|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 28, 25178-25186, July 12, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Department of Biochemistry and Molecular Biology,
University of North Dakota School of Medicine and Health Sciences,
Grand Forks, North Dakota 58202
Received for publication, January 10, 2002, and in revised form, April 9, 2002
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.
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+ and Cl Recent studies (5-9) have shown that DATs and other neurotransmitter
transporters are phosphoproteins whose functions are acutely regulated
by protein kinases, providing a mechanism for temporal and spatial
control of synaptic neurotransmitter levels and neural signaling.
Activators of protein kinase C (PKC) and inhibitors of protein
phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) cause an increase
in DAT phosphorylation with a concomitant reduction in dopamine
transport activity (5, 10, 11). A common theme emerging from these
studies is that phosphorylation and functional regulation of
transporters are accompanied by changes in their cell surface density
(6, 7, 12-14). Phorbol esters stimulate clathrin-mediated endocytosis
of DAT that results in intracellular sequestration and trafficking
through recycling and degradative pathways (12, 13). However, it is not
known if phosphorylation of DAT is required for these processes and/or contributes to other aspects of transport regulation or function.
Our current goal is to identify the sites of phosphorylation on DAT and
elucidate their functional significance. The presumed intracellular
aspects of rDAT contain numerous serines, threonines, and
tyrosines, many of which are present in PKC, protein kinase A, and
Ca2+-calmodulin-dependent protein kinase
consensus motifs (Fig. 1). These residues
are found in both N- and C-terminal cytoplasmic tails and in all
intracellular loops (ILs) except IL1. The large number of potential
phosphorylation sites, in conjunction with the possibility of multisite
phosphorylation, has made determination of phosphorylation sites by
molecular approaches problematic. Attempts to identify sites
contributing to regulation of DAT, GAT1, and Glyt1 using mutagenesis of
PKC consensus motifs and/or sites highly conserved throughout the
neurotransmitter transporter family have not been successful (14-16),
as phorbol ester-induced regulation was maintained after mutagenesis.
It is possible that the sites examined were not those involved in
phosphorylation or that multiple sites are involved, and mutagenesis of
the examined sites was insufficient to produce a result.
In this study we used phosphoamino acid analysis and protease-based
peptide mapping strategies to identify directly the region of DAT that
undergoes phosphorylation in rat brain tissue. The results show that
one or more serines at the extreme end of the N-terminal tail are the
major sites of both basal and stimulated phosphorylation. These results
have implications for the molecular basis of transport regulation and
provide strong direction for mutagenesis approaches.
Materials--
[125I]DEEP and
[125I]RTI 82 were synthesized by Dr. John Lever,
University of Missouri. Carrier-free 32PO4 was
from ICN; okadaic acid (OA), 1-oleoyl-2-acetyl-sn-glycerol (OAG), and phorbol 12-myristate 13-acetate (PMA) were from Calbiochem; endoproteinase asp-N was from Roche Molecular Biochemicals; high and
low range Rainbow molecular weight markers were from Amersham Biosciences; phosphoamino acid standards, aminopeptidase 1, carboxypeptidase Y, and TPCK-treated trypsin were from Sigma;
phosphopeptide standards were synthesized by Cell Essentials (Boston,
MA); and Tricine gel reagents were from Bio-Rad. Rats were purchased
from Charles River Laboratories and were housed and treated in
accordance with regulations established by the National Institutes of
Health and the University of North Dakota Animal Care and Use Committee.
Metabolic Phosphorylation of DAT--
For most experiments
phosphorylated DATs were prepared from rat striatal slices
metabolically labeled with 32PO4 using
procedures adapted from Halpain et al. (17). Male Sprague-Dawley rats (175-300 g) were decapitated, and the striata were
rapidly removed and weighed. The tissue was sliced into 350-µm slices
using a McElvain Tissue Chopper, and equivalent amounts of tissue (4-8
slices) were placed into wells of a 12-well culture plate containing
oxygenated Krebs-bicarbonate buffer (KBB) consisting of 25 mM NaHCO3, 125 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 5 mM MgSO4, and 10 mM glucose, pH
7.3. Slices were preincubated for 30 min at 30 °C, with shaking at
105 rpm, followed by exchange with fresh buffer containing 1 mCi/ml
32PO4, and continued incubation with shaking at
30 °C for 90-120 min. Oxygen (95% O2, 5%
CO2) was gently blown across the top of the plate during
the incubation, and test compounds or vehicles were added for the final
30 min. Test compounds (OA, OAG, and PMA) were dissolved at high
concentrations in dimethyl sulfoxide (Me2SO)
followed by dilution in the incubation mixture to a final Me2SO concentration of 0.1%. In all experiments control
tissue received 0.1% Me2SO, which by itself had no effect
on the phosphorylation state of DAT. At the end of labeling, tissue
slices were transferred to a microcentrifuge tube and centrifuged at
4 °C at 800 × g for 4 min. The supernatant
fractions were removed, and 1 ml of ice-cold KBB was added to
the slices. The tissue was disrupted by four passages through a
26-gauge needle; samples were centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatant fractions were removed from the sedimented membranes. For in situ
proteolysis procedures, membranes were resuspended in 50 mM
Tris-HCl, pH 8.0, at 50 mg/ml original wet weight, and for
immunoprecipitation and gel purification, membranes were solubilized
with 0.5% SDS at 20 mg/ml original wet weight. For both procedures,
aliquots were removed for protein assay using the Pierce protein assay
kit with bovine serum albumin as the standard, and sample volumes were adjusted to equalize protein content. For one experiment phosphorylated DAT was prepared from rat striatal synaptosomes metabolically labeled
with 32PO4 and treated with vehicle, PMA, or OA
as described previously (5). Briefly, synaptosomes suspended in
KBB were labeled with 1 mCi/ml 32PO4 at
30 °C for 45 min with drug treatments given for the final 15 min.
All phosphorylation and peptide mapping results were verified in two or
more independent experiments.
Photoaffinity Labeling and Immunoprecipitation--
Rat striatal
DATs were photoaffinity-labeled with [125I]DEEP or
[125I]RTI 82 as described previously (18) and prepared
for in situ proteolysis or gel purification in parallel with
32PO4-labeled DATs. Immunoprecipitations of
phosphorylated or photoaffinity-labeled samples were performed with
rabbit polyclonal antisera 16 or 18 generated against N-terminal amino
acids 42-59 (peptide 16) or C-terminal amino acids 580-608 (peptide
18) of the rDAT protein sequence (18). Samples were electrophoresed on
SDS-polyacrylamide gels, and dried gels were subjected to
autoradiography using Kodak Biomax film for 12-48 h. The region of the
gel containing DAT was excised for processing as described below, and
in some experiments radioactivity in excised
32PO4-labeled DAT samples was assessed by
Cerenkov counting. The identity of novel fragments immunoprecipitated
with serum 16 was verified by inclusion of 50 µg/ml peptide 16 or
peptide 1 (residues 6-21) during immunoprecipitation. High and low
range Rainbow molecular weight markers were used as standards on all gels.
Phosphoamino Acid Analysis--
Phosphoamino acid analysis was
performed using the method of Boyle et al. (19).
32PO4-Labeled striatal slices were treated with
vehicle or test compounds, and membranes were solubilized,
immunoprecipitated with antiserum 16, and electrophoresed on 8% gels.
After drying and autoradiography, the region of the gel containing DAT
was excised, and the protein was eluted overnight at 22 °C in 0.1 M ammonium bicarbonate, pH 8.0. Samples were oxidized with
performic acid followed by precipitation with trichloroacetic acid and
hydrolysis for 2 h at 110 °C with 5.7 M HCl.
Unlabeled phosphoamino acid standards (Ser(P), Thr(P), and
Tyr(P)) were dissolved in pH 1.9 buffer (acetic acid 7.8%, formic acid
2.5%) and added to the unknowns at 1 mg/ml. Samples were spotted onto
cellulose thin layer plates and electrophoresed using a Hunter thin
layer electrophoresis unit at 1.5 kV for 35 min at pH 1.9 (acetic acid
7.8%, formic acid 2.5%) in the first dimension and at 1.3 kV for 20 min at pH 3.5 (pyridine 0.5%, acetic acid 5%) in the second
dimension. Standards were visualized with ninhydrin, and the plates
were subjected to autoradiography for 10-14 days.
In Situ Proteolysis and Immunoblotting--
Striatal membrane
suspensions labeled with 32PO4 were subjected
to in situ proteolysis as described previously (18) followed by analysis of DAT by immunoprecipitation and Western blotting. Briefly, membranes were treated with trypsin or endoproteinase asp-N
for 10 or 60 min, respectively, at 22 °C and sedimented by
centrifugation, and the supernatants were transferred to fresh tubes.
Membranes were solubilized and subjected to immunoprecipitation with
antisera 16 or 18, followed by electrophoresis and autoradiography on
9% SDS-PAGE gels or 8-16% Tris-Tricine gradient gels (20). In some
experiments, the supernatant fractions were also subjected to
immunoprecipitation to assay for fragments released from membranes. For
Western blotting, trypsin-treated membranes were electrophoresed on 8%
SDS-PAGE gels followed by electrophoretic transfer to 0.45-µm polyvinylidene difluoride membranes. Dried membranes were blocked with
3% BSA prepared in 10 mM phosphate-buffered saline (PBS), pH 7.4, for 1.5 h followed by 5% non-fat dry milk in 10 mM PBS, pH 7.4, for 0.5 h, washed extensively, and
probed with antiserum 16 diluted 1:100 in 3% BSA, 10 mM
PBS, pH 7.4. Blots were washed and then incubated for 1 h at room
temperature with goat anti-rabbit IgG-linked alkaline phosphatase
diluted 1:5000 in a 3% BSA, 10 mM PBS solution. After
extensive washing, blots were developed colorimetrically using the
alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. Dried blots were scanned and quantitated with Molecular Analyst software (Bio-Rad).
Aminopeptidase and Carboxypeptidase Treatment--
DATs from
32PO4-labeled striatal synaptosomes or striatal
slices were immunoprecipitated, gel-purified on 8% SDS-PAGE gels, and
electroeluted as described previously (21). Aliquots of the
electroeluted samples were treated with 1-100 µg/ml of
aminopeptidase 1 or carboxypeptidase Y prepared in 50 mM
Tris-HCl, pH 8.0, for 1 h at 22 °C, followed by addition of
SDS-PAGE sample buffer and electrophoresis and autoradiography on 8%
SDS-PAGE gels. In some experiments DATs labeled with
[125I]DEEP were treated and analyzed in parallel.
Phosphopeptide Analysis--
Limit tryptic digestion and
phosphopeptide analysis were performed using the method of Boyle
et al. (19). 32PO4-Labeled striatal
slices were treated with 1 µM OA, and membranes were
solubilized, immunoprecipitated with antiserum 16, and subjected to
electrophoresis on 8% SDS-PAGE gels. After drying and autoradiography, the region of the gel containing DAT was excised, and protein was
eluted overnight at 22 °C in a solution containing 0.1% SDS, 0.5%
2-mercaptoethanol, and 50 mM ammonium bicarbonate, pH 8.0. The extracted protein was precipitated with trichloroacetic acid in the
presence of carrier protein (20 µg of RNase A), and the pelleted
protein was washed with absolute ethanol. When trypsin was used as the
digestive agent, the DAT sample was oxidized prior to digestion using
performic acid. The final protein sample was resuspended in 50 µl of
50 mM ammonium bicarbonate, pH 8.3, and incubated at
37 °C for 5 h in the presence of 10 µg of TPCK-trypsin followed by a second 10-µg dose of TPCK-trypsin and incubation overnight at 37 °C. After digestion, ammonium bicarbonate was removed by repeated lyophilization, and peptides were dissolved in 10 µl of running buffer (acetic acid 7.8%, formic acid 2.5%, pH 1.9).
A 5-µl aliquot of this sample was analyzed by electrophoresis and
autoradiography on a 15% Tris-Tricine gel. The remaining 5-µl aliquot was spotted onto 20 × 20-cm microcrystalline cellulose glass-backed plates and electrophoresed using a Hunter thin layer electrophoresis unit at 1.0 kV for 35 min at pH 1.9 (acetic acid 7.8%,
formic acid 2.5%) in the first dimension followed by ascending chromatography in the second dimension (39.3% n-butanol,
30.4% pyridine, 6.1% acetic acid, pH 3.5). Synthetic phosphopeptide standards (10 µg) corresponding to tryptic fragments of the first five amino acids of DAT (MpSK and pSK, where p indicates
phosphoserine) were included with the unknowns and were detected by
ninhydrin staining. The standards were analyzed separately and in
combination to determine their mobility patterns.
For CNBr cleavage (22), 32PO4-labeled DAT
prepared as above, but without oxidation, was subjected to SDS-PAGE on
8% gels and electroblotted to a 0.45-µm polyvinylidene difluoride
membrane. After drying and autoradiography, the region of the membrane
containing DAT was excised and feathered by cutting with a razor blade.
Immobilized DAT was immersed in 150 µl of 0.25 M CNBr in
70% formic acid. The sample was capped, mixed by vortexing, and
covered with aluminum foil. The resulting digestion mixture was
incubated with agitation at room temperature for 24 h in a
chemical fume hood followed by lyophilization and then the addition of
50 µl of purified water and repeated lyophilization. The lyophilized
sample was resuspended in 10 µl of running buffer (7.8% acetic acid,
2.5% formic acid, pH 1.9) and subjected to Tris-Tricine gel electrophoresis.
Characterization of DAT Phosphorylation in Striatal
Slices--
Most of the experiments in this study were performed using
striatal slices for metabolic phosphorylation of DAT, because they can
be labeled for much longer times than synaptosomes and produce highly
radiolabeled DAT suitable for additional analyses. Phosphate incorporation into DAT was easily detectable from slices treated with
vehicle only, demonstrating the presence of basal phosphorylation, and
the level of phosphorylation increased when the tissue was treated with
the PKC activator OAG, the PP1/PP2A inhibitor OA, or OA plus OAG (Fig.
2, A and B). This
mimics the pattern of DAT phosphorylation found in striatal
synaptosomes and cultured cell expression systems (5, 10) and indicates
the likelihood that the preparations correspond to those that exhibit
functional regulation. For most studies slices were stimulated with OA
plus OAG to ensure maximal phosphate incorporation, although some
analyses were performed using slices or synaptosomes treated
individually with test compounds to independently examine kinase and
phosphatase effects.
To determine which amino acids were phosphorylated under our labeling
and stimulation conditions, 32PO4-labeled DAT
samples were prepared from striatal slices that were treated with
vehicle, OA, or OAG. DATs were purified by immunoprecipitation and gel
electrophoresis and subjected to phosphoamino acid analysis on
two-dimensional thin layer plates. The results show that in slices
treated individually with vehicle, OA, or OAG, DATs undergo phosphorylation primarily on serine (Fig.
3), as did tissue treated with OA and OAG
together (not shown). A low level of phosphothreonine was consistently
observed in all samples, but phosphotyrosine was not detected, even
with longer film exposures. We have not attempted to detect
phosphotyrosine on DAT by immunoblotting with anti-phosphotyrosine
antibodies.
Immunoprecipitation of Trypsin Fragments--
Our initial attempts
to map phosphorylation sites utilized procedures that have been
successful in identifying sites of photoaffinity label attachment (18,
21). For these experiments 32PO4-labeled DATs
prepared from OA/OAG-treated slices were proteolyzed with trypsin,
which cleaves at lysine and arginine residues, and the digests were
immunoprecipitated with antibodies 16 and 18 specific for N- and
C-terminal domain epitopes (Fig. 1). When performed on DATs
photoaffinity-labeled with [125I]DEEP or
[125I]RTI 82, these procedures produce easily detectable
peptide fragments of 45 and 32 kDa that correspond to large portions of
the N- and C-terminal halves of the protein (Fig.
4, right two lanes). The 45-kDa fragment (arrow a) contains epitope 16 in
the N-terminal tail and extends through extracellular loop 2 (EL2),
whereas the 32-kDa fragment (arrow b) begins in
EL2 and extends through epitope 18 in the C-terminal tail (18, 21). The
14-kDa fragment (arrow c) is a product of the
45-kDa fragment that retains epitope 16 but terminates in IL1. However,
although antisera 16 and 18 immunoprecipitated these
photoaffinity-labeled fragments and non-proteolyzed
32PO4-labeled DAT (left two lanes),
no convincing phosphopeptide fragments were extracted by either of
these antisera (middle two lanes) or by antisera 15 and 5 (not shown) that recognize other epitopes in the N-terminal tail and
EL2 (18). This suggested that tryptic proteolysis separated the
phosphorylated residues from the antibody epitopes or generated
phosphopeptide fragments too small to be analyzed with these
techniques. Because the 45- and 32-kDa photoaffinity-labeled fragments
collectively contain all the transmembrane domains and internal
interhelical loops of DAT (18), the inability to generate comparable
32PO4-labeled fragments is strong evidence that
serines 261, 333, 421, 428, and 504 in internal loops 2-5 (Fig. 1) are
not major phosphorylation sites.
Immunoprecipitation of Asp-N Fragments--
Because these results
indicated that the bulk of the 32PO4
radiolabeling was occurring on residues in the cytoplasmic tails that
were being proteolytically separated from antibody epitopes, we sought
to minimize this possibility by digesting DAT with endoproteinase asp-N
(asp-N), which cleaves on the N-terminal side of aspartic acid
residues. In contrast to the numerous trypsin sites present in these
domains, the N-terminal tail of rDAT has no aspartates upstream of
epitope 16, whereas two aspartates in the C-terminal tail are
downstream of serines 581 and 585 and most of epitope 18 (Fig. 1). For
this experiment membranes prepared from basal and OA/OAG-stimulated
32PO4-labeled striatal slices were digested
with asp-N, followed by solubilization and immunoprecipitation with
antisera 16 or 18. Fig. 5 shows that
asp-N treatment cleaved a substantial portion (~80%) of the protein
and produced strongly labeled phosphopeptide fragments of about 19 and
16 kDa from both basal and stimulated tissue (Fig. 5A,
arrow). Both fragments were immunoprecipitated with serum
16, and precipitation was blocked by inclusion of peptide 16 but not an
irrelevant peptide (Fig. 5B), verifying the presence of
epitope 16. These results therefore provide positive identification of
the N-terminal cytoplasmic tail of DAT as a major site of both basal
and OA/OAG-stimulated phosphorylation.
The masses of the asp-N fragments suggest that they are not generated
by proteolysis at Asp-68 or Asp-79 in or near TM1 (Fig. 1),
which would produce fragments of ~7-8 kDa. No fragments of this mass
were found in either treated membranes (Fig. 5) or asp-N supernatants
(not shown), indicating that asp-N access to Asp-68 and Asp-79 during
in situ proteolysis is prevented by the lipid bilayer or the
tertiary structure of the protein. The next aspartates in the DAT
sequence are in EL2, at positions 174 and 191 (Fig. 1). The masses of
the 16- and 19-kDa fragments are consistent with cleavage at one or
both of these residues or other aspartates in EL2, and the fragments
therefore contain the N-terminal tail, TMs 1-3, EL1, IL1, and part of
EL2. However, because there are no serines in IL1, the N-terminal tail
serines must represent the sites of phosphorylation of these fragments.
Aminopeptidase Analysis--
We did not find phosphopeptide
fragments in asp-N digestion experiments using C-terminal antibody 18 for immunoprecipitation (not shown). Nevertheless, because some
full-length DAT protein remained after asp-N treatment, we could not
exclude the possibility that some of the labeled sites were present
outside the NH2 tail. We further investigated this issue by
treating 32PO4-labeled gel-purified DAT with
aminopeptidase (AP) and carboxypeptidase, which non-specifically cleave
inward from protein N or C termini. Fig.
6A shows that AP treatment
removed essentially all 32PO4 from
OA/OAG-treated DAT, whereas carboxypeptidase had no obvious effect. To
monitor the extent of AP-induced proteolysis in conjunction with
phosphorylation site loss, we examined the effect of AP on DATs labeled
with [125I]DEEP in parallel with
32PO4-labeled sample. AP digestion of
OA/OAG-treated 32PO4-labeled DAT produced
dose-dependent hydrolysis of essentially all
32PO4 from the protein with the reciprocal
appearance of radiolabel at the gel dye front, whereas the same
treatment of [125I]DEEP-labeled DAT produced a loss of
~5-7 kDa in mass (Fig. 6B), consistent with proteolytic
digestion of most of the N-terminal tail. [125I]DEEP is
incorporated in DAT in TMs 1-2 (18), and the retention of most of the
photolabel on the AP-treated protein demonstrates that proteolysis did
not progress past this region.
To assess the location of sites phosphorylated independently in
response to PKC activation and phosphatase inhibition, we performed a
similar analysis on DATs prepared from
32PO4-labeled striatal synaptosomes treated
separately with PMA or OA (Fig. 6C). Both treatments
stimulated DAT phosphorylation relative to vehicle-treated controls
(left three lanes). AP treatment removed most of the
32PO4 label from both stimulated samples
(middle three lanes) with the small loss of mass indicated
by analysis of [125I]DEEP-labeled DATs (right two
lanes), showing that phosphate incorporation induced by
independent PMA and OA treatments occurs on the same general N-terminal
domain as that seen for the combination OA/OAG treatment. Thus, the
aminopeptidase results confirm the N-terminal localization of both PKC
and OA-induced phosphorylation sites on DAT and provide additional
evidence that significant 32PO4 incorporation
does not occur in the internal loops or C-terminal tail. Several
serines are present in the N-terminal tail, and whether the specific
sites phosphorylated in response to separate and combined PKC and OA
treatments are the same, different, or overlapping remains to be
ascertained. Although the phosphorylation intensity of the basal sample
(shown for comparison to stimulated samples) is too low to be analyzed
in this experiment, the N-terminal localization of basal sites is
clearly shown in asp-N digestion experiments in Fig. 5.
In Situ Proteolysis--
There are eight serines in the rDAT
N-terminal tail, a cluster of six near the N terminus at residues 2, 4, 7, 12, 13, and 21, and two more internally at positions 45 and 64 (Figs. 1 and 7C). We used an
in situ proteolysis approach based on the position of
antibody epitope 16 at residues 42-59 to distinguish between these
groups of serines. For this experiment, membranes from OA-stimulated 32PO4-labeled striatal slices were treated with
increasing concentrations of trypsin, and serum 16 was used to either
Western blot or immunoprecipitate aliquots of each sample to
independently assess the retention of the antibody epitope and the
32PO4 label. The Western blot of these samples
shows only a negligible loss of epitope 16 immunoreactivity on the
full-length protein compared with untreated controls (Fig. 7,
A, bottom, and B), whereas even the
lowest doses of trypsin removed over 75% of the
32PO4 label (Fig. 7A,
top, and B). This indicates that serines 45 and
64, which are within or C-terminal to epitope 16 (Fig. 7C), are not the major phosphorylation sites, and that most of the 32PO4 label is found N-terminal to the epitope.
Separation of phosphorylated residues from the epitope could occur by
cleavage at one or more of several potential tryptic proteolysis sites
present between the first six serines and epitope 16 (Fig.
7C). This result is also compatible with those shown in Fig.
4, as 32PO4-labeled fragments comparable with
those labeled by [125I]DEEP would presumably be produced
and precipitated with serum 16 if DAT underwent substantial
phosphorylation on serines 45 or 64. The small fraction of
32PO4 label remaining on the protein after
in situ treatment with trypsin could indicate that a minor
amount of phosphorylation occurs on these more internal serines or
could indicate a pool of DATs inaccessible to cleavage.
One- and Two-dimensional Analysis of Purified DAT
Fragments--
We have also subjected OA-stimulated and
-phosphorylated DAT to limit digestion with trypsin and CNBr. Extensive
proteolysis of immunopurified 32PO4-labeled DAT
with trypsin produced three strongly labeled peptide fragments with
masses of about 18, 4, and 1.5 kDa, and some less intensely labeled
bands (Fig. 8A). The
calculated masses of fragments resulting from complete tryptic
proteolysis of the DAT N terminus (not including mass potentially
provided by phosphate incorporation) is shown in Fig. 8C. We
do not know if the 1.5- and 4-kDa fragments represent independent or
overlapping phosphopeptides generated from digestion at these sites. By
taking into account the limitations of gel electrophoresis for
molecular mass estimation, the mass of the smallest fragment is
reasonably compatible with various combinations of complete or
incomplete digestion of the N terminus, whereas the 4-kDa fragment is
likely to represent an incomplete digest, for example a fragment
encompassing residues 1-35 or 20-60. The 18-kDa fragment is likely to
result from digestion at residues outside the N-terminal tail and was
probably obtained in this experiment but not others because the acid
precipitation used during sample preparation denatured the protein in a
way that prevented proteolysis at sites that were cleaved in other
preparations.
An aliquot of the trypsin digest analyzed in Fig. 8A was
subjected to two-dimensional thin layer electrophoresis and
chromatography (Fig. 8B), which produced three intensely
labeled spots and some minor spots that correspond to the bands on the
Tris-Tricine gel. This sample was electrophoresed in the presence of
synthetic phosphopeptides corresponding to trypsin fragments from the
first five residues of the DAT N terminus (MpSK and pSK, where
pS is phosphoserine), but the major
32PO4-labeled fragments did not co-migrate with
either peptide. This result may be evidence that serines 2 and 4 are
not phosphorylated, but this would also occur if DAT failed to undergo
cleavage at lysines 3 and 5 or if other modifications such as
acetylation were present on the endogenous peptides. A minor
32PO4-labeled spot partially overlapped the
serine 4 phosphopeptide standard, possibly representing a
stoichiometrically minor amount of phosphorylation at Ser-4 or a minor
amount of production of this fragment.
We also digested 32PO4-labeled DAT with CNBr,
which cleaves proteins on the C-terminal side of unoxidized methionine
residues. This treatment produced one distinct phosphopeptide of about
12 kDa (Fig. 8A), a mass that corresponds to cleavage of the
DAT N-terminal sequence at residues Met-106 or Met-111 in TM2 (Fig. 1).
We did not observe a 32PO4-labeled peptide
fragment of ~1 kDa which would correspond to cleavage of DAT at
Met-11 (Fig. 1 and Fig. 8C, arrow). This result
could indicate that serines 2, 4, and 7 are not phosphorylated, consistent with the results of the two-dimensional thin layer analysis
of the tryptic fragments, although again this result would also be
obtained if cleavage at Met-11 did not occur.
Although DAT has now been characterized as a phosphorylated
protein in several studies, many questions remain regarding its properties and significance. For example, it is not known if basal phosphorylation is mediated by PKC or other kinases or if PKC-induced phosphorylation is due to direct phosphorylation of DAT by PKC or by
activation of an intermediate kinase. It is also not known if
OA-stimulated phosphorylation represents accumulation of basal phosphorylation via direct inhibition of DAT phosphatase(s) or if OA
stimulates phosphorylation by an indirect mechanism. Multiple electrophoretic mobility isoforms of DAT can be visualized on SDS gels;
this is most easily observed in immunoblots (23) but is also apparent
at times by photoaffinity labeling and phosphorylation. We do not know
if these electrophoretic mobility isoforms are caused by heterogeneous
phosphorylation, glycosylation, or other factors. Western blotting of
immunoprecipitated 32PO4-labeled DAT
demonstrates substantial overlap between both signals,2 indicating that
most bands visualized by immunostaining are phosphorylated. However, in
some but not all experiments PMA or OA stimulation results in an upward
shift of the phosphorylated band or concentration of
32PO4 radiolabeling near the trailing edge of
the DAT complex (5), but we do not know if this indicates preferential
phosphorylation of some isoforms. Clarification of these issues will
clarify our understanding of the role of phosphorylation in DAT
physiology and function.
The goal of this study was to determine which regions of DAT undergo
phosphorylation in brain tissue. For this purpose we characterized DAT
phosphorylation in rat striatal slices, finding that phosphate
incorporation was stimulated by PKC activators and PP1/PP2A inhibitors,
as in synaptosomes and cells, and was sufficiently robust for
additional sample manipulation. These preparations were therefore
suitable for phosphopeptide mapping analyses with correlation to our
previous findings of DAT functional regulation.
For all basal and stimulated conditions, we found that DAT is
phosphorylated primarily on serine, indicating the potential for these
residues to be major determinants of DAT properties regulated by
kinases and phosphatases. In addition, a low level of phosphothreonine
was consistently observed, and many threonines are found within PKC
consensus sequences that are highly conserved throughout the monoamine
transporter family (2). Recent studies (9, 24, 25) have reported the
presence of phosphotyrosine on GAT1 and the functional regulation of
DAT and GAT1 by tyrosine kinase inhibitors. We found no detectable
phosphotyrosine from DATs prepared under the described conditions, but
we did not employ treatments such as tyrosine kinase activators or
phosphotyrosine phosphatase inhibitors specific for generation or
preservation of phosphotyrosine.
Several lines of evidence demonstrate that the N-terminal
tail of DAT is the major, and possibly sole, domain that undergoes basal, PKC-induced, and OA-induced phosphorylation in rat striatum. Basal and OA/OAG-stimulated 32PO4-labeled asp-N
fragments were immunoprecipitated with N-terminal antiserum 16, and
aminopeptidase but not carboxypeptidase removed PKC- and/or
OA-stimulated 32PO4 label from DAT without
proteolysis extending past TMs 1-2. In conjunction with the inability
to immunoprecipitate tryptic or asp-N-phosphorylated fragments with
C-terminal antisera, these results indicate that the N terminus of DAT
is the primary site of basal, PKC-stimulated, and OA-stimulated
32PO4 incorporation.
In situ proteolysis of the N-terminal tail strongly
implicates the region N-terminal to antibody epitope 16 (residues
42-59), as opposed to more internal regions, as the major site of
stimulated phosphorylation. The distal end of the N-terminal tail
contains 6 serines, some closely spaced, within the first 21 residues. The stoichiometry of DAT phosphorylation is unknown, and within this
cluster of serines we do not know which combination of sites is
utilized. PKC consensus motifs are present at Ser-7 and Ser-21, although it is not known if PKC phosphorylates DAT directly or acts by
an indirect mechanism that would lead to phosphorylation at
non-canonical sites. We also do not know if phosphorylation induced by
PKC activators and phosphatase inhibitors occurs on the same,
different, or overlapping sites within this domain. Limit digestion of
phosphorylated DAT with trypsin produced two low molecular weight
phosphopeptides, but because of the number of serines, lysines, and
arginines in the N-terminal tail, we cannot determine whether the
fragments represent distinct or overlapping peptides. The major
endogenous phosphopeptides did not co-migrate with synthetic
phosphopeptides representing tryptic digests of the first five rDAT
residues containing serines 2 and 4, potential evidence that these
sites are not phosphorylated. Digestion with CNBr also failed to
generate a phosphorylated fragment consistent with cleavage at Met-11
that would contain serines 2, 4, and 7. Taken together, all of our
results are compatible with phosphorylation of DAT occurring on some
combination of serines 12, 13, and 21, but evidence from additional
strategies will be required to identify definitively the sites
utilized. Our current efforts are directed toward the use of
mutagenesis and mass spectrometry to address these questions.
To our knowledge these results are the first to demonstrate
phosphorylation of a neurotransmitter transporter on the N-terminal domain. A recent study (16) reported that serine to glycine mutagenesis
of residues Ser-262 in IL2 and Ser-586 and Thr-613 in the C-terminal
tail of human DAT (corresponding to Ser-261, Ser-585, and Thr-612 of
rDAT) resulted in the loss of PMA-stimulated phosphorylation. These
results directly conflict with our results that positively demonstrate
the presence of phosphorylation sites on the N-terminal tail after
stimulation by PMA alone or with OA/OAG together and show no evidence
for PKC-stimulated phosphorylation in IL2 or the C-terminal tail. The
discrepancies could result from methodological differences, as the
mutagenesis study examined human DAT expressed in a cell line, whereas
our mapping was done using the rat isoform phosphorylated in striatal
tissue. The positions of serines and threonines in the N-terminal tail
of rat and human DAT are very similar, with serines in both isoforms at
positions 2, 4, 7, 12, 13, and 45 and threonines at positions 43, 46, and 62. Differences are present at residues 21 (Ser in rat and Pro in
human), 44 (Asn in rat and Ser in human), 48 (Asn in rat and Thr in
human), 53 (Thr in rat and Ser in human), and 64 (Ser in rat and Gly in
human). Alternatively, the mutations may have induced secondary effects
on protein conformation that indirectly prevented N-terminal domain phosphorylation.
The potential for similarities of our results to phosphorylation sites
and PKC-dependent regulation of other plasma membrane neurotransmitter transporters is not known. The N-terminal tail of SERT
has 7 serines and 10 threonines, some within PKC consensus sites, but
phosphorylation site analysis of SERT has not yet been reported.
Interestingly, norepinephrine, which displays PKC-dependent and -independent functional regulation (26), has three threonines but
no serines in its N-terminal domain. Our results differ from the
phosphorylation pattern found for synaptic vesicle acetylcholine and
monoamine transporters, which undergo phosphorylation exclusively on
C-terminal tail residues (27, 28). In the case of the vesicular acetylcholine transporter, mutation of serine 480 resulted in altered
intracellular sorting that may impact neurotransmitter packaging (29).
This difference in phosphorylation patterns of plasma membrane and
vesicular neurotransmitter transporters may indicate the potential for
these classes of proteins to be subject to different molecular
mechanisms of kinase-induced regulation.
The finding that basal, PKC-stimulated, and OA-stimulated DAT
phosphorylation all occur on N-terminal serines is compatible with
basal phosphorylation resulting from tonic PKC and PP1/PP2A action,
which may function to define base-line dopamine clearance. DAT
dephosphorylation in the brain is extremely robust and may serve to
regulate tightly the DAT phosphorylation state and functional activity
(30). DATs are found in complexes with PP2A (31) and undergo in
vitro dephosphorylation by PP1 (32), and regulation of basal and
stimulated DAT phosphorylation may occur through physiological control
of these phosphatases as well as through activation of kinases.
At present, the function of DAT phosphorylation is not known. A
correlation exists between the dose and kinetics of PKC- and OA-induced
DAT phosphorylation, down-regulation, and transporter internalization
(5, 10-13, 33). Transporter phosphorylation could serve as a marker
for recruitment of endocytic adaptor proteins in a manner analogous to
that of G protein-coupled receptors (34). Other potential functions of
phosphorylation could include recognition by other targeting proteins
or involvement in an autoregulatory mechanism. For example, the N
terminus of GAT1 interacts with the soluble
N-ethylmaleimide-sensitive factor-attachment protein receptor protein syntaxin 1 in a PKC-specific manner that results in
regulation of We thank Heather Holden for technical
assistance and Dr. John Shabb for suggesting the endoproteinase asp-N experiments.
*
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.
§
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.
Published, JBC Papers in Press, May 6, 2002, DOI 10.1074/jbc.M200294200
2
R. A. Vaughan, unpublished results.
The abbreviations used are:
DAT, dopamine
transporter;
rDAT, rat dopamine transporter;
SERT, serotonin
transporter;
TM, transmembrane domain;
EL, extracellular loop, IL,
intracellular loop;
asp-N, endoproteinase asp-N;
AP, aminopeptidase 1;
[125I]DEEP, [125I]1- [2-(diphenylmethoxy)ethyl]-4-[2-(4-azido-3-iodophenyl)ethyl]piperazine;
[125I]RTI 82, [125I]3
Dopamine Transporters Are Phosphorylated on N-terminal Serines in
Rat Striatum*
, and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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, cytoplasmically
oriented N and C termini, and numerous potential phosphorylation sites for various protein kinases.
View larger version (36K):
[in a new window]
Fig. 1.
Schematic diagram of the rat dopamine
transporter showing antibody epitopes and potential phosphorylation and
proteolysis sites. Intracellularly oriented serines that represent
potential phosphorylation sites are indicated with arrows
designated with the residue number. Epitopes for antisera 16 and 18 are
outlined and labeled. Aspartic acid and
methionine residues near the N and C termini that represent potential
cleavage sites for endoproteinase asp-N and cyanogen bromide are
enclosed and shaded. Numerous potential sites of
trypsin proteolysis at lysine and arginine residues in the N and C
termini are present but not highlighted.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 2.
DAT phosphorylation in striatal slices.
Rat striatal slices labeled with 32PO4 were
treated with vehicle, 10 µM OAG, 10 µM OA,
or 10 µM OA plus 10 µM OAG, followed by
immunoprecipitation of DAT with serum 16, SDS-PAGE, and
autoradiography. A, quantitation of DAT phosphorylation.
Immunoprecipitated DAT bands were excised and counted for Cerenkov
radioactivity. The data from three independent experiments are
normalized, averaged, and expressed as the ratio of
32PO4 incorporation relative to the basal
sample ± S.E. B, autoradiogram of phosphorylated DAT.
Equal amounts of sample from treated and untreated tissue were
subjected to immunoprecipitation, electrophoresis, and autoradiography.
[125I]DEEP-Labeled DATs were immunoprecipitated and
electrophoresed in parallel as a control. Molecular mass
standards for all gels are shown in kDa.
View larger version (30K):
[in a new window]
Fig. 3.
Phosphoamino acid analysis of striatal
DATs. 32PO4-labeled striatal slices were
treated with vehicle (no treatment), 10 µM OA, or 10 µM OAG, and DATs were purified by immunoprecipitation and
gel electrophoresis. Autoradiographs displayed the phosphorylation
pattern demonstrated in Fig. 1A. DAT bands were excised,
eluted, and subjected to acid hydrolysis. Cerenkov counting of the
hydrolysates showed counts/min of 41, 90, and 129 in the basal, OAG,
and OA samples, respectively. Aliquots of the hydrolysates were mixed
with phosphoamino acid standards, and amino acids were separated by
two-dimensional electrophoresis on thin layer cellulose plates. Plates
were subjected to autoradiography, and phosphoamino acid standards were
visualized with ninhydrin (dotted circles). S,
phosphoserine; T, phosphothreonine; Y,
phosphotyrosine.
View larger version (43K):
[in a new window]
Fig. 4.
Tryptic proteolysis and epitope-specific
immunoprecipitation of phosphorylated and photoaffinity-labeled
DAT. Striatal slices were labeled with
32PO4 and stimulated with 10 µM
OA plus 10 µM OAG or were photoaffinity-labeled with
[125I]DEEP or [125I]RTI 82 as indicated.
DATs were gel-purified, treated with or without trypsin, and
immunoprecipitated with antiserum 16 or 18 as indicated at the
top of the lanes, followed by electrophoresis and
autoradiography on 14% SDS-PAGE gels. Full-length DAT is present at
~80 kDa; arrows a and c denote the
positions of 45- and 14-kDa [125I]DEEP-labeled fragments
that immunoprecipitate with antiserum 16, and arrow b
denotes a 32-kDa [125I]RTI 82-labeled fragment that
immunoprecipitates with antiserum 18. df, gel dye
front.
View larger version (57K):
[in a new window]
Fig. 5.
Immunoprecipitation of asp-N phosphopeptide
fragments with antiserum 16. A, membranes from striatal
slices labeled with 32PO4 and treated with or
without 10 µM OA plus 10 µM OAG were
subjected to proteolysis with the indicated concentration of
endoproteinase asp-N for 1 h at 22 °C. The membranes were
sedimented, immunoprecipitated with antibody 16, and analyzed by
electrophoresis and autoradiography on an 8-16% Tris-Tricine gel. The
arrow on the right denotes the position of
immunoprecipitated phosphopeptide fragments at ~16 and 19 kDa.
B, asp-N fragments prepared from basal and OA/OAG-stimulated
32PO4-labeled striatal slices were
immunoprecipitated with serum 16 in the presence of no addition, 50 µg/ml peptide 16, or 50 µg/ml peptide 1 as indicated.
View larger version (31K):
[in a new window]
Fig. 6.
Aminopeptidase treatment of
32PO4 and [125I]DEEP-labeled
DATs. DATs from 32PO4-labeled striatal
slices treated with 10 µM OA plus 10 µM OAG
(A and B) or
32PO4-labeled striatal synaptosomes treated
with vehicle, 10 µM PMA, or 1 µM OA
(C) were prepared by immunoprecipitation and gel
purification. A, DATs were treated with the indicated doses
of aminopeptidase 1 or carboxypeptidase Y for 1 h at 22 °C
followed by electrophoresis and autoradiography on an 8% SDS-PAGE gel.
B, 32PO4 or
[125I]DEEP-labeled DATs were treated with the indicated
doses of aminopeptidase, followed by electrophoresis and
autoradiography on an 8% gel. C,
32PO4 or [125I]DEEP-labeled DATs
were treated with or without aminopeptidase as indicated, followed by
electrophoresis and autoradiography.
View larger version (17K):
[in a new window]
Fig. 7.
Analysis of phosphorylation sites by in
situ proteolysis. A,
32PO4-labeled membranes prepared from striatal
slices treated with 1 µM OA were incubated with the
indicated concentrations of trypsin at 22 °C for 10 min. Following
proteolysis, samples were divided into 2 aliquots as follows: one set
was immunoprecipitated with serum 16 to assess the retention of
32PO4 on the protein (top panel),
and the other set was immunoblotted with antibody 16 detected
colorimetrically to assess the retention of the epitope (bottom
panel). B, quantitation of band densities relative to
control samples after immunoprecipitation (open circles) or
immunoblotting (filled circles). C, sequence of
rDAT N-terminal tail up to the approximate point of entry into TM1
showing relative positions of epitope 16 and serine, lysine, and
arginine residues. Reference residue numbers are indicated
above the sequence; serines are indicated in
boldface, and spaces after lysine and arginine residues
indicate potential tryptic proteolysis sites and resulting
fragments.
View larger version (42K):
[in a new window]
Fig. 8.
Tryptic and CNBr phosphopeptide
fragments. A, 32PO4-labeled
DATs from striatal slices treated with 1 µM OA were
isolated by immunoprecipitation and gel purification. Trichloroacetic
acid-precipitated samples were extensively digested with trypsin or
CNBr as indicated and analyzed by electrophoresis and autoradiography
on 15% Tris-Tricine SDS gels. B, an aliquot of the trypsin
digest shown in A was subjected to two-dimensional thin
layer separation in the presence of two synthetic phosphopeptide
standards corresponding to tryptic fragments from the first five
residues of DAT (sequences indicated at right). Plates were
subjected to autoradiography, and phosphopeptide standards were
visualized with ninhydrin (dotted ovals). C,
sequence of the rDAT N-terminal tail showing predicted tryptic
fragments (separated by spaces) with calculated masses denoted
below each sequence. Reference residue numbers are indicated
above the sequence, and serines are indicated in
boldface. The arrow indicates the position of
methionine 11.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid transport (35-37), although it is
not known if this is mediated by GAT1 phosphorylation. Recent evidence
also indicates the potential for DAT and SERT to exist as dimers or
oligomers (38, 39), a process controlled for some proteins by
phosphorylation (40). Our current efforts are aimed at mutating
N-terminal DAT serines to assess their usage as phosphorylation sites
and examine their involvement in down-regulation and other processes.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Dept. of Molecular Pharmacology and Toxicology,
School of Pharmacy, University of Southern California, Los Angeles, CA 90089.
ABBREVIATIONS
-(p-chlorophenyl)tropane-2-carboxylic
acid,4'-azido-3'-iodophenylethyl ester;
OA, okadaic acid;
OAG, 1-oleoyl-2-acetyl-sn-glycerol;
PMA, phorbol 12-myristate
13-acetate;
TPCK, N-tosylphenylalanine chloromethyl ketone;
PKC, protein kinase C;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
PBS, phosphate-buffered saline.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Giros, B.,
Jaber, M.,
Jones, S. R.,
Wightman, R. M.,
and Caron, M. G.
(1996)
Nature
379,
606-612[CrossRef][Medline]
[Order article via Infotrieve]
2.
Amara, S. G.,
and Kuhar, M. J.
(1993)
Annu. Rev. Neurosci.
16,
73-93[Medline]
[Order article via Infotrieve]
3.
Povlock, S. L.,
and Amara, S. G.
(1997)
in
Neurotransmitter Transporters, Structure, Function, and Regulation
(Reith, M. E. A., ed)
, pp. 1-28, Humana Press, Inc., Totowa, NJ
4.
Schuldiner, S.,
Lebendiker, M.,
and Yerushalmi, H.
(1997)
J. Exp. Biol.
200,
335-341[Abstract]
5.
Vaughan, R. A.,
Huff, R. A.,
Uhl, G. R.,
and Kuhar, M. J.
(1997)
J. Biol. Chem.
272,
15541-15546 6.
Qian, Y.,
Galli, A.,
Ramamoorthy, S.,
Risso, S.,
DeFelice, L. J.,
and Blakely, R. D.
(1997)
J. Neurosci.
17,
45-57 7.
Ramamoorthy, S.,
and Blakely, R. D.
(1999)
Science
285,
763-766 8.
Ramamoorthy, S.,
Giovanetti, E.,
Qian, Y.,
and Blakely, R. D.
(1998)
J. Biol. Chem.
273,
2458-2466 9.
Law, R. M.,
Stafford, A.,
and Quick, M. W.
(2000)
J. Biol. Chem.
275,
23986-23991 10.
Huff, R. A.,
Vaughan, R. A.,
Kuhar, M. J.,
and Uhl, G. R.
(1997)
J. Neurochem.
68,
225-232[Medline]
[Order article via Infotrieve]
11.
Copeland, B. J.,
Vogelsberg, V.,
Neff, N. H.,
and Hadjiconstantinou, M.
(1996)
J. Pharmacol. Exp. Ther.
277,
1527-1532 12.
Melikian, H. E.,
and Buckley, K. M.
(1999)
J. Neurosci.
19,
7699-7710 13.
Daniels, G. M.,
and Amara, S. G.
(1999)
J. Biol. Chem.
274,
35794-35801 14.
Corey, J. L.,
Davidson, N.,
Lester, H. A.,
Brecha, N.,
and Quick, M. W.
(1994)
J. Biol. Chem.
269,
14759-14767 15.
Sato, K.,
Adams, R.,
Betz, H.,
and Schloss, P.
(1995)
J. Neurochem.
65,
1967-1973[Medline]
[Order article via Infotrieve]
16.
Chang, M. Y.,
Lee, S. H.,
Kim, J. H.,
Lee, K. H.,
Kim, Y. S.,
Son, H.,
and Lee, Y. S.
(2001)
J. Neurochem.
77,
754-761[CrossRef][Medline]
[Order article via Infotrieve]
17.
Halpain, S.,
Girault, J. A.,
and Greengard, P.
(1990)
Nature
343,
369-372[CrossRef][Medline]
[Order article via Infotrieve]
18.
Vaughan, R. A.,
and Kuhar, M. J.
(1996)
J. Biol. Chem.
271,
21672-21680 19.
Boyle, W. J.,
van der Geer, P.,
and Hunter, T.
(1991)
Methods Enzymol.
201,
110-149[Medline]
[Order article via Infotrieve]
20.
Schagger, H.,
and von Jagow, G.
(1987)
Anal. Biochem.
166,
368-379[CrossRef][Medline]
[Order article via Infotrieve]
21.
Vaughan, R. A.
(1995)
Mol. Pharmacol.
47,
956-964[Abstract]
22.
Fontana, A.,
and Gross, E.
(1986)
in
Practical Protein Chemistry: A Handbook
(Darbre, A., ed)
, pp. 68-120, John Wiley & Sons, Inc., New York
23.
Thompson, A. C.,
Zapata, A.,
Justice, J. B., Jr.,
Vaughan, R. A.,
Sharpe, L. G.,
and Shippenberg, T. S.
(2000)
J. Neurosci.
20,
9333-9340 24.
Doolen, S.,
and Zahniser, N. R.
(2001)
J. Pharmacol. Exp. Ther.
296,
931-938 25.
Simon, J. R.,
Bare, D. J.,
Ghetti, B.,
and Richter, J. A.
(1997)
Neurosci. Lett.
224,
201-205[CrossRef][Medline]
[Order article via Infotrieve]
26.
Apparsundaram, S.,
Galli, A.,
DeFelice, L. J.,
Hartzell, H. C.,
and Blakely, R. D.
(1998)
J. Pharmacol. Exp. Ther.
287,
733-743 27.
Cho, G. W.,
Kim, M. H.,
Chai, Y. G.,
Gilmor, M. L.,
Levey, A. I.,
and Hersh, L. B.
(2000)
J. Biol. Chem.
275,
19942-19948 28.
Krantz, D. E.,
Peter, D.,
Liu, Y.,
and Edwards, R. H.
(1997)
J. Biol. Chem.
272,
6752-6759 29.
Krantz, D. E.,
Waites, C.,
Oorschot, V.,
Liu, Y.,
Wilson, R. I.,
Tan, P. K.,
Klumperman, J.,
and Edwards, R. H.
(2000)
J. Cell Biol.
149,
379-396 30.
Vaughan, R. A.
(2000)
in
Cerebral Signal Transduction: From First to Fourth Messengers
(Reith, M. E. A., ed)
, pp. 375-400, Humana Press Inc., Totowa, NJ
31.
Bauman, A. L.,
Apparsundaram, S.,
Ramamoorthy, S.,
Wadzinski, B. E.,
Vaughan, R. A.,
and Blakely, R. D.
(2000)
J. Neurosci.
20,
7571-7578 32.
Vaughan, R. A.,
and Pananusorn, B.
(2001)
Soc. Neurosci. Abstr.
27,
1865
33.
Buckley, K. M.,
Melikian, H. E.,
Provoda, C. J.,
and Waring, M. T.
(2000)
J. Physiol. (Lond.)
525,
11-19 34.
Ferguson, S. S.
(2001)
Pharmacol. Rev.
53,
1-24 35.
Beckman, M. L.,
Bernstein, E. M.,
and Quick, M. W.
(1998)
J. Neurosci.
18,
6103-6112 36.
Deken, S. L.,
Beckman, M. L.,
Boos, L.,
and Quick, M. W.
(2000)
Nat. Neurosci.
3,
998-1003[CrossRef][Medline]
[Order article via Infotrieve]
37.
Horton, N.,
and Quick, M. W.
(2001)
Mol. Membr. Biol.
18,
39-44[Medline]
[Order article via Infotrieve]
38.
Hastrup, H.,
Karlin, A.,
and Javitch, J. A.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
10055-10060 39.
Kilic, F.,
and Rudnick, G.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3106-3111 40.
Kamsteeg, E. J.,
Heijnen, I.,
van Os, C. H.,
and Deen, P. M.
(2000)
J. Cell Biol.
151,
919-930
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Guptaroy, M. Zhang, E. Bowton, F. Binda, L. Shi, H. Weinstein, A. Galli, J. A. Javitch, R. R. Neubig, and M. E. Gnegy A Juxtamembrane Mutation in the N Terminus of the Dopamine Transporter Induces Preference for an Inward-Facing Conformation Mol. Pharmacol., March 1, 2009; 75(3): 514 - 524. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Goodwin, G. A. Larson, J. Swant, N. Sen, J. A. Javitch, N. R. Zahniser, L. J. De Felice, and H. Khoshbouei Amphetamine and Methamphetamine Differentially Affect Dopamine Transporters in Vitro and in Vivo J. Biol. Chem., January 30, 2009; 284(5): 2978 - 2989. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Samuvel, L. D. Jayanthi, S. Manohar, K. Kaliyaperumal, R. E. See, and S. Ramamoorthy Dysregulation of Dopamine Transporter Trafficking and Function after Abstinence from Cocaine Self-Administration in Rats: Evidence for Differential Regulation in Caudate Putamen and Nucleus Accumbens J. Pharmacol. Exp. Ther., April 1, 2008; 325(1): 293 - 301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Parnas, J. D. Gaffaney, M. F. Zou, J. R. Lever, A. H. Newman, and R. A. Vaughan Labeling of Dopamine Transporter Transmembrane Domain 1 with the Tropane Ligand N-[4-(4-Azido-3-[125I]iodophenyl)butyl]-2{beta}-carbomethoxy-3{beta}-(4-chlorophenyl)tropane Implicates Proximity of Cocaine and Substrate Active Sites Mol. Pharmacol., April 1, 2008; 73(4): 1141 - 1150. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Zapata, B. Kivell, Y. Han, J. A. Javitch, E. A. Bolan, D. Kuraguntla, V. Jaligam, M. Oz, L. D. Jayanthi, D. J. Samuvel, et al. Regulation of Dopamine Transporter Function and Cell Surface Expression by D3 Dopamine Receptors J. Biol. Chem., December 7, 2007; 282(49): 35842 - 35854. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Bolan, B. Kivell, V. Jaligam, M. Oz, L. D. Jayanthi, Y. Han, N. Sen, E. Urizar, I. Gomes, L. A. Devi, et al. D2 Receptors Regulate Dopamine Transporter Function via an Extracellular Signal-Regulated Kinases 1 and 2-Dependent and Phosphoinositide 3 Kinase-Independent Mechanism Mol. Pharmacol., May 1, 2007; 71(5): 1222 - 1232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Miranda, K. R. Dionne, T. Sorkina, and A. Sorkin Three Ubiquitin Conjugation Sites in the Amino Terminus of the Dopamine Transporter Mediate Protein Kinase C-dependent Endocytosis of the Transporter Mol. Biol. Cell, January 1, 2007; 18(1): 313 - 323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. D. Jayanthi, B. Annamalai, D. J. Samuvel, U. Gether, and S. Ramamoorthy Phosphorylation of the Norepinephrine Transporter at Threonine 258 and Serine 259 Is Linked to Protein Kinase C-mediated Transporter Internalization J. Biol. Chem., August 18, 2006; 281(33): 23326 - 23340. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Cervinski, J. D. Foster, and R. A. Vaughan Psychoactive Substrates Stimulate Dopamine Transporter Phosphorylation and Down-regulation by Cocaine-sensitive and Protein Kinase C-dependent Mechanisms J. Biol. Chem., December 9, 2005; 280(49): 40442 - 40449. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Miranda, C. C. Wu, T. Sorkina, D. R. Korstjens, and A. Sorkin Enhanced Ubiquitylation and Accelerated Degradation of the Dopamine Transporter Mediated by Protein Kinase C J. Biol. Chem., October 21, 2005; 280(42): 35617 - 35624. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. D. Blakely, L. J. DeFelice, and A. Galli Biogenic Amine Neurotransmitter Transporters: Just When You Thought You Knew Them Physiology, August 1, 2005; 20(4): 225 - 231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. D. Jayanthi, D. J. Samuvel, R. D. Blakely, and S. Ramamoorthy Evidence for Biphasic Effects of Protein Kinase C on Serotonin Transporter Function, Endocytosis, and Phosphorylation Mol. Pharmacol., June 1, 2005; 67(6): 2077 - 2087. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Johnson, B. Guptaroy, D. Lund, S. Shamban, and M. E. Gnegy Regulation of Amphetamine-stimulated Dopamine Efflux by Protein Kinase C {beta} J. Biol. Chem., March 25, 2005; 280(12): 10914 - 10919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Vaughan Phosphorylation and Regulation of Psychostimulant-Sensitive Neurotransmitter Transporters J. Pharmacol. Exp. Ther., July 1, 2004; 310(1): 1 - 7. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Gnegy, H. Khoshbouei, K. A. Berg, J. A. Javitch, W. P. Clarke, M. Zhang, and A. Galli Intracellular Ca2+ Regulates Amphetamine-Induced Dopamine Efflux and Currents Mediated by the Human Dopamine Transporter Mol. Pharmacol., July 1, 2004; 66(1): 137 - 143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. D. Jayanthi, D. J. Samuvel, and S. Ramamoorthy Regulated Internalization and Phosphorylation of the Native Norepinephrine Transporter in Response to Phorbol Esters: EVIDENCE FOR LOCALIZATION IN LIPID RAFTS AND LIPID RAFT-MEDIATED INTERNALIZATION J. Biol. Chem., April 30, 2004; 279(18): 19315 - 19326. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. I. Gonzalez, P. G. Bannerman, and M. B. Robinson Phorbol Myristate Acetate-Dependent Interaction of Protein Kinase C{alpha} and the Neuronal Glutamate Transporter EAAC1 J. Neurosci., July 2, 2003; 23(13): 5589 - 5593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. K. Loder and H. E. Melikian The Dopamine Transporter Constitutively Internalizes and Recycles in a Protein Kinase C-regulated Manner in Stably Transfected PC12 Cell Lines J. Biol. Chem., June 6, 2003; 278(24): 22168 - 22174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Lin, P.-W. Zhang, X. Zhu, J.-M. Melgari, R. Huff, R. L. Spieldoch, and G. R. Uhl Phosphatidylinositol 3-Kinase, Protein Kinase C, and MEK1/2 Kinase Regulation of Dopamine Transporters (DAT) Require N-terminal DAT Phosphoacceptor Sites J. Biol. Chem., May 23, 2003; 278(22): 20162 - 20170. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Granas, J. Ferrer, C. J. Loland, J. A. Javitch, and U. Gether N-terminal Truncation of the Dopamine Transporter Abolishes Phorbol Ester- and Substance P Receptor-stimulated Phosphorylation without Impairing Transporter Internalization J. Biol. Chem., February 7, 2003; 278(7): 4990 - 5000. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |