Regulation of human organic anion transporter 4 by protein kinase C and NHERF-1: altering the endocytosis of the transporter

Abstract

Purpose: Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs. We have previously shown that the activity of hOAT4 was down-regulated by activation of PKC and up-regulated by PDZ protein NHERF-1. Here, we investigated the mechanisms underlying such regulations.

Methods: COS-7 cells expressing hOAT4 were treated with PKC activator phorbol 12-myristate 13-acetate (PMA) or transfected with dominant negative mutants of dynamin-2 or Eps15 or transfected with NHERF-1. The internalization and the function of hOAT4 were then determined.

Results: We showed that hOAT4 constitutively internalized from and recycled back to plasma membrane. Transfection of dominant negative mutants of dynamin-2 or Eps15 into the cells, all of which block clathrin-dependent endocytotic pathway, significantly blocked hOAT4 internalization. Treatment of cells with PMA accelerated hOAT4 internalization, whereas transfection of cells with NHERF-1 attenuated hOAT4 internalization.

Conclusion: Our studies demonstrated that i) hOAT4 undergoes constitutive trafficking between cell surface and intracellular compartments, ii) hOAT4 internalization partly occurs through clathrin-dependent pathway, iii) the down-regulation of hOAT4 activity by activation of PKC and the up-regulation of hOAT4 activity by NHERF-1 are mediated through alteration of hOAT4 internalization.

Fig. 1

Biotinylation analysis of constitutive hOAT4 internalization in COS-7 cells. a. hOAT4 internalization was analyzed as described in “Materials and Methods” section followed by Western blotting using anti-myc antibody (1:100). b. Densitometry plot of results from Fig. 1a as well as from other experiments. Internalized hOAT4 was expressed as % of total initial cell surface hOAT4 pool. Values are mean ± S.E. (n=3).

Fig. 2

Biotinylation analysis of constitutive hOAT4 recycling. a. 30-min recycling of transferrin receptor (TfR) was analyzed as described in “Materials and Methods” section followed by Western blotting using anti-TfR antibody (1:100). b. hOAT4 recycling was analyzed at a 5-min time point followed by Western blotting using anti-myc antibody (1:100). c. Densitometry plot of results from Fig. 2b as well as from other experiments. Total biotin-labeled hOAT4 was expressed as % of hOAT4 biotinylated at 4°C (before initiation of recycling). Values are mean ± S.E. (n=3).

Fig. 3

Dominant negative mutant of dynamin 2 blocked constitutive hOAT4 internalization. a. Top panel: cells were transfected with control plasmid (pcDNA vector). 48 h later, hOAT4 internalization (15 min) was analyzed as described in “Materials and Methods” section followed by Western blotting using anti-myc antibody (1:100). Bottom panel: Densitometry analysis of results from top panel as well as other experiments. Internalized hOAT4 was expressed as % of total initial cell surface hOAT4 pool. Values are mean ± S.E. (n=3). b. Top panel: Cells were transfected with cDNA encoding dominant negative mutant of dynamin-2 (Dyn-2 mutant). 48 h later, hOAT4 internalization (15 min) was analyzed as described in “Materials and Methods” section followed by Western blotting using anti-myc antibody (1:100). Bottom panel: Densitometry analysis of results from top panel as well as other experiments. Internalized hOAT4 was expressed as % of total initial cell surface hOAT4 pool. Values are mean ± S.E. (n=3). c. Top panel: Steady-state surface expression of hOAT4 in cells transfected with or without Dyn-2 mutant was determined by biotinylation analyses. Bottom panel: Densitometry analysis of results from top panel as well as other experiments. Surface expression of hOAT4 in cell transfected with Dyn-2 mutant was expressed as % of total cell surface hOAT4 pool in control cells. Values are mean ± S.E. (n=3). d. 4-min uptake of [³H] estrone sulfate (100 nM) into cells transfected with Dyn-2 mutant. Uptake activity was expressed as a percentage of the uptake measured in control cells. Values are mean ± S.E. (n=3).

Fig. 4

Dominant negative mutant of Eps15 blocked constitutive hOAT4 internalization. a. Top panel: cells were transfected with control plasmid (pcDNAvector). 48 h later, hOAT4 internalization (15 min) was analyzed as described in “Materials and Methods” section followed by Western blotting using anti-myc antibody (1:100). Bottom panel: Densitometry analysis of results from top panel as well as other experiments. Internalized hOAT4 was expressed as % of total initial cell surface hOAT4 pool. Values are mean ± S.E. (n=3). b. Top panel: Cells were transfected with cDNA encoding dominant negative mutant of Eps15. 48 h later, hOAT4 internalization (15 min) was analyzed as described in “Materials and Methods” section followed by Western blotting using anti-myc antibody (1:100). Bottom panel: Densitometry analysis of results from top panel as well as other experiments. Internalized hOAT4 was expressed as % of total initial cell surface hOAT4 pool. Values are mean ± S.E. (n=3). c. Top panel: Steady-state surface expression of hOAT4 in cells transfected with or without Eps15 mutant was determined by biotinylation analyses. Bottom panel: Densitometry analysis of results from top panel as well as other experiments. Surface expression of hOAT4 in cell transfected with Eps15 mutant was expressed as % of total cell surface hOAT4 pool in control cells. Values are mean ± S.E. (n=3). d. 4-min uptake of [³H] estrone sulfate (100 nM) into cells transfected with Eps15 mutant. Uptake activity was expressed as a percentage of the uptake measured in control cells. Values are mean ± S.E. (n=3).

Fig. 5

Immuno-localization of hOAT4 and EEA1. The cells were immunostained for hOAT4, and early endosome marker EEA1. Fluorescence images were taken for hOAT4 (red), and EEA1 (green). The merged image of hOAT4 and EEA1 was shown as orange/yellow. Bar=~10 μm.

Fig. 6

Biotinylation analysis of PKC-modulated hOAT4 internalization and steady-state cell surface expression. a. Top panel: hOAT4 internalization (15 min) was analyzed as described in “Materials and Methods” section in the presence and the absence of 1 μM PMA followed by Western blotting using anti-myc antibody (1:100). Bottom panel: Densitometry analyses of results from top panel as well as from other experiments. Internalized hOAT4 was expressed as % of total initial cell surface hOAT4 pool. Values are mean ± S.E. (n=3). b. Top panel: The effect of PMA on the steady-state expression of hOAT4 at cell surface was analyzed by biotinylation approach. Bottom panel: Densitometry analyses of results from top panel as well as from other experiments. The cell surface expression of hOAT4 in the presence of PMA was expressed as % of the cell surface hOAT4 in the absence of PMA. Values are mean ± S.E. (n=3).

Fig. 7

Biotinylation analysis of NHERF-1-modulated hOAT4 internalization and steady-state cell surface expression. a. Top panel: Cells were transfected with control plasmid (pcDNA vector). 48 h later, hOAT4 internalization (15 min) was analyzed as described in “Materials and Methods” section followed by Western blotting using anti-myc antibody (1:100). Bottom panel: Densitometry analysis of results from top panel as well as other experiments. Internalized hOAT4 was expressed as % of total initial cell surface hOAT4 pool. Values are mean ± S.E. (n=3). b. Top panel: Cells were transfected with cDNA encoding NHERF-1. 48 h later, hOAT4 internalization (15 min) was analyzed as described in “Materials and Methods” section followed by Western blotting using anti-myc antibody (1:100). Bottom panel: Densitometry analysis of results from top panel as well as other experiments. Internalized hOAT4 was expressed as % of total initial cell surface hOAT4 pool. Values are mean ± S.E. (n=3). c. Top panel: Steady-state surface expression of hOAT4 in cells transfected with or without NHERF-1 mutant was determined by biotinylation analyses. Bottom panel: Densitometry analysis of results from top panel as well as other experiments. Surface expression of hOAT4 in cell transfected with NHERF-1 mutant was expressed as % of total cell surface hOAT4 pool in control cells. Values are mean ± S.E. (n=3).