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Regulation of Dopamine Transporter Function and Cell
Surface Expression by D3 Dopamine Receptors
*
Received for publication, December 22, 2006, and in revised form, September 28, 2007 Published, JBC Papers in Press, October 8, 2007, DOI 10.1074/jbc.M611758200
Agustin Zapata
‡1
, Bronwyn Kivell
‡1
, Yang Han
§
, Jonathan A. Javitch
§
, Elizabeth A. Bolan
‡
, David Kuraguntla
‡
,
Vanaja Jaligam
‡
, Murat Oz
‡
, Lankupalle D. Jayanthi
¶
, Devadoss J. Samuvel
¶
, Sammanda Ramamoorthy
¶2
,
and Toni S. Shippenberg
‡2,3
From the
‡
Integrative Neuroscience Section, National Institutes of Health/National Institute on Drug Abuse Intramural Research
Program/Department of Health and Human Services, Baltimore, Maryland 21224, the
§
Departments of Psychiatry and
Pharmacology, Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University,
New York, New York 10032, and the
¶
Department of Neurosciences, Division of Neuroscience Research,
Medical University of South Carolina, Charleston, South Carolina 29425
D
3
dopamine receptors are expressed by dopamine neurons
and are implicated in the modulation of presynaptic dopamine
neurotransmission. The mechanisms underlying this modulation
remain ill defined. The dopamine transporter, which terminates
dopamine transmission via reuptake of released neurotransmitter,
is regulated by receptor- and second messenger-linked sig-
naling pathways. Whether D3 receptors regulate dopamine
transporter function is unknown. We addressed this issue
using a fluorescent imaging technique that permits real time
quantification of dopamine transporter function in living sin-
gle cells. Accumulation of the fluorescent dopamine trans-
porter substrate trans-4-[4-(dimethylamino)styryl]-1-meth-
ylpyridinium (ASP
ⴙ
) in human embryonic kidney cells
expressing human dopamine transporter was saturable and
temperature-dependent. In cells co-expressing dopamine
transporter and D3 receptors, the D2/D3 agonist quinpirole
produced a rapid, concentration-dependent, and pertussis
toxin-sensitive increase of ASP
ⴙ
uptake. Similar agonist
effects were observed in Neuro2A cells and replicated in
human embryonic kidney cells using a radioligand uptake
assay in which binding to and activation of D3 receptors by
[
3
H]dopamine was prevented. D3 receptor stimulation acti-
vated phosphoinositide 3-kinase and MAPK. Inhibition of
either kinase prevented the quinpirole-induced increase in
uptake. D3 receptor activation differentially affected dopa-
mine transporter function and subcellular distribution
depending on the duration of agonist exposure. Biotinylation
experiments revealed that the rapid increase of uptake was
associated with increased cell surface and decreased intracel-
lular expression and increased dopamine transporter exocy-
tosis. In contrast, prolonged agonist exposure reduced
uptake and transporter cell surface expression. These results
demonstrate that D3 receptors regulate dopamine trans-
porter function and identify a novel mechanism by which D3
receptors regulate extracellular dopamine concentrations.
The D
3
dopamine (DA)
4
receptor, a member of the D2-like
family of DA receptors, is expressed in limbic brain regions,
both presynaptically on DA neurons as well as postsynaptically.
The D3 receptor has gained increasing attention as a target for
the treatment of schizophrenia, psycho-stimulant abuse, and
DA cell neurodegeneration (1– 4). Its restricted central nervous
system distribution, relative to D2 receptors, suggests that D3
receptor ligands may have fewer side effects than currently
available therapeutic agents.
Studies using D3 receptor knock-out mice (5–7) or D3 anti-
sense (8, 9) revealed that D3 receptors regulate extracellular DA
in ventral striatum. This effect was attributed to D3 regulation
of a long negative feedback loop in which postsynaptic D3
receptors on medium spiny neurons modulate the activity of
accumbens output neurons projecting to DA cell bodies in
mid-brain (5). However, this hypothesis is incompatible with
the effects of DA receptor ligands in tissue preparations in
which efferent projections to midbrain DA nuclei are dis-
rupted; modulation of extracellular DA by D3 receptors has
been demonstrated in striatal slices (7) and tissue suspen-
sions (10). Pharmacological studies examining the mecha-
nism of such regulation have been precluded by the lack of
selective ligands that discriminate between D2 and D3
receptors in vivo (5, 11–13).
DA signaling is terminated by the DA transporter (DAT), an
integral membrane protein that re-uptakes DA released into
the extracellular space (14). Receptor and second messenger-
linked kinase cascades regulate DAT function and cell surface
* This work was supported by the National Institutes of Health/National Insti-
tute on Drug Abuse (NIDA) Intramural Research Program and National
Institute on Drug Abuse and National Institutes of Health Grants
P50DA015369, MH062612 (to S. R.), GM081054 (to L. D. J.), MH57324,
MH54137, and DA11495 (to J. A. J.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
1
Both authors contributed equally and should be considered first authors.
2
Both authors contributed equally and should be considered senior authors.
3
To whom correspondence should be addressed: Integrative Neuroscience
Section, NIDA, 5500 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-550-
1514; E-mail: tshippen@intra.nida.nih.gov.
4
The abbreviations used are: DA, dopamine; DAT, DA transporter; ANOVA,
analysis of variance; ELISA, enzyme-linked immunosorbent assay; PKC,
protein kinase C; MAPK, mitogen-activated protein kinase; PI3K, phosphoi-
nositide 3-kinase; DMEM, Dulbecco’s modified Eagle’s medium; BSA,
bovine serum albumin; PBS, phosphate-buffered saline; MesNa, sodium
2-mercaptoethanesulfonate; TfR, transferrin receptor; h, human; ASP
⫹
,
trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium; YFP, yellow fluo-
rescent protein; GFP, green fluorescent protein; df, degrees of freedom;
MEK, MAPK/ERK kinase.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 49, pp. 35842–35854, December 7, 2007
Printed in the U.S.A.
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expression (15). An involvement of D2 DA receptors in regu-
lating DAT function was suggested previously (16 –18). Con-
sistent with this hypothesis, DA uptake and transporter-asso-
ciated currents are increased in oocytes (19) co-expressing
human DAT (hDAT) and D2 receptors, and D2 receptor stim-
ulation increases DAT activity in transfected HEK293 cells (20).
Recently, modulation of DA uptake by low concentrations of
D3-preferring ligands was reported in vivo (10). Such findings
suggest that D3 receptors may regulate DA neurotransmission
via a DAT-mediated mechanism. However, because the brain
regions examined contain D2 and D3 receptors and the ligands
employed bind to both receptor subtypes, the role of D3 recep-
tors remains unclear.
The present studies aimed to determine whether D3 recep-
tors regulate DAT function in heterologous expression systems
and to identify the intracellular mechanisms mediating this
effect. We show that acute D3 receptor activation increases
DAT activity in human embryonic kidney and mouse neuro-
blastoma cells co-expressing hDAT and human D3 receptors
(hD3). This acute effect is pertussis toxin-sensitive and requires
the activation of phosphoinositide 3-kinase (PI3K) and mito-
gen-activated protein kinase (MAPK). Interestingly, sustained
D3 receptor activation has opposite effects, decreasing DAT
activity. Furthermore, biotinylation studies show that D3
receptor stimulation regulates DAT trafficking by affecting
both DAT endocytosis and exocytosis.
EXPERIMENTAL PROCEDURES
Materials—2-(2-Amino-3-methyoxyphenyl)-4H-1-benzopy-
ran-4-one (PD98059), (S)-(⫹)-(4aR10bR)-3,4,4a,10b-tetra-
hydro- 4- propyl - 2 H,5 H[ 1] benzopyrano [4 ,3b] - 1,4 -oxazin-9-
ol hydrochloride (PD128907), 2-(4-morpholinyl)-8-phenyl-
4H-1-benzopyran-4-one hydrochloride (LY294002), 2-[1-
(3-dimethylamino-propyl)indol-3-yl]-3-(indol-3-yl) maleimide
(GF109203X), nomifensine, spiperone, and quinpirole were
obtained from Tocris Cookson Inc. (Ellisville, MO). ASP
⫹
and
R(⫹)-6-bromo-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5 tetrahy-
dro-1H-3-benzazepine hydrobromide (B135) were obtained from
Sigma.
Cell Culture—Experiments were conducted in EM4 cells (R.
Horlick, Pharmacopeia, Cranberry, NJ), HEK293 cells stably
expressing macrophage scavenger receptor (21), and Neuro2A
cells (N2A, American Type Culture Collection) unless other-
wise indicated. ASP
⫹
experiments were performed in EM4 cells
stably transfected with FLAG-hDAT (22) and transiently trans-
fected with GFP-tagged human D3 (hD3) receptors (M. G.
Caron, Duke University Medical Center, Durham, NC) and in
EM4 or N2A cells transiently co-transfected with GFP-hD3
receptors and either FLAG-hDAT or YFP-hDAT (23). Previous
studies have shown that the addition of these tags does not alter
the function of these proteins (22–26).
EM4 and N2A cells were maintained in DMEM/F-12 and
minimum Eagle’s medium (Cellgro威, Mediatech, Inc, Herndon,
VA), respectively, and supplemented with 10% fetal bovine
serum (Invitrogen). Cells were seeded on day 1 at 1.5 ⫻ 10
5
cells/35-mm glass-bottomed Petri dish (BiOptechs, Butler,
PA), and transient transfections were performed on day 2
using Lipofectamine 2000. Experiments were performed
32–48 h after transfection when cells were 70 –90% conflu-
ent. Cells were grown in a humidified atmosphere at 37 °C
and 5% CO
2
.
[
3
H]DA Uptake—EM4 cells were transiently transfected with
the appropriate vector (FLAG-DAT, myc-hD3, and FLAG-
DAT plus myc-hD3 receptor and pcDNA3 plasmids) as indi-
cated in figures and legends. DA uptake was performed as
described. Briefly, cells were washed with 1.0 ml of Krebs-Ring-
er-HEPES (KRH) buffer, pH 7.4 (120 m
M NaCl, 4.7 mM KCl, 2.2
m
M CaCl
2
,10mM HEPES, 1.2 mM MgSO
4
, 1.2 mM KH
2
PO
4
,5
m
M Tris, and 10 mMD-glucose), containing 0.1 mM ascorbic
acid and 0.1 m
M pargyline. Cells were then preincubated with
the modulators for the indicated times followed by the addition
of 20 n
M [
3
H]DA to initiate DA uptake for 1 min. Uptake was
terminated after 1.0 min of incubation at 22 °C by rapid wash-
ings with cold KRH assay buffer. Cells were lysed in 0.1% SDS,
and accumulated radioactivity was measured by liquid scintil-
lation. Initial studies revealed that part of the [
3
H]DA signal in
cells transfected with hDAT plus hD3 was insensitive to the
DAT blocker nomifensine but was blocked by the D3 antago-
nist spiperone or agonist quinpirole (Fig. 1, A and B, see
“Results”). Therefore, for each experiment specific [
3
H]DA
binding to the hD3 receptor was obtained from cells transfected
with hD3 receptor plasmid alone or DAT plus hD3 receptor
plasmids in the presence of 50
M nomifensine. DAT-specific
[
3
H]DA uptake was defined as the accumulation of [
3
H]DA in
the presence of 0.1
M spiperone (no receptor stimulation) or
10
M quinpirole (maximal receptor stimulation). Note that
[
3
H]DA signal obtained in the presence of spiperone or quinpi
-
role was sensitive to DAT blocker nomifensine indicative of
DAT-specific [
3
H]DA uptake. Nonspecific [
3
H]DA uptake and
binding was defined as the accumulation/binding in the pres-
ence of both spiperone (or quinpirole) and nomifensine and
was subtracted from total counts. Nonspecific background was
also compared with cells transfected with pcDNA3 vector
alone.
Cell-based ELISA for pERK Activation—A phosphospecific
cell-based ELISA (modified from Versteeg et al. (71)) for phos-
phorylated ERK1/2 (p-ERK) was used to determine whether
DA (1 p
M to 10
M) or the DAT substrates, tyramine (1 pM to
100
M) and ASP
⫹
(1 pM to 100
M), activate D3 receptors.
Flp-in T-rex 293 cells (Invitrogen) stably expressing FLAG-
tagged hD3 receptors were used. Cells were seeded in 96-well
plates (50,000 cells/cm
2
) in DMEM supplemented with 10%
fetal bovine serum and grown at 37 °C and 5% CO
2
for 24 h.
Tetracycline (0.1
M) was added for an additional 16 –20 h to
induce hD3 receptor expression. Cells were serum-starved in
serum-free media for 2 h prior to substrate addition (100
l).
After 3 min, media were aspirated, and cells were immediately
fixed by adding 150
l of 4% formaldehyde/PBS solution for 30
min. Cells were permeabilized by addition of 0.1% Triton
X-100/PBS solution (three times for 10 min). After blocking
with 10% BSA in Triton/PBS solution for 1 h, cells were washed
with Triton/PBS solution. Primary p-ERK monoclonal anti-
body (Cell Signaling Technology) was diluted 1:400 in Triton/
PBS solution containing 5% BSA, and cells were incubated with
primary antibody for1hatroom temperature. After washing
cells three times with PBS/Triton solution, and1hofincuba-
D3 Receptor Regulation of Dopamine Transporter
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tion with horseradish peroxidase-conjugated goat anti-mouse
2° antibody (1:1000 dilution; Santa Cruz Biotechnology) in
PBS/Triton solution containing 5% BSA, cells were again
washed with Triton/PBS solution. Pierce supersignal ELISA
Pico substrate (100
l) was added to each well for 2 min, and
the luminescence signal was measured with a BMG PolarStar
reader. The values from each experiment were normalized to
the basal level for that experiment. The means ⫾ S.E. are
shown for these determinations from three independent
experiments, each performed in triplicate. The data were
analyzed with GraphPad Prism using a one-site sigmoidal
dose-response curve.
Live Cell Imaging of ASP
⫹
Uptake—Time-resolved quantifi
-
cation of DAT function in single cells was achieved using the
fluorescent, high affinity DAT substrate ASP
⫹
(27). Immedi
-
ately prior to experiments, growth media were removed, and
cells were washed two times in Krebs-Ringer/HEPES medium
(KRH (in m
M): 130 NaCl, 1.3 KCl, 2.2 CaCl
2
, 1.2 MgSO
4
, 1.2
KH
2
PO
4
, 10 HEPES, and 1.8 g/liter glucose, pH 7.4). After
washing, fresh KRH was added, and the culture dish was
mounted on an UltraVIEW
TM
LCI spinning-disk confocal
microscope fitted with a ⫻60 water objective lens (PerkinElmer
Life Sciences).
A within cell design was used to assess the effects of the
D2/D3 agonists, quinpirole (0.1–10
M) and PD12897 (10
M)
on ASP
⫹
uptake. Immediately after mounting of the culture
dish, the microscope was focused on the center of a monolayer
of cells, and background auto-fluorescence was determined by
collecting an image immediately prior to replacement of the
KRH buffer with buffer containing ASP
⫹
(10
M). Vehicle or
agonist was added 5 min later, and the slope of ASP
⫹
accumu
-
lation was determined over a 1-min period both before and
after their addition. Control studies showed that ASP
⫹
uptake
by DAT was linear over the first 10 min after ASP
⫹
addition
(28). Images were collected every 20 s for 10 min to enable
capture of either GFP or YFP (excitation, 488 nm; emission,
525–575 nm) and ASP
⫹
fluorescence (excitation, 488 nm;
emission, 607– 652 nm). A between cell design was used for
control experiments assessing saturability, temperature
dependence, and the influence of DAT substrates and blockers
on ASP
⫹
uptake. Cells were preincubated with test drug or
vehicle for the designated time periods, and total intracellular
ASP
⫹
accumulation over a 5-min time period was quantified.
Saturation of ASP
⫹
uptake was tested using a range of ASP
⫹
concentrations (5–70
M), and temperature dependence was
determined by measuring total ASP
⫹
accumulation at 22 and
6 °C. The influence of the DAT substrates d-amphetamine
(0.1–10
M), dopamine (0.1–10
M), or cocaine (3–15
M)on
ASP
⫹
uptake was examined by preincubating cells for 15 min
and then adding ASP
⫹
and measuring total intracellular ASP
⫹
accumulation for 5 min in the continued presence of substrate
or inhibitor. The sodium dependence was investigated by
measuring ASP
⫹
uptake in the presence or absence of the
monovalent cation channel-forming peptide gramicidin (10
g/ml, 10 min of preincubation) (29). The influence of the
MAPK inhibitor PD98059 (30), the PI3K inhibitor LY294002
(31), and the protein kinase C inhibitor GF109203X (32) on
quinpirole (10
M)-evoked ASP
⫹
uptake was assessed by pre
-
incubating cells with vehicle or inhibitor for 15 min. ASP
⫹
was
then added, and the effects of the D3 agonist on the rate of
intracellular ASP
⫹
accumulation were determined using a
within cell design as described above, in the presence or
absence of the corresponding kinase inhibitor. The concentra-
tions of inhibitors used were chosen based on reported effective
concentrations (30 –32).
Fluorescent images were processed using MetaMorph or
ImageJ software (W. Rasband, National Institutes of Health).
For ASP
⫹
uptake, fluorescent accumulation within the cell was
measured as the average pixel intensity of time-resolved
images. The intracellular fluorescence is the signal contained
inside the cell as defined by the plasma membrane. The plasma
membrane was identified by either GFP-D3 or YFP-DAT visu-
alization (which are localized mainly in the plasma membrane)
or bright field optics (in the case of FLAG-hDAT or N2A cells).
Data are expressed as arbitrary fluorescence units or as a per-
cent change in the rate of ASP
⫹
uptake after drug addition. The
total intracellular ASP
⫹
fluorescence 5 min after addition of
ASP
⫹
was used as a measure of basal uptake (ASP
⫹
accumula
-
tion over 5 min, see Figs. 2 and 3). To measure drug-induced
effects on uptake, the rate of ASP
⫹
fluorescence accumulation
during the minutes before and after drug addition was calculated
as the increase in intracellular ASP
⫹
signal over that min. The
effect of the drug was calculated as the percent change in the rate of
ASP
⫹
accumulation before and after the drug addition. Typically
10–100 cells were used for each experiment. These cells were
from at least two culture dishes from three separate transfec-
tion experiments. Data are expressed as mean ⫾ S.E. and ana-
lyzed by ANOVA followed by the Student-Newman-Keuls test
for multiple comparisons between groups. Statistical signifi-
cance was achieved at p ⬍ 0.05.
Immunoblotting of Kinases—Cells were transfected as
described above and serum-starved in serum-free media 16 –18
h before the assay. On the day of the assay, cells were treated for
15 min with kinase inhibitor or vehicle prior to stimulation with
quinpirole (10
M) for 1 min at 37 °C. After incubation, media
were aspirated, and boiling Laemmli buffer (62.5 m
M Tris, pH
6.8, 20% glycerol, 2% SDS, 5%

-mercaptoethanol, and 0.01%
bromphenol blue) was added directly to the wells. Lysates were
then collected and boiled for 10 min. For DAT protein immu-
noblot analysis, samples were incubated with Laemmli buffer
for 30 min at room temperature (see below). Proteins were sep-
arated by SDS-PAGE (4 –20% Duramide gradient gel; Cambrex,
Walkersville, MD) and blotted onto polyvinylidene difluoride
(Millipore, Bellerica, MA). The membranes were then incu-
bated for1hatroom temperature in TBS-T containing 5%
nonfat milk. Phosphorylated MAPK was detected using a rabbit
polyclonal antibody specific for p44/42 MAPK phosphorylated
at residues threonine 202 and tyrosine 204 (p-ERK). Phospho-
rylated Akt was detected utilizing a rabbit polyclonal antibody
that detects levels of Akt only when phosphorylated at serine
473 (p-Akt). To control for differences in protein loading, blots
were stripped with 2% SDS, 100 m
M

-mercaptoethanol in 62.5
m
M Tris, pH 6.8, for1hat50°Candreprobed with a polyclonal
antibody that recognizes total p44/42 MAPK or total Akt. All
kinase antibodies were purchased from Cell Signaling Tech-
nology, Inc. (Beverly, MA). Blots were visualized using a
D3 Receptor Regulation of Dopamine Transporter
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horseradish peroxidase-conjugated secondary antibody
(Upstate Biotechnology, Inc., Lake Placid, NY) with
enhanced chemiluminescence reagents.
For quantification, films were scanned at high resolution
using a Scanjet 7400c (Hewlett-Packard, Palo Alto, CA), and
band densities were quantified using MetaMorph software
(Molecular Devices, Downingtown, PA). The relative amounts
of phosphorylated kinases were normalized against those of the
corresponding total kinase. Data were analyzed by one-way
ANOVA followed by Student-Newman-Keuls Multiple Com-
parison post hoc test using the GraphPad Prism version 4.0.
The significance level for all analyses was p ⱕ 0.05.
Surface Biotinylation of DAT—Cell surface biotinylation and
immunoblot analyses were performed as described (33) to
quantify the amount of plasma membrane DAT protein. EM4
cells (1 ⫻ 10
5
cells/well) were seeded into 12-well plates con
-
taining DMEM/F-12 medium supplemented with 10% fetal
bovine serum and penicillin (100 units/ml) and streptomycin
(100
g/ml) in an atmosphere of 5% CO
2
and 95% O
2
at 37 °C.
After 24 h, cells were transfected with FLAG-hDAT and myc-
hD3 receptor cDNA plasmids (0.9
g of D3 receptor and 0.3
g
of DAT) using Lipofectamine
TM
2000 according to the manu
-
facturer’s protocol. In all wells, the total amount of plasmid
DNA was adjusted with the corresponding empty vector.
Where indicated, cells were treated with different modulators
24 h after transfection, as described in the figure legends. At the
end of the treatment, cells were washed two times with ice-cold
PBS/Ca-Mg (138 m
M NaCl, 2.7 mM KCl, 1.5 mM KH
2
PO
4
, 9.6
m
M Na
2
HPO
4
,1mM MgCl
2
, 0.1 mM CaCl
2
, pH 7.3) and incu
-
bated with EZ link NHS-Sulfo-SS-biotin (1 mg/ml) in PBS/
Ca-Mg for 30 min at 4 °C. The reaction was quenched by two
washes with cold 100 m
M glycine in PBS/Ca-Mg and further
incubation with 100 m
M glycine in PBS/Ca-Mg at 4 °C for 20
min. The cells were then lysed in 500
l of radioimmunopre-
cipitation assay (RIPA) buffer (10 m
M Tris-HCl, pH 7.5, 150 mM
NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, and 0.1%
sodium deoxycholic acid) containing protease inhibitors (1
M
pepstatin A, 250
M phenylmethylsulfonyl fluoride, 1
g/ml
leupeptin, and 1
g/ml aprotinin) for1hat4°Cwith constant
shaking. Lysates were centrifuged at 25,000 ⫻ g for 30 min at
4 °C, and supernatants were incubated with streptavidin beads
(100
l of beads/400
l of cell lysates from one well) for1hat
room temperature. Beads were washed three times with RIPA
buffer, and bound proteins were eluted with 50
l of Laemmli
buffer (62.5 m
M Tris, pH 6.8, 20% glycerol, 2% SDS, 5%

-mer-
captoethanol, and 0.01% bromphenol blue) for 30 min at 22 °C.
Aliquots from total cell lysates (50
l) and unbound fractions
(100
l), and all (50
l) of the streptavidin-bound samples were
analyzed by immunoblotting with a monoclonal DAT-specific
antibody (Chemicon, Temecula, CA). To validate the surface
localization of biotinylated DAT protein, blots were stripped
and reprobed with anticalnexin antibody (StressGen Biotech-
nologies, Victoria, British Columbia, Canada). Band intensities
were quantified using NIH Image J software (version 1.32j).
Exposures were precalibrated to ensure quantitation within the
linear range of the film, and multiple exposures were taken to
validate linearity of quantitation. DAT densities from total,
nonbiotinylated (representing the intracellular pool), and bio-
tinylated fractions (representing the surface pool) were normal-
ized using levels of calnexin in the total extract, and values were
averaged across five experiments. Data were analyzed by
ANOVA followed by the Student-Newman-Keuls test for com-
parisons between groups.
Reversible Biotinylation to Determine DAT Internalization—
A reversible biotinylation strategy was used to quantify DAT
internalization (34, 35). The rationale for this method is based
on initial labeling of the extracellular DAT pool (under traffick-
ing inhibiting conditions at 4 °C). Then after washing out the
free biotin label, incubations are conducted under trafficking
permissive conditions (22 °C) to allow for internalization of the
biotin-labeled DAT. Finally, at the end of the incubation, traf-
ficking is stopped (at 4 °C), and the extracellular DAT-bound
biotin is stripped with a reducing agent (MesNa), leaving only
the intracellular biotinylated DAT pool, which represents DAT
that was initially on the plasma membrane and was internalized
during the incubation. Briefly, EM4 cells transiently transfected
with FLAG-DAT and myc-hD3 plasmids were prepared as
described under surface biotinylation. Cells were cooled rapidly
to 4 °C to inhibit protein trafficking by washing with cold PBS.
Cell surface proteins were biotinylated with a disulfide-cleav-
able biotin (sulfo-NHS-SS-biotin; Pierce), and free biotinylat-
ing reagent was removed by quenching with glycine. DAT
internalization was initiated by incubating the cells with pre-
warmed KRH buffer containing the vehicle or quinpirole (10
M) for 1 or 30 min at room temperature (in the absence of
NHS-SS-biotin). At the end of incubation, the reagents were
removed, and fresh pre-chilled KRH buffer was added to stop
trafficking. The cells were then washed and incubated twice
with 250
M MesNa, a reducing agent, in PBS/Ca-Mg for 20
min to dissociate the biotin from cell surface resident proteins
via disulfide exchange. To determine the total biotinylated
DATs, one dish of biotinylated cells per condition was not sub-
jected to reduction with MesNa and directly processed for
extraction followed by isolation by avidin beads. To determine
MesNa-accessible DAT proteins, another dish of cells was
treated with MesNa immediately (at 0 time) following biotiny-
lation at 4 °C to reveal the quantity of surface DAT biotinyla-
tion that MesNa can reverse efficiently. Cells were then sol-
ubilized in RIPA, and biotinylated DAT were separated by
using monomeric avidin beads. Biotinylated proteins were
eluted from beads and resolved by SDS-PAGE. DAT proteins
in the fractions were visualized as described under surface
DAT biotinylation.
Assay of DAT Insertion into the Plasma Membrane by
Biotinylation—The levels of DAT newly inserted into the
plasma membrane were measured using protocols similar to
those described previously (36). EM4 cells co-expressing
FLAG-hDAT and GFP-hD3 receptors were washed with PBS/
Ca-Mg and incubated twice with 1 mg/ml sulfo-NHS-acetate
(Pierce) in PBS/Ca-Mg for1hat4°Ctoblock all the free amino
groups under nonpermissive trafficking conditions (at 4 °C)
(37). After washing away the sulfo-NHS-acetate with cold PBS/
Ca-Mg, cell membrane-impermeable sulfo-NHS-biotin in PBS/
Ca-Mg containing vehicle or quinpirole (10
M) was added to
the cells and incubated further for 1 or 30 min at room temper-
ature. Because previously existing surface DAT has been
D3 Receptor Regulation of Dopamine Transporter
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blocked in the previous step, sulfo-NHS-biotin will label only
DAT newly delivered to the plasma membrane. To investigate
the influence of quinpirole on DAT plasma membrane inser-
tion, sulfo-NHS-acetate-treated cells were incubated with
either vehicle or quinpirole (10
M, 1 or 30 min) with NHS-SS-
sulfo-biotin present throughout the incubation period (34, 38).
At the end of the incubation period, cells were washed with cold
PBS/Ca-Mg containing glycine to quench the excess unbound
NHS-SS-biotin, and biotinylated DAT proteins were isolated in
streptavidin beads and analyzed as described above. Following
determination of DAT in the biotinylated fraction, the blots
were stripped and reprobed with transferrin receptor (TfR)
(Zymed Laboratories Inc.) and calnexin antibodies (StressGen,
Victoria, Canada). Levels of biotinylated TfR, a protein known
to recycle rapidly to and from the cell surface, were quantified
to confirm that the 22 °C condition permits protein trafficking.
Biotinylated calnexin levels were quantified to assess biotinyla-
tion of intracellular proteins (the presence of which would indi-
cate loss of plasma membrane integrity).
RESULTS
[
3
H]DA Uptake Studies—We first determined the ability of
D3 receptor agonist activation to modulate DAT activity as
determined by accumulation of [
3
H]DA in EM4 cells co-trans
-
fected with hD3 receptors and DAT. DA binds with high affin-
ity to D3 and D2 receptors (39). Therefore, a potential problem
for studies using [
3
H]DA for assessing DAT activity is that a
fraction of the radioligand is likely to bind and activate D3
receptors as well. To circumvent this problem, [
3
H]DA uptake
was determined in the presence (no receptor activation) or
absence (receptor activation by [
3
H]DA) of a saturating con
-
centration of a D3 receptor antagonist as described by Mayfield
and Zahniser (19). Incubation of cells with spiperone decreased
[
3
H]DA uptake relative to vehicle-treated cells suggesting that
D3 receptor activation (by [
3
H]DA) increased DAT activity
(Fig. 1A, F
5,18
⫽ 117.8, p ⬍ 0.0001; ANOVA, p ⬍ 0.001 spiper
-
one versus vehicle group, Student-Newman-Keuls). However,
incubation of cells with the DAT inhibitor, nomifensine, only
partially abolished [
3
H]DA uptake in vehicle-treated cells sug
-
gesting a non-DAT-mediated component. Control experi-
ments in cells transfected with either hD3 receptor or DAT
alone indicated that a significant part of the [
3
H] signal in cell
lysates was because of binding to D3 receptors rather than
DAT-mediated uptake, because it was present in cells trans-
fected with hD3 receptors alone (no DAT) and was completely
displaced by spiperone or quinpirole (Fig. 1B, F
5,18
⫽ 331.4, p ⬍
0.0001; ANOVA, p ⬍ 0.001 spiperone or quinpirole versus vehi-
cle, Student-Newman-Keuls). Thus, these findings suggest that
the decreased [
3
H]DA uptake observed in the presence of spip
-
erone in hD3 and DAT co-transfected cells may reflect dis-
placement of [
3
H]DA binding to D3 receptors by spiperone,
rather than an actual decrease in DAT activity. To address this
issue, uptake experiments were conducted in the presence of
either 0.1
M spiperone or 1
M quinpirole. We reasoned that
under these conditions hD3 receptor should be maximally
stimulated in the presence of quinpirole, and the presence of
spiperone should prevent receptor stimulation induced by the
addition of [
3
H]DA. Experiments in D3 only transfected cells
confirmed that the concentrations of spiperone and quinpirole
used were sufficient to displace all [
3
H]DA binding to D3 recep
-
tors (Fig. 1B). Using this protocol, we found that acute (1 min)
D3 receptor stimulation significantly increased DAT activity
(Fig. 1C, t ⫽⫺13.76, df ⫽ 6, p ⬍ 0.0001, t test). In contrast,
prolonged (30 min) receptor stimulation had the opposite
effect, resulting in a significant decrease in DAT activity (Fig.
1C, t ⫽⫺14.51, df ⫽ 6, p ⬍ 0.0001, t test).
Although we were able to demonstrate modulation of DAT
by D3 receptor activation using [
3
H]DA uptake, a DAT sub
-
strate that binds to and activates the receptor being investi-
gated, the complicated experimental design needed to rule out
confounding effects of substrate-receptor interactions is of lim-
ited utility. ASP
⫹
is a fluorescent analog of MPP
⫹
that binds
with high affinity to monoamine transporters. Recent studies
have shown that it can be used to monitor transporter function
in real time in single cells. Therefore, we tested whether ASP
⫹
activates D3 receptors by measuring MAPK activation. Addi-
tion of ASP
⫹
to Flp-in T-rex 293 cells stably expressing FLAG-
tagged hD3 receptors did not activate hD3 receptors at concen-
trations up to 100
M (Fig. 1D). Consistent with our [
3
H]DA
FIGURE 1. [
3
H]DA uptake in EM4 cells co-expressing hDAT and hD3 recep
-
tor. [
3
H]DA accumulation was measured during 1 min in the presence of the
specified drugs. Initial experiments identified two components of the
3
H sig
-
nal. One component is nomifensine-sensitive (uptake), and the other is spip-
erone-sensitive (binding to D3 receptors) (A). This was confirmed in EM4 cells
transfected with either the hD3 receptor or hDAT alone, indicating that a
significant fraction of the [
3
H]DA added bound to hD3 receptors (B). To avoid
confounding effects of this D3 receptor binding, subsequent [
3
H]DA uptake
experiments were carried out in the presence of 0.1
M spiperone (no recep-
tor activation) or 10
M quinpirole (maximal D3 receptor activation). These
experiments indicated that short (1 min) D3 receptor stimulation increased,
whereas sustained (30 min) stimulation decreased [
3
H]DA uptake (C). Charac
-
terization experiments with different DAT substrates using MAPK phospho-
rylation as an index of D3 receptor activation indicated that ASP
⫹
, in contrast
to either DA or tyramine, did not activate D3 receptors at concentrations up to
100
M (D). *, p ⬍ 0.05, versus control group, Newman-Keuls post hoc test;
**, p ⬍ 0.01.
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uptake studies, addition of DA resulted in a concentration-de-
pendent increase in pERK1/2 (EC
50
⫽ 4.7 nM; Fig. 1D). The
DAT substrate tyramine showed a similar maximal effect but
lower potency (EC
50
⫽ 1.4
M; Fig. 1D). Given these findings,
the ASP
⫹
imaging technique was used for subsequent studies.
Characterization of ASP
⫹
Uptake—ASP
⫹
rapidly accumu
-
lated in the cytoplasm of EM4 cells stably transfected with GFP-
hDAT (Fig. 2A) or FLAG-hDAT (not shown). Two distinct
phases of incorporation of the ASP
⫹
signal into DAT-express
-
ing cells are observed. Binding of ASP
⫹
to transporters located
on the cell surface is rapid (milliseconds to seconds) and is
followed by a second slower (seconds to minutes) phase of
accumulation of ASP
⫹
signal in the cytoplasm (Fig. 2, A and D,
and Fig. 3D, vehicle group). Intracellular accumulation is linear
for at least 10 min (Figs. 2D and 3D, vehicle group) and is neg-
ligible in control EM4 cells not expressing DAT (Fig. 2C). Anal-
ogous to [
3
H]DA assays, ASP
⫹
accumulation was saturable
(Fig. 2B) and temperature-dependent (Fig. 2C). Disruption of
the sodium gradient with gramicidin (10
g/ml) blocked
ASP
⫹
uptake (Fig. 2, D and E). These data indicate the
involvement of an active sodium-dependent uptake system.
Incubation of cells with either DA or d-amphetamine
resulted in a concentration-dependent decrease in ASP
⫹
uptake (Fig. 3A). The transporter blocker cocaine decreased
ASP
⫹
accumulation in a concentration-dependent manner
(Fig. 3B). These data are in accord with recent findings (40) and
confirm the utility of ASP
⫹
as a measure of DAT activity.
D3 Receptor Activation Modulates ASP
⫹
Uptake—To deter
-
mine whether D3 receptor activation alters DAT activity, EM4
cells stably expressing FLAG-hDAT were transiently trans-
fected with GFP-tagged hD3 receptors to enable receptor visu-
alization. The rate of accumulation of ASP
⫹
was measured
immediately before and after receptor activation. GFP-hD3
receptors were readily expressed and targeted to the plasma mem-
brane (Fig. 3C). Addition of quinpirole (10
M, Fig. 3D) induced a
rapid increase in ASP
⫹
uptake that was apparent within 1 min of
agonistexposure(Fig.3D).Quinpirolealsoinducedaconcentration-
dependent increase in the rate of ASP
⫹
accumulation in EM4 cells
transiently co-transfected with YFP-hDAT and myc-hD3 recep-
tors (Fig. 4A, F
3,155
⫽ 4.99, p ⫽ 0.0025; ANOVA). A structurally
different D2/D3 agonist, PD128907 (10
M), also induced a signif-
icant increase in ASP
⫹
uptake (33 ⫾ 8%; t ⫽ 4.81, df ⫽ 65, p ⬍
0.0001 versus vehicle, t test; data not shown). The quinpirole (10
M)-evoked increase in ASP
⫹
uptake was completely blocked by
0.1
M spiperone (significant quinpirole ⫻ spiperone interaction,
F
1,227
⫽ 19.64, p ⬍ 0.001, 2-way ANOVA; Fig. 4C). Moreover,
quinpirole failed to alter ASP
⫹
uptake in EM4 cells transfected
with YFP-hDAT but lacking the D3 receptor indicating that D3
receptor expression is required for this effect. The percent
change ⫾ S.E. in the rate of intracellular ASP
⫹
accumulation after
addition of 10
M quinpirole was 33.7 ⫾ 7.2% (n ⫽ 56) and ⫺7.5 ⫾
3.4% (n ⫽ 22) in hD3 receptor-transfected and nontransfected
cells, respectively (data not shown). EM4 cells transiently trans-
fected with FLAG-hDAT and human D1 DA receptors (D. R. Sib-
ley, NINDS, Bethesda) and the addition of the selective D1 agonist
B135 (R(⫹)-6-bromo-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tet-
rahydro-1H-3-benzazepine hydrobomide, 10
M) (41) did not
alter ASP
⫹
accumulation. The percent change ⫾ S.E. in the rate of
intracellular ASP
⫹
accumulation after addition of vehicle or ago
-
nist was 5.8 ⫾ 3.2% (n ⫽ 54) and 0.8 ⫾ 2.9% (n ⫽ 29) for vehicle and
B135, respectively, indicating that, in contrast to D3 receptors, D1
receptor activation is ineffective in modulating DAT activity (data
not shown).
FIGURE 2. ASP
ⴙ
uptake in hDAT-expressing EM4 cells. ASP
⫹
(in red) rapidly
accumulates in the intracellular compartment of EM4 cells stably expressing
GFP-hDAT (green). Note co-localization of ASP
⫹
is bound to DAT (as a yellow
signal) and differentiated from the intracellularly accumulated ASP
⫹
(A).
ASP
⫹
uptake is saturable with increasing substrate concentrations (B). ASP
⫹
uptake is temperature-dependent in EM4 cells expressing hDAT and is negli-
gible in EM4 cells not expressing hDAT (C). Disruption of the sodium gradient
with gramicidin inhibits ASP
⫹
uptake indicating a sodium-dependent mech
-
anism. A representative experiment (eight cells/group) is shown in D. The
average data of three replicate experiments is shown in E. AFU, arbitrary flu-
orescence units.
D3 Receptor Regulation of Dopamine Transporter
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To determine whether hD3 receptor modulation of DAT
occurs in a neuronal cell line, ASP
⫹
uptake was examined in
N2A cells transiently co-transfected with YFP-hDAT and
myc-D3 receptors. Analogous to EM4 cells, addition of quinpi-
role (10
M) to N2A cells co-expressing these proteins signifi-
cantly increased ASP
⫹
uptake (Fig. 4B; F
3,93
⫽ 5.11, p ⫽ 0.003
ANOVA).
Our [
3
H]DA uptake experiments suggested that prolonged
D3 receptor activation down-regulates DAT activity. To deter-
mine whether prolonged D3 receptor stimulation decreases
DAT activity, as opposed to the effects observed after acute
receptor stimulation, EM4 cells transiently transfected with
YFP-DAT and myc-D3 were preincubated with either vehicle
or quinpirole (10
M) for 30 min. After preincubation, cells
were washed, and ASP
⫹
was added. The rate of accumulation
was then measured over 1 min. Consistent with our radioligand
uptake and biotinylation studies (see below), preincubation
with quinpirole for 30 min induced a significant (t ⫽ 2.018, df ⫽
79, p ⫽ 0.047, t test) 28.5% decrease in the rate of ASP
⫹
uptake.
The mean arbitrary fluorescence units in vehicle and quinpi-
role-treated cells was 0.508 ⫾ 0.054 (n ⫽ 37) and 0.363 ⫾ 0.047
(n ⫽ 54 cells), respectively (data not shown).
The D3-mediated Increase in DAT Function Is Pertussis
Toxin-sensitive—D3 receptors are G protein-coupled receptors
that signal primarily through the G
i
/G
o
class of heterotrimeric
G proteins. Pertussis toxin catalyzes ADP-ribosylation of
␣
sub-
units in the G
i
and G
o
subfamilies of heterotrimeric G-proteins,
thereby preventing G
i
/G
o
proteins from interacting with recep
-
tors (42). To test whether the hD3 receptor-mediated modula-
tion of DAT activity requires G
i
/G
o
, EM4 cells expressing
FLAG-hDAT and GFP-hD3 receptors were incubated with per-
tussis toxin (100 ng/ml) for 16 –24 h, and the ability of quinpi-
role to increase ASP
⫹
uptake was assessed. Preincubation with
pertussis toxin prevented the quinpirole-evoked increase in
ASP
⫹
uptake (Fig. 4D, F
2,97
⫽ 6.11, p ⫽ 0.0032, ANOVA).
D3 Activation Stimulates PI3K and MAPK—D3 receptors
signal via MAPK and PI3K in brain and various cell lines (43,
44). The ability of the D3 receptor to stimulate MAPK was
evaluated in EM4 cells transiently expressing GFP-hD3 recep-
tors and YFP-hDAT by measuring agonist-induced (quinpirole
10
M, 1 min incubation) ERK phosphorylation (Fig. 5A). Con-
sistent with our findings in the whole cell ELISA (Fig. 1D), quin-
pirole evoked a 2.5-fold increase in p-ERK/total MAPK, indi-
cating activation of the MAPK pathway. This increase in
p-ERK/total MAPK was prevented by the MEK inhibitor
PD98059 (10
M, 15 min preincubation) (30) but not by the
PI3K inhibitor LY294002 (10
M, 15 min preincubation; data
not shown) (31). Stimulation of the PI3K pathway via the D3
receptor was measured by assessing phosphorylation of the ser-
ine/threonine protein kinase Akt (Fig. 5B). Quinpirole
increased p-Akt/total Akt 2.8-fold above vehicle in D3 recep-
tor-transfected cells. This effect was blocked by preincubation
with LY294002 (10
M (15 min preincubation) but not by the
FIGURE 3. ASP
ⴙ
uptake in hDAT-expressing EM4 cells. ASP
⫹
uptake is
inhibited by the DAT substrates d-amphetamine and DA (A) and the trans-
porter blocker cocaine (B) in EM4 cells stably expressing YFP-hDAT. EM4 cells
stably expressing FLAG-hDAT and transiently transfected with GFP-hD3
cDNA readily express the receptor, which is targeted to the plasma mem-
brane (C). The time course of ASP
⫹
uptake in a representative within the cell
experiment is shown in D. Note the initial rapid binding phase after ASP
⫹
addition, followed by the linear uptake phase. Addition of quinpirole
increases the rate of ASP
⫹
uptake relative to pre-drug values. The increase in
DAT function is measured as a change in the slope of uptake measured within
1 min before and after addition of drug. AFU, arbitrary fluorescence units.
FIGURE 4. Regulation of DAT function by activation of D3 receptors. EM4
cells transiently transfected with YFP-hDAT and myc-hD3 receptors showed a
concentration-dependent increase in ASP
⫹
uptake upon quinpirole (quin)
exposure (A). The quinpirole-evoked increase in DAT-mediated ASP
⫹
uptake
is also seen in N2A cells co-transfected with YFP-hDAT and myc-hD3 recep-
tors (B). Quinpirole effects on ASP
⫹
uptake are blocked by the antagonist
spiperone (C). The increase in agonist-induced ASP
⫹
uptake was prevented
by preincubation of cells for 16 –24 h with pertussis toxin (PTX) (100 ng/ml)
(D). *, p ⬍ 0.05; **, p ⬍ 0.01 versus vehicle (Veh) group; #, p ⬍ 0.05 versus
quinpirole stimulated group, Newman-Keuls post hoc test. n ⫽ 13– 63
cells/group).
D3 Receptor Regulation of Dopamine Transporter
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MAPK inhibitor PD98059 (data not shown). Thus, hD3 recep-
tor activation stimulates both MAPK and PI3K pathways in our
cell system.
D3 Receptor Regulates DAT Function via PI3K- and MAPK-
dependent Mechanisms—To determine the involvement of
these kinases in mediating the effects of hD3 receptor activa-
tion on DAT activity, EM4 cells co-transfected with YFP-hDAT
and GFP-hD3 receptor were incubated with either LY294002 or
PD98059 for 15 min prior to quinpirole (10
M) exposure. The
influence of the PKC inhibitor GF109203X on D3 receptor ago-
nist-evoked ASP
⫹
uptake was also assessed because D3 recep
-
tor-mediated MAPK phosphorylation involves an atypical iso-
form of PKC in some cell lines (45). The concentration of
GF109203X used (10
M) was that previously shown to result in
the inhibition of PKC isoforms
␣
,

1,
␦
,
⑀
, and
(32). ANOVA
revealed a significant effect of the various kinase inhibitors
on quinpirole-evoked ASP
⫹
accumulation (F
4,361
⫽ 14.21,
p ⬍ 0.0001 ANOVA). Post hoc analysis showed that inhibi-
tion of either PI3K or MAPK prevented the quinpirole-in-
duced increase in DAT activity (Fig. 6A ). In contrast, the
PKC inhibitor was without effect. The effects of both
LY294002 (F
4,297
⫽ 8.09, p ⬍ 0.0001 ANOVA) and PD98059
were concentration-dependent (F
4,258
⫽ 6.88, p ⬍ 0.0001
ANOVA; see Fig. 6, C and D).
D3 Receptor Regulation of DAT Function Does Not Require an
Intact DAT N Terminus—The DAT N-terminal domain con-
tains several target phosphorylation sites for kinases, includ-
ing MAPK and PKC (46 – 48). Therefore, we investigated
whether the N-terminal domain of DAT was required for the
effects of D3 receptors on DAT activity. Quinpirole
increased the rate of ASP
⫹
uptake in EM4 cells transfected
with hD3 receptor and a truncated FLAG-hDAT lacking the
first 55 N-terminal amino acids (⌬N
1–55
). The effect of quin
-
pirole on uptake was similar to that observed in cells express-
ing hD3 receptors and wild type FLAG-hDAT (Fig. 6B,
22.2 ⫾ 2.8% versus 25.6 ⫾ 2.1% in N-terminal truncated
versus wild type FLAG-hDAT, respectively). The ⌬N
1–55
mutant DAT shows normal uptake activity and insertion of
the transporter in the cell membrane.
5
5
N. Sen and J. A. Javitch, unpublished results.
FIGURE 5. Agonist stimulation of D3 receptors activates the PI3K and
MAPK pathways. A, immunoblot analysis of ERK phosphorylation in EM4
cells transiently expressing GFP-hD3 receptors and YFP-DAT. Cells were pre-
incubated for 15 min with vehicle (Veh) or PD98059 (PD) (10
M) before stim-
ulation with quinpirole (Quin) (10
M) for 1 min at 37 °C. The extent of phos-
phorylation was determined as described under “Experimental Procedures.”
Statistical analyses are as follows: ANOVA F
(3, 35)
⫽ 53.79 p ⬍ 0.0001; Newman-
Keuls post hoc test, significance from vehicle, unless otherwise noted (**, p ⬍
0.01; ***, p ⬍ 0.001). Additional experiments testing lower doses showed that
1
M PD98059 inhibited quinpirole (10
M) and evoked ERK phosphorylation
by 70% (data not shown). B, immunoblot analysis of Akt phosphorylation in
EM4 cells transfected as in A and preincubated 15 min with either vehicle or
LY294002 (10
M) before addition of quinpirole (10
M) for 1 min. Statistical
analyses are as follows: ANOVA F
(3, 31)
⫽ 44.59 p ⬍ 0.0001; Newman-Keuls
post hoc test, significance from vehicle, unless otherwise noted (*, p ⬍ 0.05;
**, p ⬍ 0.01; ***, p ⬍ 0.001). Representative immunoblots from at least three
experiments are shown.
FIGURE 6. Intracellular pathways involved in D3-mediated modulation of
DAT function. EM4 cells were transiently transfected with YFP-hDAT and
myc-hD3 receptors. Cells were preincubated for 15 min with the correspond-
ing inhibitor before addition of quinpirole (Quin) (10
M). The agonist-in-
duced increase in ASP
⫹
uptake was blocked by the PI3K inhibitor LY294002
(LY) (30
M) and the MAPK inhibitor PD98059 (PD) (50
M) but not the PKC
inhibitor GF109203X (GF) (10
M)(A). D3 receptor stimulation by quinpirole
evoked a similar increase in ASP
⫹
uptake in EM4 cells transfected with wild
type (WT) or a mutant N-terminal truncated DAT lacking amino acids 1–55
(⌬N
1–55
)(B). The effects of the PI3K inhibitor (C) and the MAPK inhibitor (D)
were concentration-dependent. *, p ⬍ 0.05 versus control (con) group; #, p ⬍
0.05 versus vehicle (veh) quinpirole-treated group, Newman-Keuls post hoc
test. n ⫽ 11–70 cells/group.
D3 Receptor Regulation of Dopamine Transporter
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D3 Receptor Activation Regulates DAT Subcellular
Distribution—Transporter trafficking allows rapid up- and
down-regulation of transporter expression at the cell surface
and is implicated in the regulation of transport activity (49).
Because D3 receptor activation is able to either increase (1 min
of activation) or decrease (30 min of activation) uptake of DA
and the fluorescent DAT substrate ASP
⫹
, we examined
whether D3 receptor activation regulates DAT trafficking.
To determine the effects of short (1 min) versus prolonged
(30 min) hD3 receptor stimulation on DAT trafficking, biotiny-
lation studies, which permit quantification of cell surface and
intracellular DAT, were conducted. Consistent with the uptake
data, incubation of cells for 1 min with the D3 receptor agonist
significantly increased biotinylated DAT (52.3 ⫾ 4.4%, p ⬍
0.05) and reduced nonbiotinylated DAT, indicating an increase
in cell surface DAT (Fig. 7A). Whereas DAT surface expression
and activity were higher after 1 min of exposure to quinpirole
(10
M), DAT surface expression and activity significantly
decreased following 30 min of quinpirole exposure. In agree-
ment with [
3
H]DA and ASP
⫹
uptake data, prolonged (30 min)
incubation with quinpirole decreased the amount of biotiny-
lated DAT (26.4 ⫾ 4.2%, p ⬍ 0.05), and this was accompanied
by an increase in nonbiotinylated DAT protein. Less than 0.5%
of total calnexin was present in streptavidin-bound fractions
suggesting that cells were intact and intracellular proteins were
not significantly biotinylated. Treatment with quinpirole did
FIGURE 7. D3 receptor activation modulates DAT trafficking. A, time-dependent effects of D3 receptor activation on DAT surface expression. EM4 cells were
co-transfected with FLAG-hDAT and GFP-hD3 receptors. After 24 h, cells were washed once with KRH buffer and incubated with vehicle or quinpirole (10
M)
for 1 or 30 min at room temperature. Surface DAT was marked by biotinylation. Immunoblot analysis of DAT expression using biotinylation procedures (top
panel, representative blot from six independent experiments shown) and quantitative DAT surface and intracellular band density (bottom panel) were per-
formed as described under “Experimental Procedures.” The data represent the mean ⫾ S.E. of six separate experiments. Incubation with quinpirole induced a
rapid (1 min) increase in cell surface DAT. However, prolonged incubation (30 min) produced opposite effects, decreasing the amount of cell surface DAT and
increasing its redistribution to the intracellular compartment. *, significantly different from vehicle-treated controls cells (p ⬍ 0.05, Newman-Keuls). #, signif-
icantly different from 1-min quinpirole-treated cells (p ⬍ 0.05, Newman-Keuls). B, time-dependent effects of D3 receptor activation on DAT plasma membrane
insertion. EM4 cells were co-transfected with FLAG-hDAT and GFP-hD3 receptors. After 24 h, cells were washed once with PBS/Ca-Mg, and free amino groups
were blocked with sulfo-NHS-acetate at 4 °C. After this blocking step, cells were incubated with biotinylating reagent at 22 °C in the presence or absence of
quinpirole (10
M) for 1 or 30 min. The isolation and quantification of biotinylated DAT newly inserted to the plasma membrane were performed as described
under “Experimental Procedures.” Western blots of DAT, TfR, and calnexin from avidin beads eluates are also shown. Each blot is representative of three
separate experiments. Quantitative analysis of DAT band densities for three experiments is given as mean ⫾ S.E. (bottom panel). Asterisks indicate significant
changes in biotinylated DAT compared with vehicle treatment at 22 °C. (p ⬍ 0.05, one-way ANOVA with Bonferroni post-hoc analysis.) Note that the blots
corresponding to 1-min incubations were exposed for longer times than the ones incubated 30 min to be able to visualize DAT proteins. Therefore, direct
quantitative comparison of DAT protein insertion between 1 and 30 min is not possible. C, time-dependent effects of D3 receptor activation on DAT internal-
ization. EM4 cells were co-transfected with FLAG-hDAT and GFP-hD3 receptors. After 24 h, cells were biotinylated with sulfo-NHS-SS-biotin at 4 °C and
incubated with quinpirole (10
M) or the vehicle for 1 or 30 min. Following MesNa treatment, biotinylated (internalized) DATs were isolated and analyzed as
described under “Experimental Procedures.” A representative immunoblot from five separate experiments is shown (top panel). The internalized DAT band
densities from five separate experiments are presented as mean ⫾ S.E. (bottom panel). Asterisks indicate a significant change in DAT internalization by
quinpirole treatment compared with vehicle treatment (p ⬍ 0.05, one-way ANOVA with Bonferroni post hoc analysis). In parallel experiments, cells were kept
at 4 °C throughout the procedure, and biotinylated DAT was analyzed before (total DAT biotinylated on the surface) and after MesNa treatment (background,
the efficiency of MesNa cleaving surface biotin-SS-linked proteins). Note that MesNa treatment in control experiments performed at 4 °C where endocytosis
was arrested revealed a background signal of ⬃5–10% from total amount of DAT (top panel).
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not alter the total amount of DAT protein as measured by
immunoblotting.
Altered plasma membrane expression levels of DAT follow-
ing D3 activation could arise from altering DAT endocytosis or
exocytosis or both. To examine the influence of hD3 receptor
activation on DAT plasma membrane insertion (exocytosis),
cells were biotinylated in the presence and absence of quinpi-
role for either 1 or 30 min after previously blocking existing
surface DAT. Cells were first treated with sulfo-NHS-acetate to
block all sulfo-NHS-biotin-reactive amino groups on the cell
surface, thereby preventing labeling of DAT already present on
the cell surface. Cells were then biotinylated in the presence and
absence of quinpirole for either 1 or 30 min under trafficking
permissive temperature (22 °C). Thus the amount of biotiny-
lated DAT represents DAT that was newly inserted (exocytosis)
on the plasma membrane. The sulfo-NHS-acetate preincuba-
tion step was effective in preventing biotinylation of preexisting
surface DAT at the beginning of the incubation (Fig. 7B, lanes 2
versus 1 and 6 versus 5) indicating that biotinylated DAT rep-
resents transporter newly delivered to the plasma membrane
during the incubation. In vehicle-treated cells, the amounts of
biotinylated DAT and TfR were greater at 22 °C (Fig. 7B, lanes 3
and 7) as compared with biotinylation performed at 4 °C (Fig.
7B, lanes 2 and 6). The changes in DAT and TfR biotinylation at
22 °C were not because of biotinylation of intracellular proteins
(because of loss of plasma membrane integrity) because lev-
els of calnexin, an intracellular protein, were unaltered in the
biotinylated fraction (Fig. 7B, top panel). Thus, the changes
in biotinylated DAT observed at different time points after
warming the cells to 22 °C represents DAT that was newly
delivered to the plasma membrane. Quinpirole did not affect
TfR insertion. When biotinylation was performed at 22 °C in
the presence of quinpirole for 1 min, a significant increase in the
amount of biotinylated DAT was seen (Fig. 7B, top panel, lane 3
versus 4, and bottom panel). However, after 30 min of incuba-
tion with quinpirole, the amount of biotinylated DAT was
reduced relative to vehicle (Fig. 7B, top panel, lanes 7 versus 8,
and bottom panel). These results suggest that whereas shorter
(1 min) quinpirole treatment increases DAT plasma membrane
insertion, following prolonged quinpirole exposure, less DAT is
ultimately located at the plasma membrane. Although this pro-
cedure is routinely used to study transporter insertion into the
plasma membrane (50), it should be noted that this procedure
assumes that DAT inserted in the plasma membrane is biotin-
ylated before it is again internalized. If the efficiency of biotiny-
lation is low then changes in the rate of internalization could
potentially increase the time a DAT molecule is on the surface
and thereby increase the efficiency of biotinylation. Finally,
although it is tempting to infer greater amounts of biotinylated
DAT and TfR after 30 min as compared with 1 min of incuba-
tion (Fig. 7B, lanes 3 and 4 versus 7 and 8), different exposure
times of both blots preclude direct comparisons between
time points. For the same reasons, we cannot directly com-
pare DAT protein levels across different experiments ( i.e.
across Fig. 7, A–C).
Next we sought to examine the internalization of DAT after 1
and 30 min of quinpirole exposure. Using a reversible biotiny-
lation strategy, DAT internalization was determined by esti-
mating surface biotinylated DAT that moves to an intracellular
compartment. The amount of biotinylated DAT protein was
determined for the surface (cells left at 4 °C without cleavage of
surface biotin, total biotinylated DAT (Fig. 7C, top panel, lane
1)) and intracellular pools (after MesNa treatment, internalized
biotinylated DAT (Fig. 7C, top panel, lanes 3– 6)). Control
experiments performed at 4 °C, a condition in which endocyto-
sis was arrested, revealed a background signal of ⬃2–4% after
MesNa treatment (Fig. 7C, top panel, lane 2). This likely repre-
sents surface DAT from which biotin was not completely
cleaved by MesNa. Following 1 min of quinpirole exposure, the
level of biotinylated DAT in MesNa-treated fractions was
decreased compared with vehicle-treated control (Fig. 7C, top
panel, lanes 5 and 6, and bottom panel). However, this effect did
not reach statistical significance. This may be due to the low
level of DAT internalization relative to background at the
1-min time period. However, a significant increase in the
amount of biotinylated DAT in MesNa-resistant fractions was
observed in cells treated with quinpirole for 30 min at 22 °C
compared with the respective vehicle-treated control (Fig. 7C,
top panel lanes 3 and 4, and bottom panel), indicating an
increase in internalized DAT.
The amount of biotinylated protein that is internalized is
affected not only by the rate of endocytosis but also by the rate
at which internalized protein is recycled back to the plasma
membrane. For this reason it is important to interpret the
results of the internalization assay together with the results
obtained in the assay for DAT plasma membrane insertion.
Taken together these data demonstrate that acute D3 receptor
activation increases cell surface DAT, DAT plasma membrane
insertion, and increases uptake activity, whereas prolonged
receptor stimulation results in decrease in uptake activity by
decreasing DAT plasma membrane insertion and enhancing
DAT internalization.
DISCUSSION
D3 receptors regulate extracellular DA concentrations in
limbic brain regions, an effect that has been attributed to alter-
ations in DA release (7). Evidence that D3 receptor ligands
modulate DA uptake was provided recently (10). In vivo and in
vitro studies showed that the preferential D3 agonist,
PD128907, enhanced the rate of DA clearance in nucleus
accumbens by increasing the maximal transport velocity. Fur-
thermore, in vitro studies revealed decreased DA uptake in
response to a preferential D3 antagonist. However, the pres-
ence of D2 and D3 receptor types in brain and the limited spec-
ificity of the ligands used precluded definitive identification of
the receptor types mediating this effect.
By using cell expression systems in which D2 receptors are
absent, we show that D3 receptors regulate DAT function. The
D2/D3 receptor agonist, quinpirole, induced a concentration-
dependent increase in uptake of the high affinity DAT sub-
strate, ASP
⫹
, in EM4 cells co-expressing DAT and hD3 recep
-
tors. Similar effects were observed in a neuronal cell line
co-expressing these proteins and in response to PD128907; a
structurally different D3 agonist. In cells lacking D3 receptors,
no agonist-induced increase in ASP
⫹
accumulation was seen.
The results obtained with the ASP
⫹
technique were con
-
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firmed using a [
3
H]DA uptake assay in which the DA recep
-
tor antagonist, spiperone, was used to prevent binding to and
activation of D3 receptors by [
3
H]DA. These data provide
the first clear evidence that D3 receptor stimulation is suffi-
cient to regulate DAT function. Moreover, the results indi-
cate that the effects of D3 receptor activation on DAT activity
are time-dependent.
Pertussis toxin prevented the increase in ASP
⫹
uptake pro
-
duced by quinpirole indicating that D3 receptors regulate DAT
via coupling to G
i
/G
o
proteins. D2/D3 receptor agonists acti
-
vate MAPK via a pertussis toxin-sensitive mechanism (44, 51).
D3 receptors also activate PI3K and PKC. Cross-talk between
these kinase cascades is implicated in D3 receptor-mediated
MAPK activation (43, 45). Evidence that these kinases regulate
DAT function and cell surface expression (52–56) prompted us
to hypothesize that their activation may be one mechanism
mediating the increase in DAT function produced by D3 recep-
tor stimulation.
Immunoblotting experiments revealed that quinpirole
increased phosphorylated ERK1/2 confirming that hD3 recep-
tor stimulation activates MAPK in our cell system. In agree-
ment with previous findings, D3 receptor stimulation resulted
in phosphorylation of Akt, a major target of PI3K (57, 58). Akt
phosphorylation was prevented by the PI3K inhibitor,
LY294002 (31), confirming PI3K mediation. LY294002 pre-
treatment did not prevent quinpirole-evoked MAPK phospho-
rylation suggesting that D3 receptor activation can stimulate
MAPK in a PI3K-independent manner.
Pretreatment with PD98059, a selective MEK inhibitor (30,
59), abolished the quinpirole-evoked increase in ASP
⫹
uptake.
The PI3K inhibitor produced a similar effect. It is important to
note that a within cell design was used such that uptake after
agonist addition was compared with that immediately before
agonist addition in the same cell, with the kinase inhibitor pres-
ent during the whole experiment. Furthermore, quantification
of YFP-DAT during ASP
⫹
studies revealed no alteration in cell
surface localization following incubation of cells with the kinase
inhibitors alone. Therefore, the decreased response to hD3
receptor activation cannot be attributed to effects of inhibitors
on basal DAT function. Rather it reflects the ability of inhibitors
to prevent the effects of D3 receptor stimulation and indicates
that the observed functional interaction of the D3 receptor with
DAT is dependent on both MAPK and PI3K activation. Block-
ade of conventional and atypical PKC isoforms by GF109203X
did not alter significantly the effects of D3 receptor activation.
Together, these findings indicate a specific involvement of
MAPK and PI3K in mediating D3 regulation of DAT and that
parallel activation of both MAPK and PI3K pathways is neces-
sary for the D3 receptor-induced increase in DAT activity. Fur-
thermore, although D3 receptors may couple to PKC (45), this
action is not critical for the effect of D3 receptor activation on
DAT activity.
DAT may be directly phosphorylated by these kinases,
resulting in modulation of transport activity. Alternatively, the
kinases may phosphorylate other regulatory proteins that mod-
ulate DAT. The DAT N terminus contains several target phos-
phorylation sites (47, 48) and is phosphorylated in response to
PKC activation (46, 47) and amphetamine (60). However, this
phosphorylation does not play a role in DAT trafficking in
response to PKC activation or amphetamine (47, 60). Similarly,
our studies with an N-terminal truncated DAT show that an
intact N terminus is not essential for the modulation of DAT
function by hD3 receptor activation.
Increased ASP
⫹
uptake occurred seconds after agonist addi
-
tion, suggesting mediation by a post-translational mechanism.
Transporter activity can be post-translationally modulated
resulting in changes in apparent substrate affinity and/or the
maximal transport rate. Although these mechanisms may con-
tribute to monoamine transporter regulation (61), a more com-
mon post-translational mechanism of DAT regulation involves
its redistribution to or from the plasma membrane (62– 64).
Constitutive DAT trafficking has been reported (50). It has
been suggested that under basal conditions DAT traffics
between the membrane and cytosol. Following internalization,
it is either degraded or recycled back to the membrane. There-
fore, we examined the influence of D3 receptor activation on
DAT trafficking.
Biotinylation experiments, which used an identical protocol
to that in ASP
⫹
uptake experiments, confirmed that brief D3
stimulation resulted in a significant increase in cell surface
DAT that paralleled the increase in DAT function. These
increases were accompanied by a significant decrease in intra-
cellular DAT. These data suggest that acute D3 receptor acti-
vation increases DAT redistribution from the intracellular
compartment to the cell surface, resulting in increased trans-
port capacity.
We observed that prolonged hD3 receptor activation
decreased cell surface DAT, as determined by cell surface bioti-
nylation, and decreased ASP
⫹
and [
3
H]DA uptake. Thus,
although acute D3 activation leads to increased DAT function,
prolonged D3 receptor activation leads to DAT down-regula-
tion. A recent study found that repeated systemic administra-
tion of a preferential D3 agonist decreased maximal DA trans-
port velocity. DAT binding density was unchanged (65).
Interestingly, this effect was absent in D3 knock-out mice sug-
gesting D3 receptor mediation. In addition, there is evidence
that D3 receptor stimulation can block the toxic effects of
high doses of cocaine in vivo (66). Together with our data,
these findings suggest that repeated or sustained activation
of D3 receptors may have effects opposite from those of
acute activation.
The effect of acute D3 agonist activation on DAT activity was
very rapid. Similarly, in electrochemical studies where DAT
regulation by D2/D3 receptor agonists has been observed, very
short (30 s) incubation times were used (17, 67). With longer
exposure periods, as is typically the case with studies employing
radiolabeled substrates, inconsistent effects are seen (15, 68).
Therefore, sustained D3 receptor stimulation may induce
adaptations that oppose those of acute receptor stimulation.
The mechanisms underlying these adaptations will require fur-
ther study.
The amount of cell surface DAT is determined by the balance
between transporter internalization and recycling back to the
plasma membrane (50). Changes in cell surface DAT can be
caused by altering the endocytotic pathway, the exocytotic
pathway, or both. For example, a recent study suggested that
D3 Receptor Regulation of Dopamine Transporter
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amphetamine induces a rapid increase in surface DAT that is
associated with its increased delivery to the plasma membrane
(69) and is followed by a redistribution away from the cell sur-
face at later time points (22). In contrast, the PKC-evoked
decreases in surface DAT involves both increased internaliza-
tion and reduced recycling (50). We investigated the contribu-
tion of the endocytotic and exocytotic recycling pathways to D3
receptor-induced DAT cellular redistribution. Long (30 min)
agonist exposure increased DAT internalization and decreased
DAT recycling. These findings are consistent with the cell sur-
face labeling studies and indicate that prolonged D3 receptor
stimulation decreases cell surface DAT by increasing the rate of
DAT internalization and decreasing the rate of recycling back
to the plasma membrane. By contrast, after 1 min of agonist
exposure, an increased exocytosis rate was observed, with a
nonsignificant decrease in internalization. It is important to
note that the biotinylation technique may lack sufficient sensi-
tivity or time resolution to detect significant decreases in endo-
cytosis that occur within a 1-min time scale. This caveat not-
withstanding, our results suggest that different mechanisms
underlie the effects of short and prolonged D3 receptor stimu-
lation on DAT trafficking. Furthermore, the increase in cell
surface expression observed in response to brief receptor stim-
ulation is in accord with an increasing body of evidence that
transporter trafficking can be rapidly regulated (69, 70).
In summary, these data provide the first unequivocal evi-
dence that D3 receptors up-regulate DAT activity, an effect that
requires both MAPK and PI3K activation. The D3 receptor-
mediated enhancement of DAT activity is associated with
redistribution of DAT from the intracellular compartment to
the cell surface, an effect that results, at least in part, from
increased DAT exocytosis. Prolonged receptor stimulation
decreases DAT function and cell surface expression. The rate of
DAT endocytosis is increased and exocytosis is decreased.
These data indicate that different mechanisms may be
recruited following acute versus sustained D3 receptor activa-
tion resulting in opposite effects on DAT function.
These findings provide potential insights regarding the cel-
lular mechanisms by which D3 receptors regulate DA transmis-
sion and maintain DA homeostasis. Transporter substrates
promote DAT redistribution from the plasma membrane to the
cytosol (22). This effect appears paradoxical because decreased
cell surface DAT expression decreases DA clearance from the
extracellular fluid. Our studies, however, suggest that if DA is
available to transiently stimulate D3 receptors, transporter
internalization will decrease in the vicinity of these receptors,
resulting in rapid clearance of extracellular DA and prevention
of prolonged receptor activation. When DA release occurs in
areas in which D3 receptors are lacking or at low density, sub-
strate-induced DAT internalization would be unopposed,
resulting in a greater half-life of extracellular DA, facilitating
diffusion farther from its release site.
Acknowledgments—We thank Namita Sen for making the ⌬N
1–55
FLAG-DAT construct. We thank Drs. L. J. DeFelice and J. W. Schwartz
for helpful advice on the ASP
⫹
uptake technique.
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