N-terminal truncation of the dopamine transporter abolishes phorbol ester- and substance P receptor-stimulated phosphorylation without impairing transporter internalization.

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.