Abstract
The structural basis of phosphorylation and its putative role in internalization were investigated in the human dopamine transporter (hDAT). Activation of protein kinase C (PKC) was achieved either directly by treatment with 4-alpha-phorbol 12-myristate 13-acetate (PMA) or by activating the Galpha(q)-coupled human substance P receptor (hNK-1) co-expressed with hDAT in HEK293 cells and in N2A neuroblastoma cells. In both cell lines, activation of the hNK-1 receptor by substance P reduced the V(max) for [(3)H]dopamine uptake to the same degree as did PMA ( approximately 50 and approximately 20% in HEK293 and N2A cells, respectively). In HEK293 cells, the reduction in transport capacity could be accounted for by internalization of the transporter, as assessed by cell surface biotinylation experiments, and by fluorescence microscopy using enhanced green fluorescent protein-tagged hDAT. In HEK293 cells, hNK-1 receptor activation, as well as direct PKC activation by PMA, was accompanied by a marked increase in transporter phosphorylation. However, truncation of the first 22 N-terminal residues almost abolished detectable phosphorylation without affecting the SP- or PMA-induced reduction in transport capacity and internalization. In this background truncation construct, systematic mutation of all the phosphorylation consensus serines and threonines in hDAT, alone and in various combinations, did also not alter the effect of hNK-1 receptor activation or PMA treatment in either HEK293 or N2A cells. Mutation of a dileucine and of two tyrosine-based motifs in hDAT was similarly without effect. We conclude that the major phosphorylation sites in hDAT are within the distal N terminus, which contains several serines. Moreover, the present data strongly suggest that neither this phosphorylation, nor the phosphorylation of any other sites within hDAT, is required for either receptor-mediated or direct PKC-mediated internalization of the hDAT.
Highlights
The dopamine transporter (DAT)1 is situated in the presynaptic membrane of dopaminergic nerve terminals and is responsible for the rapid removal of dopamine released into the synaptic cleft upon neuronal stimulation [1,2,3]
The effect of hNK-1 receptor activation on human dopamine transporter (hDAT) activity in comparison to that of direct protein kinase C (PKC) activation by phorbol esters was investigated upon transient expression of the hDAT tagged at the N terminus with the FLAG epitope (FLAG-hDAT) in the hNK-1 receptorexpressing cells
Addition of the PKC inhibitor staurosporine markedly inhibited the response to substance P, not as efficiently as it inhibited the response to phorbol 12-myristate 13-acetate (PMA)
Summary
Site-directed Mutagenesis—The cDNA encoding the human dopamine transporter (hDAT) in pRC/CMV [20] was kindly provided by Dr Marc G. Binding assays were performed in a final volume of 100 l of binding buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 5 mM MnCl2, 0.1% bovine serum albumin, and 40 g/ml bacitracin) with 0.4 nM 125I-labeled substance P plus increasing concentrations of non-labeled substance P for determination of Bmax and KD values. Upon the addition of phosphatase inhibitors, 1 M okadaic acid and 50 M Na3VO4, the cells were stimulated with 200 nM SP, 1 M PMA, or vehicle for 60 min followed by washing in 5 ml ice-cold PBS and subsequent lysis by shaking for 15 min on ice in lysis buffer (25 mM Tris-HCl, pH 7.6, with 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 50 mM sodium pyrophosphate, 50 mM NaF, 1 M okadaic acid, and 1% Triton X-100) supplemented with protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride and a protease inhibitor mixture tablet (Roche Diagnostics)). One-way analysis of variance followed by Dunnett’s post hoc test was used for statistical comparisons
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