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J. Biol. Chem., Vol. 277, Issue 28, 25178-25186, July 12, 2002
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From the Department of Biochemistry and Molecular Biology,
University of North Dakota School of Medicine and Health Sciences,
Grand Forks, North Dakota 58202
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
32PO4-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 32PO4-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
32PO4 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.
Dopamine Transporters Are Phosphorylated on N-terminal Serines in
Rat Striatum*
, and
*
This work was supported by National Institutes of Health
Grant DA13147 (to R. A. V.) and National Science Foundation Grant ND
EPSCoR.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Dept. of Molecular Pharmacology and Toxicology,
School of Pharmacy, University of Southern California, Los Angeles, CA 90089.
§
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Heath Science, 501 N. Columbia Rd., Grand Forks, ND
58203. Tel.: 701-777-3419; Fax: 701-777-2382; E-mail:
rvaughan@medicine.nodak.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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