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, 23 (24), 8480-8

Mitogen-activated Protein Kinase Regulates Dopamine Transporter Surface Expression and Dopamine Transport Capacity

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Mitogen-activated Protein Kinase Regulates Dopamine Transporter Surface Expression and Dopamine Transport Capacity

José A Morón et al. J Neurosci.

Abstract

The dopamine transporter (DAT) regulates the clearance of dopamine (DA) released into the extracellular space and is an important site on which psychostimulants act to produce their effects. Here, we show that mitogen-activated protein kinase (MAPK) regulates the transport capacity and intracellular trafficking of DAT. Incubation of striatal synaptosomes or epitope-tagged human DAT (hDAT) human embryonic kidney (HEK) 293 cells with the MAPK kinase (MEK) inhibitors 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one decreased DA uptake in a concentration- and time-dependent manner. Kinetic studies revealed a decrease in the capacity of transport (Vmax) but no change in Km. Immunoblotting confirmed labeling of p42 and p44 MAPK in untreated striatal synaptosomes and HEK 293 cells, consistent with constitutive MAPK activation, and the inhibitors used decreased MAPK phosphorylation. Biotinylation and confocal imaging studies showed that MAPK inhibition promoted the clathrin-associated redistribution of hDAT from the plasma membrane to the cytosol. In contrast, transient transfection of hDAT-expressing cells with constitutively active MEK increased the Vmax of DA transport without altering Km. However, only a small increase in hDAT cell surface expression was seen. These data demonstrate an involvement of the MAPK cascade in regulating DAT transport capacity in striatum and that inhibition of this cascade decreases DAT cell surface expression in HEK 293 cells. Furthermore, they highlight the potential role of MAPK as a presynaptic mechanism that regulates DA signaling.

Figures

Figure 1.
Figure 1.
MAPK phosphorylation in striatal synaptosomes. Synaptosomes were incubated with vehicle, U0126 (50 μm), PD98059 (50 μm), or LY294002 (30 μm), for 30 min at 37°C. Solubilized synaptosomes were immunoblotted with anti-phospho-MAPK (ERK1 and ERK2) antibody. Total MAPK levels were detected with an antibody that recognized both native and phosphorylated forms of MAPK (a representative blot is shown). Quantification of MAPK phosphorylation was performed related to total MAPK as described in Materials and Methods. Data from three separate experiments were averaged, and mean values ± SEM were plotted. *Significant difference compared with vehicle-treated controls (p < 0.01, Student's t test).
Figure 2.
Figure 2.
[3H]DA uptake by U0126 or PD98059. A, Striatal synaptosomes were treated for the indicated times with U0126 (50 μm) or PD98059 (50 μm) before [3H]DA uptake determination. B, Striatal synaptosomes were treated with the indicated concentrations of U0126 or PD98059 for 30 min before determination of uptake. Results are the mean ± SEM of three experiments. *,**Significant difference compared with vehicle-treated controls (*p < 0.05; Student's t test).
Figure 3.
Figure 3.
PD98059 induces a loss of hDAT cell surface expression. A, B, Confocal microscopic images of FLAG-hDAT cells in the absence (A) or presence (B) of 50 μm PD98059 added to the bath solution for 30 min. C, Z-sections of FLAG-hDAT cells treated with PD98059 go from the top to the bottom of the cells; the six confocal planes of 1μm in thickness show extensive intracellular immunofluorescence. The confocal microscopic images shown are representative of four different experiments.
Figure 4.
Figure 4.
PD98059-induced hDAT cell surface redistribution is an internalization event. A, B, Confocal microscopic images of FLAG-hDAT cells incubated in the absence (A) or presence (B)of 50 μm PD98059. C, Preincubation of FLAG-hDAT cells with ConA (250 μg/ml for 20 min) reduced the PD98059-induced redistribution of FLAG-hDAT from the plasma membrane to the cystosol. The confocal microscopic images shown are representative of four different experiments (PD98059 vs control) and three experiments (PD98059 vs CoA).
Figure 5.
Figure 5.
PD98059 induces an hDAT redistribution from the cell surface to an intracellular pool. Cells were treated with 50 μm PD98059 or vehicle for 30 min before biotinylation with sulfo-NHS-SS-biotin. Biotinylated (cell surface) and nonbiotinylated (intracellular) proteins were separated with streptavidin beads and analyzed by immunoblot with an hDAT-specific antibody as described in Material and Methods. A, Representative immunoblot. B, Immunoblots from four separate experiments were scanned densitometrically, and mean values ± SEM were plotted. Data are expressed as a percentage of vehicle-treated cells. *Significant difference compared with vehicle-treated controls (p < 0.01, Student's t test).
Figure 6.
Figure 6.
PD98059-induced internalization of FLAG-hDAT is clathrin-mediated. FLAG-hDAT cells were transiently transfected with the neuronal-specific GFP-clathrin light chain. Double-staining experiments (red fluorescence for the FLAG-hDAT, green fluorescence for clathrin) were performed to determine colocalization of FLAG-hDAT and GFP-clathrin. Main panel, Confocal microscopy revealed little or no puncta of colocalization under control conditions. Inset, In FLAG-hDAT cells exposed for 30 min to 50μm PD9805, there is extensive colocalization (yellow) between GFP-clathrin and FLAG-hDAT, presumably at the level of endocytotic compartments.

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