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J. Biol. Chem., Vol. 278, Issue 7, 4990-5000, February 14, 2003
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From the 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-
N-terminal Truncation of the Dopamine Transporter Abolishes
Phorbol Ester- and Substance P Receptor-stimulated Phosphorylation
without Impairing Transporter Internalization*
,,¶
Molecular Neuropharmacology Group,
Department of Pharmacology, Panum Institute, University of Copenhagen,
DK-2200 Copenhagen, Denmark and the § Center for Molecular
Recognition and the Departments of Psychiatry and Pharmacology,
Columbia University College of Physicians & Surgeons,
New York, New York 10032
-phorbol 12-myristate 13-acetate (PMA) or by activating the
Gq-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 Vmax for [3H]dopamine
uptake to the same degree as did PMA (~50 and ~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.
*
This work was supported in part by National Institutes of
Health Grants DA 11495 and MH 57324 (to J. A. J.) and by grants from the Lundbeck Foundation (to U. G.), the Danish Health Science Research Council (to U. G.), the Novo Nordic Foundation (to
U. G.), and the Lebovitz Trust (to J. A. J.).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.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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