Dopamine transporters (DATs) are neuronal phosphoproteins that clear dopamine from the synaptic cleft. Activation of protein kinase C (PKC) and inhibition of protein phosphatases by okadaic acid (OA) increase phosphorylation of DAT and lead to concomitant reduction in DAT activity and cell surface expression. Numerous potential sites for phosphorylation are present on DAT, but the sites utilized and their relationship to transport regulation are currently unknown. We used peptide mapping and epitope-specific immunoprecipitation to identify the region of DAT that undergoes phosphorylation in rat striatal tissue. Phosphoamino acid analysis revealed that basal and stimulated samples were phosphorylated primarily on serine. Digestion of 32 PO 4 -labeled DAT with trypsin and immunoprecipitation with N-or C-terminal specific antisera failed to isolate phosphopeptide fragments corresponding to photoaffinity-labeled fragments that contain all internal interhelical loops. However, digestion of 32 PO 4 -labeled DAT with endoproteinase asp-N and immunoprecipitation with an N-terminal antiserum extracted two phosphopeptide fragments from both basal and PKC/ OA-stimulated samples, demonstrating that the N-terminal cytoplasmic tail is a major site of phosphorylation. Aminopeptidase treatment of PKC-and/or OA-stimulated DAT cleaved essentially all 32 PO 4 label without proteolysis extending past transmembrane domains 1 and 2, providing further evidence that most phosphorylation sites are near the N terminus and not in intracellular loops or C-terminal domains. In situ proteolysis of the N-terminal tail indicates that the majority of stimulated phosphorylation sites are N-terminal to an antibody epitope at residues 42-59. Two-dimensional analysis of purified protein produced three tryptic phosphopeptides that may result from phosphorylation of multiple sites, but the fragments did not co-migrate with synthetic tryptic peptides phosphorylated at serines 2 and 4. These results indicate that most or all of the basal and stimulated phosphorylation of DAT in striatal tissue occurs on one or more residues in a group of six serines clustered near the distal end of the cytoplasmic N terminus.The concentration of dopamine in the synaptic and extrasynaptic space is controlled to a great extent by the action of dopamine transporters (DATs), 1 which utilize the energy of Na ϩ and Cl Ϫ ionic gradients to drive reuptake of the neurotransmitter into the presynaptic cell (1). DAT belongs to a family of transporters including those for norepinephrine and serotonin (SERT) that constitutes the major sites of action for tricyclic antidepressants and psychostimulants (2). Other neurotransmitter reuptake carriers include plasma membrane transporters for glycine (Glyt1) and ␥-aminobutyric acid (GAT1), and the synaptic vesicle monoamine and acetylcholine transporters, vesicular monoamine transporter and vesicular acetylcholine transporter (3, 4). These proteins are believed to have a similar structure of 12 transmembrane-spanning domains, cytoplasmica...

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