] E3S in OATP1A2-transfected HEK-293 cells with or without co-expressed PDZK1 or NHERF1 at 37°C. Values are means ± S.E. (<i>n</i> = 3). **: Different from control (OATP1A2 + vector): <i>P</i><0.01. (B) Western blot analysis of total cellular expression of OATP1A2-N-flag isoforms with or without co-expression of PDZK1 or NHERF1. <i>Top Panel</i>: Cells were lysed and proteins were separated by SDS-polyacrylamide gel electrophoresis, followed by Western blotting with anti-flag antibody. <i>Bottom Panel</i>: After stripping, the blot was reprobed with anti-β-actin antibody. (C) Western blot analysis of cell surface expression of OATP1A2-N-flag with or without co-expression of PDZK1 or NHERF1. Cells were biotinylated, and the labeled cell surface proteins were precipitated with streptavidin beads and separated by gel electrophoresis, followed by Western blotting with anti-flag antibody.</p

Scheme outlining the biotinylation-based internalization and recycling/membrane targeting assays.

<p>(A) Internalization assay. HEK-293 cells co-transfected with OATP1A2-N-flag and PDZK1-N-myc, NHERF1-N-myc or control vector were labeled with NHS-SS-biotin at 4°C. After quenching of residual NHS-SS-biotin with glycine (100 mM) the cells were warmed to 37°C to initialize internalization. At the end of incubations, sodium 2-mercaptoethanesulfonate (50 mM) was added to strip biotinylated, but non-internalized, proteins remaining at the cell surface. Cell lysates were prepared in lysis buffer and 500 µg protein from each sample was applied to streptavidin-agarose beads to capture biotinylated proteins. (B) Recycling/membrane targeting assay. HEK-293 cells co-transfected with OATP1A2-N-flag and PDZK1-N-myc, NHERF1-N-myc or control vector were labeled with NHS-SS-biotin at 4°C and then warmed to 37°C to initialize recycling/targeting for varying times; an additional aliquot of NHS-SS-biotin was applied to cells to optimize biotinylation of recycled/targeted proteins. At the end of incubations residual NHS-SS-biotin was quenched with glycine (100 mM) at 4°C. Cell lysates were prepared in lysis buffer and 500 µg protein from each sample was applied to streptavidin-agarose beads to capture biotinylated proteins. Electrophoresis and immunoblotting of lysate proteins bound to streptavidin/agarose beads was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094712#s2" target="_blank">Materials and Methods</a>.</p

Stability of OATP1A2 protein in HEK-293 cells in the presence or absence of co-expressed PDZK1 or NHERF1.

<p>(A) Western analysis of total cellular expression of OATP1A2-N-flag with or without co-expression of PDZK1 or NHERF1. <i>Top Panel</i>: HEK-293 cells were treated with 5 µg/ml puromycin for 24 and 48 h. Cells were harvested and lysate proteins were separated by SDS-polyacrylamide gel electrophoresis, followed by Western blotting with anti-flag antibody. <i>Bottom Panel</i>: After stripping, blots were reprobed with anti-β-actin antibody. (B) Densitometric analysis of the mature (∼95 KDa) isoform of OATP1A2-N-flag as a percentage of total OATP1A2 protein in the absence of puromycin treatment (means±S.E. of 3 individual experiments). Different from control: *<i>P</i><0.05; **<i>P</i><0.01.</p

Caveolin-dependent internalization of OATP1A2 by PDZK1 and NHERF1.

<p>(A) Disruption of the caveolin-dependent pathway impairs OATP1A2-N-flag internalization. HEK-293 cells over-expressing OATP1A2-N-flag were treated with filipin (5 µg/ml) or nystatin (25 µM) for 30 mins, relative to dimethylsulfoxide-treated control. The cells were allowed to internalize at 37°C for 5 or 10 mins as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094712#s2" target="_blank">Materials and Methods</a>” followed by immunoblotting for OATP1A2-N-flag. (B) Internalized OATP1A2-N-flag as a percentage of the total initial pool of biotinylated OATP1A2-N-flag at the cell surface (means±S.E. of 3 individual experiments) *: Different from control: <i>P</i><0.05. (C) <i>Top panel</i>: immunoblot analysis of OATP1A2-N-flag in HEK-293 cells containing co-expressed PDZK1-N-myc or NHERF1-N-myc after treatment with filipin (5 µg/ml) for 30 mins, followed by internalization at 37°C for 5 or 10 mins. <i>Bottom panel</i>: Internalized OATP1A2-N-flag as a percentage of the total initial pool of biotinylated OATP1A2-N-flag at the cell surface (means±S.E. of 3 individual experiments). *: Different from control: <i>P</i><0.05 (D) <i>Top panel</i>: immunoblot analysis of OATP1A2-N-flag in HEK-293 cells containing co-expressed PDZK1-N-myc or NHERF1-N-myc after treatment with nystatin (25 µM) for 30 mins, followed by internalization at 37°C for 5 or 10 mins. <i>Bottom panel</i>: Internalized OATP1A2-N-flag as a percentage of the total initial pool of biotinylated OATP1A2-N-flag at the cell surface (means±S.E. of 3 individual experiments). *: Different from control: <i>P</i><0.05. Blotting for β-actin in separate aliquots of total lysates was used to confirm uniform protein loading.</p