From the Molecular Neuropharmacology Group,
Department of Pharmacology, Panum Institute, University of Copenhagen,
DK-2200 Copenhagen, Denmark and the § Center for Molecular
Recognition and the Departments of Psychiatry and Pharmacology,
Columbia University College of Physicians & Surgeons,
New York, New York 10032
Received for publication, May 22, 2002, and in revised form, October 28, 2002
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ABSTRACT |
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The structural basis of
phosphorylation and its putative role in internalization were
investigated in the human dopamine transporter (hDAT). Activation of
protein kinase C (PKC) was achieved either directly by treatment with
4- The dopamine transporter
(DAT)1 is situated in the
presynaptic membrane of dopaminergic nerve terminals and is
responsible for the rapid removal of dopamine released into the
synaptic cleft upon neuronal stimulation (1-3). Accordingly, the
transporter plays a critical role in regulating the availability of
dopamine in the synaptic cleft and thus in modulating the physiological functions of dopamine, including locomotor activity, higher cognitive functions, and neuroendocrine systems (1, 2). Furthermore, the DAT is
the principle target for the action of widely abused psychostimulants
such as cocaine and amphetamine (1-3). The transporter belongs to the
family of Na+/Cl Given the critical role of the DAT in regulating dopaminergic
neurotransmission, it is not surprising that the activity and availability of the DAT at the cell surface is tightly regulated. Most
significantly, activation of protein kinase C (PKC) by phorbol esters
such as 4- The activation of PKC leads to a marked increase in phosphorylation of
DAT (8-10). It has been hypothesized, therefore, albeit without direct
evidence, that serine and/or threonine phosphorylation of the DAT is
the molecular event that drives the internalization process. In light
of the predicted high specificity by which these transporters are
regulated, it would accordingly be expected that a specific Ser/Thr
phosphorylation site (or sites) could be identified within the
intracellular domains of the transporter and that mutation of this
site(s) would result in a concomitant impairment of internalization and
phosphorylation. Alternatively, the phosphorylation could represent an
independent process and phosphorylation of unidentified associated
proteins may be the actual trigger of the internalization process.
In the present study, we have investigated the structural basis and
putative role of direct phosphorylation of DAT for its acute
down-regulation and internalization. Importantly, we examine not only
phosphorylation induced by direct PKC stimulation with phorbol esters
but also that induced upon activation of a G Site-directed Mutagenesis--
The cDNA encoding the human
dopamine transporter (hDAT) in pRC/CMV (20) was kindly provided by Dr.
Marc G. Caron (Duke University, Durham, NC). The FLAG-tag sequence
(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (Sigma) was added to the N terminus
of the hDAT cDNA using polymerase chain reaction (PCR)-derived
mutagenesis using Pfu polymerase according to the
instructions from the manufacturer (Stratagene, La Jolla, CA). The
resulting FLAG-hDAT construct was cloned into the mammalian expression
vector pcDNA3 (Invitrogen, Carlsbad, CA). The mutations Y335A (21),
D436A, D436A/L440A/L441A, and Y575A were generated by PCR-based
mutagenesis in the background of this construct
(pcDNA3- FLAG-hDAT). The truncation mutant Cell Culture and Transfection--
HEK293 cells were maintained
at 37 °C in 5% CO2 in Dulbecco's modified Eagle's
medium with Glutamax I supplemented with 10% fetal calf serum and 0.01 mg/ml gentamicin (all products from Invitrogen). N2A
neuroblastoma cells were grown at 37 °C in 5% CO2 in
Dulbecco's modified Eagle's medium with Earle's salts with Glutamax-1 supplemented with 10% fetal calf serum, 1 mM
sodium pyruvate, 0.05 g/liter streptomycin, and 0.06 g/liter penicillin G (all products from Invitrogen). For stable expression, the HEK293 or
N2A cells were seeded in 100-mm tissue culture plates, grown to ~30%
confluence, and subsequently transfected with 2 µg of the pCIN4 or
PCIhygro constructs using the
LipofectAMINETM/Opti-MEMTM (Invitrogen)
transfection system. A stably transfected pool was selected with
Geneticin (G418) (0.35 mg/ml) or hygromycin (0.35 mg/ml), respectively,
as previously described (25). For stable co-expression, a
G418-resistant cell line expressing the hNK-1 receptor was transfected
with the desired hDAT constructs and stable cell pools expressing both
the hNK-1 receptor and the appropriate hDAT construct were obtained by
combined selection with hygromycin (0.25 mg/ml) and G418 (0.1 mg/ml).
[3H]Dopamine Uptake Experiments--
Uptake assays
were modified from Giros et al. (20) using
2,5,6-[3H]dopamine (7-21 Ci/mmol) (Amersham Biosciences,
Little Chalfont, United Kingdom). Transfected cells were plated in
poly-D-lysine-coated 24-well dishes (2 × 105 to 4 × 105 cells/well). The cells
were grown for 48 h prior to the experiment. On the day of the
experiment, the cells were washed once in 500 µl of uptake buffer
(7.5 mM HEPES, pH 7.1, with 5 mM Tris base, 120 mM NaCl, 5.4 mM KCl, 1.2 mM
CaCl2, 1.2 mM MgSO4, 1 mM L-ascorbic acid, 5 mM
D-glucose, and 1 µM amount of the
catechol-O-methyltransferase inhibitor Ro 41-0960 (26).
Subsequently, indicated concentrations of 4- Radioligand Binding Assay--
Binding assays were carried out
essentially as described (27) on whole cells using
125I-labeled substance P (2000 Ci/mmol) (Amersham
Biosciences, Little Chalfont, United Kingdom) as radioligand.
Transfected cells were plated in poly-D-lysine-coated
96-well dishes (2 × 104 cells/well). The cells were
incubated for 48 h prior to the experiment. Binding assays were
performed in a final volume of 100 µl of binding buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 5 mM MnCl2, 0.1% bovine serum albumin, and 40 µg/ml bacitracin) with 0.4 nM 125I-labeled
substance P plus increasing concentrations of non-labeled substance P
for determination of Bmax and
KD values. Binding was terminated after 2 h at
4 °C by washing the cells twice in 200 µl of binding buffer. 200 µl of Opti-phase Hi Safe 3 scintillation fluid (Wallac) was added to
each sample, and the plates were counted in a Wallac Tri-Lux
Labeling with Sulfo-NHS-SS-biotin--
Biotinylation of cell
surface proteins was performed by reaction with the membrane-impermeant
amine-specific biotinylating reagent sulfo-NHS-SS-biotin (Pierce).
Transfected HEK293 cells were seeded in
poly-D-lysine-coated 100-mm cell culture dishes (Corning,
Corning, NY) at 5 × 106 cells/dish and grown for
24 h before the experiment. Cells were stimulated with 200 nM SP, 1 µM PMA, or vehicle for 1 h at
37 °C. The cells were subsequently washed with ice-cold
phosphate-buffered saline (PBS)/Ca-Mg, pH 7.3, before treatment with
sulfo-NHS-SS-biotin (1.5 mg/ml) at 4 °C for 40 min in PBS/Ca-Mg,
followed by two washes with 100 mM glycine in PBS/Ca-Mg,
and incubation with 100 mM glycine for 20 min. The cells
were washed with PBS/Ca-Mg and lysed with 3 ml of lysis buffer (25 mM Tris, pH 7.5, with 1 mM EDTA, 5 mM N-ethyl maleimide, 200 µM
phenylmethylsulfonyl fluoride, and a protease inhibitor mixture tablet
(Roche Diagnostics)), scraped off, and centrifuged at 1,000 × g for 5 min. The cell pellets were resuspended in 1 ml of
solubilization buffer (lysis buffer supplemented with 150 mM NaCl and 1.0% Triton X) and left for 30 min at 4 °C
with constant shaking. Lysates were centrifuged at 20,000 × g for 30 min at 4 °C, and the protein concentration in
the supernatants was determined using a Bio-Rad DC protein assay kit. Monomeric avidin beads (175 µl) (Pierce) were added to the
samples, 500 µg of total cell protein was used for the [32P]Orthophosphate Labeling and
Immunoprecipitation--
Transfected HEK293 cells (3.5 × 106) were seeded in poly-D-lysine-coated
25-cm2 flasks and grown for 48 h reaching ~70%
confluence. The cells were washed in phosphate-free medium before
labeling at 37 °C for 4h in phosphate-free medium containing 10%
dialyzed fetal calf serum (Invitrogen), 20 mM HEPES, pH
7.2, and 1.0 mCi/ml [32P]orthophosphate (Amersham
Biosciences). Upon the addition of phosphatase inhibitors, 1 µM okadaic acid and 50 µM
Na3VO4, the cells were stimulated with 200 nM SP, 1 µM PMA, or vehicle for 60 min
followed by washing in 5 ml ice-cold PBS and subsequent lysis by
shaking for 15 min on ice in lysis buffer (25 mM Tris-HCl, pH 7.6, with 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 50 mM sodium pyrophosphate, 50 mM NaF, 1 µM okadaic acid,
and 1% Triton X-100) supplemented with protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride and a protease inhibitor
mixture tablet (Roche Diagnostics)). The cell lysates were centrifuged
at 20,000 × g for 15 min at 4 °C and precleared
with 4 mg/ml mouse IgG and 100 µl of protein G-Sepharose before
immunoprecipitation for 1 h at 4 °C using an antibody against
the FLAG epitope (2 µg of M2 antibody, Sigma). For
immunoprecipitation of the Fluorescence Microscopy--
Stably transfected HEK293 cells
were seeded in eight-well Lab-Tek II glass chamber slides (Nalge Nunc
International, Naperville, IL). After 36 h, the medium was changed
to uptake buffer and the cells were incubated in the presence of
indicated substances or vehicle (control) for 30 min at 37 °C. Cells
were examined using a confocal microscope (Leica DM IRBE HCB S100 Fluo
TCS) microscope (magnification, ×63). The filter sets for the
epifluorescence were BP 450-490 nm for excitation of EGFP, and the
emitted light passed through a LP 515-nm filter.
Calculations--
Uptake and binding data were analyzed by
nonlinear regression analysis using Prism 3.0 from GraphPad Software,
San Diego, CA. One-way analysis of variance followed by Dunnett's
post hoc test was used for statistical comparisons.
Regulation of DAT by Phorbol Esters and Substance P--
The hNK-1
(substance P) receptor, which is a typical G
The acute down-regulation observed in response to substance P treatment
of HEK293 cells co-expressing the hDAT and the hNK-1 receptor was
further investigated. Incubation of the cells co-expressing FLAG-hDAT
and hNK-1 with increasing concentrations of substance P for 30 min
caused a dose-dependent reduction in the specific [3H]dopamine uptake with an EC50 of 5.5 ± 0.1 nM (n = 5) and a maximal inhibition
of around 40% (Fig. 1B). Addition of 1 µM
selective high affinity hNK-1 receptor antagonist LY303870 (28)
essentially eliminated the effect of substance P on
[3H]dopamine uptake (Fig. 1B), consistent with
the specific action of substance P at hNK-1. Moreover, no effect of
substance P was observed when the same experiments were performed on
HEK293 cells expressing FLAG-hDAT but not the hNK-1 receptor (data not
shown). Finally, to assess the mechanism of the inhibitory effect of
substance P and PMA on uptake capacity, saturation uptake experiments
with [3H]dopamine were carried out with the
Substance P and PMA Promote Internalization of the
hDAT--
The decrease in Vmax for
[3H]dopamine uptake produced by substance P and PMA could
either be the result of sequestration of the transporter from the cell
surface or of a decrease in the transport rate of the individual
transporter molecules (or a combination of these effects). To
investigate this, we fused EGFP to the N terminus of hDAT and stably
co-expressed the resulting construct with the hNK-1 receptor in HEK293
cells. Co-expression was verified by measurement of specific
125I-labeled substance P binding
(Bmax = 3.2 ± 1.3 fmol/10,000 cells, n = 3) and specific [3H]dopamine uptake.
In the EGFP-hDAT-expressing cells, [3H]dopamine uptake
was similar to that observed in FLAG-hDAT-expressing cells (data not
shown). Substance P and PMA inhibited [3H]DA uptake in
the HEK293 cells co-expressing the EGFP-hDAT and the hNK-1 receptor to
an extent similar to that observed in cells co-expressing the FLAG-hDAT
and the hNK-1 receptor (data not shown). Fluorescence microscopy of
vehicle-treated cells demonstrated a clear fluorescent signal
corresponding to the plasma membrane and essentially no intracellular
fluorescence (Fig. 2, upper
panel). However, a 30-min incubation with either 200 nM substance P or 1 µM PMA resulted in a
substantially reduced surface fluorescence and the appearance of
punctuate intracellular fluorescence (Fig. 2, middle and
lower panels). These data support the hypothesis that the inhibitory effect of substance P on hDAT function, similar to
PMA, is caused by rapid internalization of the transporter to an
intracellular compartment.
Mutation of Trafficking Motifs in the hDAT--
Both dileucine
motifs ((D/E)XXXLL, where D/E is an acidic residue,
X any residue, and L a leucine) and tyrosine-based motifs (YXX Mutation of Putative Phosphorylation Sites in the hDAT--
It is
the prevailing perception that phosphorylation of specific serine
and/or threonine residues is required for PKC-dependent hDAT internalization. We decided to undertake a systematic mutagenesis approach both to test this hypothesis and to identify the possible specific site or sites responsible for the internalization process. As
shown in the two-dimensional representation of hDAT in Fig. 3, DAT
contains multiple serines and threonines in the predicted intracellular
domains. In several cases, these residues are part of consensus
sequences for PKC phosphorylation (Ser-7, Ser-53, Thr-62, Ser-261,
Ser-262, Thr-339, Ser-517, Ser-586, and Thr-613) (31),
cGMP-dependent protein kinase phosphorylation (Ser-53, Thr-62, Ser-262, Thr-339, Ser-517) (31), cAMP-dependent
kinase phosphorylation (Ser-517) (31), and
calmodulin-dependent protein kinase II phosphorylation
(Thr-613) (31). An analysis of the hDAT sequence using the NetPhos
prediction server (www.cbs.dtu.dk/services/NetPhos), which produces
neural network predictions for serine, threonine, and tyrosine
phosphorylation sites in eukaryotic proteins, also confirmed that
several sites are highly likely to become phosphorylated. Notably, four
serines/threonines and one tyrosine displayed a probability larger than
0.8 for being a phosphorylation site (Ser-53, Ser-517, Ser-586,
Thr-613, and Tyr-593).
In our mutagenesis strategy, we initially generated a construct in
which the first 22 N-terminal residues were deleted and substituted
with the FLAG tag followed by a HA tag (
The mutant transporters were assayed for their sensitivity to hNK-1
receptor and PKC activation. As shown in Fig.
5, like FLAG-hDAT, all nine mutants
displayed a strong inhibition of [3H]dopamine uptake in
response to both 200 nM substance P and 1 µM
PMA (Fig. 5). We even observed a tendency to an enhanced response to
PMA in the mutants containing substitutions of residues in the
intracellular loops (ICL, N+ICL, C+ICL, and XPK8) (Fig. 5). In the ICL
mutant, for example, PMA reduced uptake more than 70% in contrast to
~50% for the WT. Likewise, PMA reduced uptake in XPK8 ~65%
despite the fact that eight consensus sites for PKC phosphorylation
were simultaneously mutated (Fig. 5). Assuming that the observed
reduced uptake capacity for each mutant reflects internalization of the
transporter, these data argue against the hypothesis that
phosphorylation of specific serines and threonines is required for
internalization promoted by either direct PKC activation or by
activation of the G protein-coupled hNK-1 receptor. In addition, the
data suggest that phosphorylation of Tyr-593 is also not critical for
this process.
Direct Evidence for PMA- and Substance P-induced Internalization of
the Mutant Transporters--
Next, we wished to verify that the
inhibition of uptake observed in response to PMA and substance P in the
mutants described above did reflect an actual sequestration of the
transporter from the surface of the cell. Therefore, we carried out
cell surface biotinylation experiments on selected mutants using the
membrane-impermeant biotinylation reagent sulfo-NHS-SS-biotin
(sulfosuccinimido-NHS-biotin) to assess whether stimulation with PMA
and substance P would alter the amount of hDAT protein in the plasma
membrane. As shown in Fig. 6, the data
from the biotinylation experiment substantiated the observations with
EGFP-tagged transporter (Fig. 2) by providing clear biochemical
evidence that the reduced uptake seen upon treatment of the
cells with substance P and PMA could be explained by sequestration of
the transporter from the cell surface (Fig. 6). The data also showed
that neither removal of the serines in the distal N terminus ( The hDAT Is Phosphorylated in Its Distal N Terminus--
To
further substantiate the conclusion that hDAT phosphorylation is not
required for internalization, we performed direct phosphorylation
assays in the transfected HEK293 cells. Specifically, we wished to
exclude the remote possibility that phosphorylation of one or more of
the few remaining non-consensus site serines and threonines that we did
not mutate might be responsible for PMA- and substance P-induced
internalization. We compared control cells expressing the hNK-1
receptor, cells co-expressing the hNK-1 receptor and FLAG-hDAT, and
cells co-expressing the hNK-1 receptor and Similar Kinetics of Substance P-mediated Down-regulation of
FLAG-hDAT and Both WT and Mutants Are Regulated by PMA and Substance P in N2A
Neuroblastoma Cells--
Finally, we wanted to investigate whether
receptor-mediated and PKC-mediated regulation of DAT is independent of
serine and threonine phosphorylation not only in HEK293 cells but also
in a neuronally derived cell line. Accordingly, the hNK-1 receptor was
stably expressed in the human neuroblastoma cell line N2A followed by
transient co-expression with FLAG-hDAT and selected hDAT mutants.
Expression of hNK-1 was assessed by radioligand binding using
125I-labeled substance P (Bmax = 2.4 ± 0.9 fmol/10,000 cells, mean ± S.E., n = 3). Transient expression of FLAG-hDAT and the mutants Considerable evidence has previously indicated a key role of PKC
in regulating the activity and availability of DAT at the cell surface
(reviewed in Refs. 5-7). In agreement with this, we show here both in
transfected HEK293 cells and N2A neuroblastoma cells that activation of
PKC by phorbol esters elicits a marked decrease in hDAT transport
activity and that this decrease results from rapid sequestration of the
transporter from the cell surface. In addition, we demonstrate in the
two cell lines that DAT activity can be regulated by the
G The acute down-regulation of the hDAT observed in response to PKC
activation was accompanied by a parallel increase in hDAT phosphorylation (Fig. 7) in concurrence with earlier studies in heterologous cells and rat synaptosomes (8-12). Likewise, we found that activation of the hNK-1 receptor expressed in the same cells enhanced phosphorylation of hDAT (Fig. 7). Truncation of the 22 N-terminal residues, which contains several serines that might be
phosphorylated, nearly abolished detectable phosphorylation without
affecting functional regulation by PKC and the hNK-1 receptor (Fig. 7).
To address the question whether phosphorylation below the level that we
could detect still played a role in the truncation mutant
( Because the present study was carried out using epitope-tagged
constructs in heterologous expression systems, it is necessary to be
somewhat cautious in extrapolating our conclusions to the in
vivo situation. However, the fact that that we were able to achieve the same result in both HEK293 cells and in the neuronally derived N2A cells does support that our data are of general importance. Moreover, as mentioned above, our data agree fully with recent in
vivo data providing evidence for phosphorylation of the distal N
terminus (19). In addition, we have no evidence that the addition of
the N-terminal FLAG epitope to the full-length transporter affects its
function or regulation. Expression and uptake are essentially
identical, as are the PMA- and substance P-induced acute reduction in
uptake capacity of both the non-tagged and the FLAG-tagged transporter
(data not shown). Another critical issue when using heterologous
expression is that the expression levels achieved may not correspond to
that found in native tissue. Without the use of inducible promoters, it
is unfortunately difficult to accurately titrate expression. It is
interesting to note, however, that despite substantial differences in
expression levels several mutants displayed the same sensitivity to PMA
and substance P, indirectly suggesting that the level of expression is
not highly critical for the observed effects. Finally, we cannot rule
out the remote possibility that the use of a heterologous expression system masks more subtle effects of phosphorylation. For example, phosphorylation under in vivo conditions, although not
essential in itself, might enhance the likelihood of internalization
when associated with other factors. Furthermore, it should be
considered that both the measurements of internalization and
phosphorylation are rather slow measurements that may not reveal minor
kinetic differences, which could be critical in vivo.
Consistent with the present data, the simultaneous mutation of Ser-262,
Ser-586, and Thr-613 to glycine in the human DAT was recently reported
not to affect PKC-mediated regulation of DAT activity (32). The authors
also studied phosphorylation of the triple mutation in comparison to
the WT (32). They found that the PMA-induced increase in
phosphorylation apparently was absent in the mutant, although basal
phosphorylation in the absence of PMA was preserved or even increased
(32). Consequently, it was concluded that direct phosphorylation of the
DAT is not responsible for PKC-mediated regulation of the transporter
(32). Although we have reached the same conclusion based on our present
data, their phosphorylation data differ substantially from both our data and the data from the very recent study of rat striatal tissue (19). The apparent discrepancy with the observations by Chang et
al. (32) may relate to differences between the phosphorylation assay protocols. Thus, Chang et al. measured phosphorylation
over an extended time frame of 12 h (32). Upon 30 min of
stimulation with PMA, the cells were incubated with
32PO4 for 12 h before the level of
phosphorylation was assessed (32). In contrast, in both our protocol
and that of Vaughan and co-workers (19), the cells were precincubated
with 32PO4 followed by PMA stimulation for
30-60 min and immediate assessment of phosphorylation. It is highly
likely that adaptive processes together with the concerted action of
both kinases and phosphatases over 12 h may cause profound changes
in the transporter phosphorylation state; accordingly, it may be
difficult to judge the relationship between the acute effect of PKC
activation and phosphorylation measured over such a long time period.
In addition to DAT, several other
Na+/Cl Only little is known about the mechanisms in vivo that
govern the modulatory effects of PKC on the activity of DAT and related transporters. A growing number of reports suggest the critical function
of co-expressed G protein-coupled receptors (37). In the NET, it has
for example been shown that activation of muscarinic receptors in
SK-N-SH cells inhibits NET uptake capacity, causes internalization of
NET, and increases phosphorylation (36). This hNET response was, like
the present hDAT response to substance P, only partially reversed by
the PKC inhibitor staurosporine in contrast to the responses to PMA,
suggesting that both PKC-dependent and -independent
pathways may be involved in regulating the function of this class of
transporters. Of further interest, activation of the metabotropic
glutamate receptor 5 (mGluR5) has recently been reported to
down-regulate the activity of DAT in rat striatal synaptosomes (42); in
this study, we describe the regulation of DAT by the hNK-1 receptor,
providing the first evidence that neurotransmitter transporter activity
can be regulated by neuropeptide systems. Importantly, previous studies
have shown that this receptor, like the DAT, is expressed in
dopaminergic neurons and that substance P and related tachykinins can
mediate dopamine release directly and/or indirectly (17, 18).
Interestingly, it is not clear from the studies performed whether this
release, at least to some degree, could involve also down-regulation of
DAT and thus subsequent inhibition of uptake rather than only true
release (17, 18). It is therefore conceivable that the effect of hNK-1
receptor activation described here in two different heterologous
expression systems also may operate in vivo. Regulation of
DAT by hNK-1 receptor activation is also intriguing in light of the
fact that hNK-1 receptor antagonists, like inhibitors of the three
monoamine transporters DAT, NET, and SERT, may be useful for treatment
of depression (43). Preliminary studies in our laboratory suggest that
hNK-1 receptor-mediated regulation is not restricted to DAT but also applies to SERT, which is the target for the most widely used antidepressants (selective serotonin re-uptake inhibitors).
If phosphorylation of DAT is not required for internalization, we are
left with two important questions. First, the actual molecular trigger
of the internalization process needs to be identified, and, second, the
functional role of N-terminal phosphorylation remains to be determined.
With regard to the molecular trigger of the internalization process, we
hypothesize that upon phosphorylation another protein either
dissociates from or binds to the transporter. In the present paper we
could not find evidence that proteins known to mediate internalization
via interaction with dileucine- or tyrosine-based trafficking motifs
are involved in this process, because the mutation of such motifs in
the DAT sequence did not alter regulation by PKC or the hNK-1 receptor.
It remains to be determined whether one or more of the very few
proteins known to be associated with DAT and the closely related
transporters SERT and NET may play a role in the internalization
process (44-46). Of these, the interaction with PP2A, which is best
described for SERT, is of particular interest. It has been reported not
only that the SERT exists in a complex with PP2Ac in transfected cells and in native tissue, but also that the interaction may be regulated by
PKC (44). Although PP2A was shown to interact with DAT, the functional
consequences of this interaction were not investigated. Thus,
clarification of a putative role of PP2Ac in regulating DAT trafficking
awaits further studies.
Recently an interaction between the PDZ domain-containing
"scaffolding" protein PICK-1 and the C-terminal PDZ-binding
sequence of DAT has been reported (45). The association between the DAT and PICK-1 not only provides the first glimpse of how DAT may be
targeted to the presynaptic membrane, but also introduces an entirely
new paradigm to this class of transporters; via PDZ domain interactions, the transporters may be part of large multiprotein complexes containing a broad spectrum of cellular proteins. Given that
several hundred different proteins are known to contain PDZ domains, we
would predict that DAT likely interacts with other PDZ
domain-containing proteins in addition to PICK-1. The identification of
yet unknown proteins within such multiprotein complexes, as well as
unraveling putative interactions with other PDZ domain-containing proteins may provide a path toward understanding the molecular mechanisms underlying DAT internalization as well as insight into the still unknown role of N-terminal phosphorylation.
-phorbol 12-myristate 13-acetate (PMA) or by activating the
G
q-coupled human substance P receptor (hNK-1)
co-expressed with hDAT in HEK293 cells and in N2A neuroblastoma cells.
In both cell lines, activation of the hNK-1 receptor by substance P
reduced the Vmax for [3H]dopamine
uptake to the same degree as did PMA (~50 and ~20% in HEK293 and
N2A cells, respectively). In HEK293 cells, the reduction in transport
capacity could be accounted for by internalization of the transporter,
as assessed by cell surface biotinylation experiments, and by
fluorescence microscopy using enhanced green fluorescent protein-tagged
hDAT. In HEK293 cells, hNK-1 receptor activation, as well as direct PKC
activation by PMA, was accompanied by a marked increase in transporter
phosphorylation. However, truncation of the first 22 N-terminal
residues almost abolished detectable phosphorylation without affecting
the SP- or PMA-induced reduction in transport capacity and
internalization. In this background truncation construct, systematic
mutation of all the phosphorylation consensus serines and threonines in
hDAT, alone and in various combinations, did also not alter the effect
of hNK-1 receptor activation or PMA treatment in either HEK293 or N2A
cells. Mutation of a dileucine and of two tyrosine-based motifs in hDAT
was similarly without effect. We conclude that the major
phosphorylation sites in hDAT are within the distal N terminus, which
contains several serines. Moreover, the present data strongly suggest
that neither this phosphorylation, nor the phosphorylation of
any other sites within hDAT, is required for either
receptor-mediated or direct PKC-mediated internalization of the
hDAT.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dependent
transporters, along with transporters for several other neurotransmitters including the norepinephrine (NET), serotonin (SERT),
-aminobutyric acid (GAT), and glycine (GLYT) transporters (3, 4).
This class of transporters is characterized structurally by 12 transmembrane segments, intracellular N and C termini, and a large
glycosylated second extracellular loop (3, 4). No high resolution
structural information is available for any related transporter, and
our insight into the packing of the 12 helices remains quite limited
(3, 4).
-phorbol 12-myristate 13-acetate (PMA) is known to lead to
an acute reduction in the activity of the dopamine transporter (for
review, see Refs. 5-7). This has been demonstrated both in
synaptosomal preparations (8, 9) as well as in transfected cell lines
and Xenopus oocytes (10-13). A similar effect of PKC activation has also been observed for other neurotransmitter
transporters within the same class including the SERT, NET, GAT-1, and
GLYT-1 (reviewed in Refs. 5-7). Compelling evidence suggests that the PKC-mediated reduction in uptake is the result of an internalization of
the transporters rather than a change in the activity of the transporter molecules residing in the membrane (5-7, 11, 13-15). The
internalization of the DAT is believed to occur via a clathrin and
dynamin-dependent mechanism, resulting in accumulation of the transporter in early endosomes where it co-localizes with transferrin (13).
q-coupled human substance P (hNK-1) receptor co-expressed with hDAT in HEK293 cells (human embryonic kidney cells) and in N2A neuroblastoma cells.
The hNK-1 receptor was chosen as a typical G
q-coupled receptor that stimulates PKC activity via
G
q-dependent activation of phospholipase C
(16), is expressed in dopaminergic neurons along with DAT (17) and
shown to involved in dopamine release (18). Our results show that
activation of the co-expressed hNK-1 receptor, as well as direct
activation of PKC by phorbol esters, markedly reduced the transport
capacity of DAT and that this reduction can be accounted for by rapid
internalization of the transporter to an intracellular compartment.
Moreover, in agreement with a recent in vivo study of
phosphorylation of the rat DAT (19), evidence is obtained that the
increase in transporter phosphorylation seen in response to both
hNK-1 receptor and PKC activation is caused almost entirely by
phosphorylation of the distal N terminus of DAT. Neither
receptor-mediated nor direct PKC-mediated internalization of DAT,
however, is impaired by truncation of the putative sites of
phosphorylation, suggesting that DAT internalization is not dependent
on DAT phosphorylation. Subsequent mutation, alone and in combination,
of multiple serines and threonines throughout the intracellular domains
of the transporter, also had no effect on hNK-1 receptor-mediated or
direct PKC-mediated internalization of DAT, thereby providing
additional support for the dissociation of internalization and
phosphorylation, at least in two heterologous expression systems.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-22FLAG-HA-hDAT (HA is hemagglutinin) was generated from a synthetic hDAT cDNA as
previously described (22) and cloned into the bicistronic expression
vector pCIhygro containing the hygromycin resistance gene (23). All
serine and threonine mutations, as well as Y593A, were generated in the
background of pCIhygro-
1-22FLAG-HA-hDAT. The cDNA encoding
enhanced green fluorescent protein (EGFP) was fused to the N terminus
of synthetic hDAT cDNA and inserted into pCIhygro resulting in
pCIhygro-EGFP-hDAT (21). The cDNA encoding the human hNK-1
(substance P) receptor was excised from pTEJ-8 (24) and cloned into the
bicistronic expression vector pCIN4 (23). For all mutations, the
generated PCR fragments were digested with the appropriate enzymes,
purified by agarose gel electrophoresis, and cloned into the
appropriate eukaryotic expression vectors. All mutations were confirmed
by restriction enzyme mapping and automated DNA sequencing.
-phorbol 12-myristate
13-acetate (PMA) (Sigma), substance P (Peninsula Laboratories, Inc.,
San Carlos, CA), okadaic acid (Sigma), staurosporine (Sigma), or
vehicle were added in a final volume of 400 µl of uptake buffer
before incubation for 30 min at 37 °C. The uptake experiment was
initiated by addition of 70 nM [3H]dopamine
to a final volume of 500 µl. Nonspecific uptake was determined in the
presence of 1 mM non-labeled dopamine (Research Biochemicals International, Natick, MA). For determination of Vmax and Km values,
increasing concentrations of non-labeled dopamine (11 different
concentrations in triplicate) were added immediately before
[3H]dopamine. After 5 min of incubation at 37 °C, the
cells were washed twice with 500 µl of uptake buffer, lysed in 300 µl of 1% SDS, and left 1 h at 37 °C. All samples were
transferred to 24-well counting plates (Wallac, Turku, Finland), 600 µl of Opti-phase Hi Safe 3 scintillation fluid (Wallac) was added,
and the plates were counted in a Wallac Tri-Lux
-scintillation
counter (Wallac). All determinations were performed in triplicate.
-scintillation counter (Wallac). Nonspecific binding was determined
in the presence of 10 µM substance P. Determinations were
made in triplicate.
1-22 and
N' mutants, whereas 1000 and 1500 µg were used for the for N+C and
ICL mutants, respectively. The volume was adjusted to 1.0 ml with
solubilization buffer, and the samples were incubated for 1 h at
room temperature. The beads were washed four times with solubilization
buffer, before elution with 50 µl of 2× loading buffer (100 mM Tris-HCl, pH 6.8, 20% glycerol, 10% SDS, 0.1 M dithiothreitol, and 0.2% bromphenol blue) for 30 min at
room temperature. The eluates (25 µl) were resolved by SDS-PAGE (10%
acrylamide) and immunoblotted with monoclonal mouse HA antibody (Sigma)
diluted 1:2000. Immunoreactive bands were visualized using goat
anti-mouse horseradish peroxidase-conjugated secondary antibody
(1:10,000) and the ECL detection method (Amersham Biosciences).
Quantification of bands was performed on a model 300A densitometer in
combination with ImageQuant software (Amersham Biosciences) by using
film exposures that were in the linear range.
1-22FLAG-HA-hDAT, 900 µg of total cell
protein was used, and, for immunoprecipitation of FLAG-hDAT, 750 µg
was used. Samples were resolved by SDS-PAGE (10%) and either transferred to a nitrocellulose membrane and blotted with a
biotinylated M2 antibody (Sigma) according to instructions from the
manufacturer or exposed to a PhosphorImager screen, quantified using a
PhosphorImager (Amersham Biosciences) and analyzed with
ImageQuant software (Amersham Biosciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q-coupled
7-transmembrane segment receptor (16), was stably expressed in HEK293
cells using the bicistronic vector pCIN4 (23). Expression of the
receptor was demonstrated by detection of specific high affinity
binding of 125I-labeled substance P in a whole cell binding
assay (KD = 0.22 ± 0.04 nM,
Bmax = 3.2 ± 1.3 fmol/10,000 cells,
n = 4). Specific binding could not be detected in
non-transfected cells (data not shown). The effect of hNK-1 receptor
activation on hDAT activity in comparison to that of direct PKC
activation by phorbol esters was investigated upon transient expression
of the hDAT tagged at the N terminus with the FLAG epitope (FLAG-hDAT)
in the hNK-1 receptor-expressing cells. Note that the addition of this
FLAG tag does not affect the functional properties of the protein.2 In agreement with
previous observations, stimulation with the phorbol ester PMA (1 µM for 30 min) resulted in a marked reduction in
transporter uptake capacity (~50%) that could be reversed by the PKC
inhibitor staurosporine (Fig.
1A). Also as expected, the phosphatase inhibitor okadaic acid reduced uptake capacity, although to
a smaller extent (~25%) (Fig. 1A). These responses were
compared with those observed in response to the hNK-1 receptor agonist substance P. As shown in Fig. 1A, stimulation with 200 nM substance P for 30 min reduced 3H]dopamine
uptake capacity to approximately the same extent as PMA. Importantly, a
similar effect of 200 nM substance P was observed for the
non-tagged hDAT (data not shown). The combined addition of PMA and
substance P as well as the combined addition of okadaic acid and
substance P resulted in only marginally higher responses than those
observed in response to the individual compounds (Fig. 1A).
Addition of the PKC inhibitor staurosporine markedly inhibited the
response to substance P, although not as efficiently as it inhibited
the response to PMA. This suggests that the inhibitory effect of
substance P on hDAT is mediated, at least in part, via PKC, consistent
with the ability of the hNK-1 receptor to activate G
q-dependent pathways.
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Fig. 1.
Regulation of hDAT activity by substance
P. A, comparison of the substance P effect with the
effect of PMA and okadaic acid. The effect of substance P (200 nM), PMA (1 µM), okadaic acid (1 µM), and/or staurosporine (1 µM) (30 min at
37 °C) on [3H]dopamine uptake is shown as percentage
of uptake relative to the uptake observed in untreated control cells
(100%). Data are presented as means ± S.E. of three to five
experiments performed in triplicate. *, values that are significantly
different from control, p < 0.05. **, values that are
significantly different from control, p < 0.01. B, antagonist sensitivity of substance P-induced inhibition
of [3H]DA uptake in HEK293 cells stably expressing the
hNK-1 (substance P) receptor and transiently expressing FLAG-hDAT. ,
dose-response curve of substance P-induced inhibition of
[3H]DA uptake.
, dose-response curve of substance
P-induced inhibition of [3H]DA uptake in the presence of
the hNK-1 receptor antagonist LY303870 (1 µM).
1-22FLAG-HA-hDAT construct, which revealed that substance P and PMA
profoundly decreased the Vmax but did not alter
the Km for [3H]dopamine uptake (Table
I).
Vmax and KM values for [3H]dopamine uptake in
HEK293 cells co-expressing hNK-1 and hDAT
1-22FLAG-HA-hDAT
were incubated before the uptake assay for 30 min at 37 °C in the
absence (control) or presence of 200 nM substance P or 1 µM PMA. The Vmax and
KM values for [3H]dopamine uptake were
calculated from non-linear regression analysis of uptake data. The mean
KM value is calculated from means of
pKM, and the S.E. interval from the
pKM ± S.E.
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Fig. 2.
EGFP-hDAT is internalized by substance P and
PMA. Fluorescence microscopy was performed on HEK293 cells
stably co-expressing EGFP-hDAT and hNK-1 after stimulation for 30 min
at 37 °C with vehicle (control, upper panel), substance P
(200 nM) (middle panel), or PMA (1 µM) (lower panel). No effect of substance P
could be seen when the experiment was performed on HEK293-EGFP-hDAT
cells not expressing the hNK-1 receptor (data not shown). The images
shown are from a representative experiment out of four separate
experiments.
where Y is a tyrosine, X is any amino
acid, and
is a residue with a bulky hydrophobic side chain) are
known to play key roles in intracellular trafficking of membrane
proteins by mediating critical protein-protein interactions (29, 30).
The hDAT contains a dileucine motif in the third intracellular loop
(Fig. 3) and two tyrosine-based motifs,
one in the second intracellular loop (Tyr-335) and one in the C
terminus (Tyr-575). As part of our effort to understand the molecular
basis for transporter internalization, all three motifs were mutated.
Asp-436 was mutated either alone (D436A) or together with the two
leucines (D436A/L440A/L441A, called DLL in Fig.
4), whereas Tyr-335 and Tyr-575 were
mutated one at a time to alanine (Y335A and Y575A). The mutants were
all transiently expressed in HEK293 cells stably expressing the hNK-1 receptor. In all four mutants (D436A/L440A/L441A, D436A, Y335A, and
Y575A), 200 nM SP and 1 µM PMA inhibited
[3H]dopamine uptake to the same extent as observed in the
WT (Fig. 4). It should be noted that in Y335A the overall specific
uptake was reduced substantially (to ~4% of the WT level, Table
II). In agreement with our previously
published results, addition of Zn2+ partially restored
uptake in the Y335A mutant (21). However, the relative effect of
substance P and PMA in the presence of Zn2+ on uptake in
cells expressing the Y335A mutant was the same as in the absence of
Zn2+ (data not shown). Altogether, these data indicate that
the trafficking motifs present in hDAT are not critical for
either PKC- or hNK-1 receptor-mediated hDAT-internalization.
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Fig. 3.
Schematic two-dimensional representation of
the human dopamine transporter. All enlarged
circles indicate residues that were mutated to alanines.
Black circles with white
letter indicate mutated Ser/Thr residues that are part of
phosphorylation consensus sites (31). Dark shaded
circles with white letter indicate
Ser/Thr residues that were mutated but that are not part of classical
consensus sites for phosphorylation. Light shaded
circles with black letter indicate
residues that are part of putative trafficking motifs (tyrosine-based
and dileucine motifs) (29, 30).
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Fig. 4.
Mutation of a dileucine motif and two
tyrosine-based motifs does not affect regulation of DAT by substance P
or PMA. The data represent the relative [3H]DA
uptake observed upon stimulation with substance P (200 nM)
or PMA (1 µM) for 30 min at 37 °C in percentage of
uptake in untreated cells (mean ± S.E. of three to five
experiments performed in triplicate). The experiment was performed in
HEK293 stably expressing hNK-1 and transiently expressing either
FLAG-hDAT (WT in figure) or indicated mutants (Table I)
generated in the background of this construct. The decrease in
[3H]DA uptake as compared with untreated control cells
was significant for all four mutants (p < 0.01).
Relative uptake activity of hDAT mutants as compared to the WT hDAT
1-22FLAG-HA-hDAT) (22).
This construct displayed uptake properties similar to the full-length
DAT (Tables I and II and Ref. 22). In the background of
1-22FLAG-HA-hDAT, we generated eight constructs containing various
Ser/Thr substitutions and a single tyrosine substitution (Table II).
Overall, we mutated all remaining serines and threonines in the N
terminus (N'), all serines and threonines plus Tyr-593 in the C
terminus (C), and four serines/threonines in the loops, which are part
of PKC consensus motifs (ICL). These mutants were combined in different
ways (N'+C, N+C, N+ICL, and C+ICL). In addition, we generated a
construct in which eight PKC consensus sites were simultaneously
mutated (XPK8). All the mutants displayed specific [3H]dopamine uptake upon transient transfection into the
HEK293 cells stably expressing the hNK-1 receptor (Table II). As a
general trend, increasing the number of mutated residues in the
transporter resulted in lower uptake capacity (Table II). The
transporter was particularly sensitive to mutations in the
intracellular loops. Accordingly, it was not possible to generate a
transporter entirely devoid of Ser/Thr residues, but only the construct
with eight PKC consensus sites mutated (XPK8). This mutant transporter
displayed a substantially reduced uptake capacity (Table II).
Importantly, surface biotinylation experiments provided evidence that
this reduction was caused by a lowered surface expression rather than a
functional change of the mutant transporters residing in the membrane
(Fig. 6 and data not shown).
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Fig. 5.
Mutation of multiple Ser/Thr residues
including all nine PKC consensus sites does not affect regulation of
DAT by substance P and PMA. The data represent the relative
[3H]DA uptake observed upon stimulation with substance P
(200 nM) or PMA (1 µM) for 30 min at 37 °C
in percentage of uptake in untreated cells (mean ± S.E. of three
to five experiments performed in triplicate). The experiment was
performed in HEK293 stably expressing hNK-1 and transiently expressing
either 1-22FLAG-HA-hDAT or indicated mutants generated in the
background of this construct (Table I). The decrease in
[3H]DA uptake as compared with untreated control cells
was significant for all four mutants (p < 0.01).
1-22FLAG-HA-hDAT), additional mutation of several N-terminal serines and threonines (N), mutation of PKC consensus sites in the
loops (ICL), nor the combined mutation of several N-terminal and all
C-terminal serines/threonines plus Tyr-593 affected sequestration of
the transporter from the cell surface. For all four analyzed constructs, substance P reduced surface expression ~ 40%,
whereas PMA reduced surface expression ~60-70%. These results
correspond well to the observed inhibition of uptake for these mutant
transporters (Fig. 5).
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Fig. 6.
Substance P and PMA reduce cell surface
expression of mutant transporters. Expression of the
1-22FLAG-HA-hDAT, N', ICL, and N+C mutants (Table I) on the cell
surface of HEK293 cells stably co-expressing the hNK-1 receptor and
indicated DAT mutants was assessed by a surface biotinylation procedure
using the biotinylation reagent sulfo-NHS-SS-biotin. The cells were
stimulated with substance P (200 nM), PMA (1 µM), or vehicle (control) at 37 °C for 30 min before
biotinylation, and immunoblotting was performed as described under
"Experimental Procedures." For the analysis, 150 µg of
avidin-purified protein from the
1-22FLAG-HA-hDAT-expressing cells
was loaded on the gel, 150 µg of N', 500 µg of ICL, and 300 µg of
N+C. Band densities were estimated using ImageQuant software (Amersham
Biosciences) and are indicated as mean density ± S.E.
(n = 3) in percentage of control. *, values that are
significantly different from control (100%), p < 0.01. The insets show representative blots from one of three
separate experiments. No immunospecific bands were detected when
immunoblotting was performed on protein from non-transfected cells
(data not shown). The stable cell lines co-expressing hNK-1 and DAT
mutants displayed the following Bmax values for
125I-labeled substance P binding:
HEK293-hNK-1(
1-22FLAG-HA-hDAT), 2.7 ± 0.9 fmol/104 cells; HEK293-hNK-1(N'), 2.8 ± 1.0 fmol/104 cells; HEK293-hNK-1(ICL), 3.5 ± 0.8 fmol/104 cells; HEK293-hNK-1(N+C), 3.3 ± 1.0 fmol/104 cells. The Km and
Vmax values for [3H]DA uptake in
the same cell lines were: HEK293-hNK-1(
1-22FLAG-HA-hDAT), 2.5 (2.3-2.7) µM and 27.1 ± 2.4 pmol/min/105 cells; HEK293-hNK-1(N'), 2.4 (1.9-3.0)
µM and 15.6 ± 0.3 pmol/min/105 cells;
HEK293-hNK-1(ICL), 2.1 (1.7-2.5) µM and 2.5 ± 0.7 pmol/min/105 cells; HEK293-hNK-1(N+C), 1.7 (1.3-2.1)
µM and 9.7 ± 3.1 pmol/min/105 cells,
respectively. The Km are means followed by the S.E.
interval, and Vmax values are expressed as
means ± S.E. from three to five experiments performed in
triplicate.
1-22FLAG-HA-hDAT. The
cells were labeled for 4 h with [32P]orthophosphate
before stimulation for 60 min with 200 nM substance P or 1 µM PMA in the presence of 1 µM okadaic
acid. The transporter was subsequently immunoprecipitated from the cell
extracts with an antibody directed against the FLAG epitope and
analyzed by SDS-PAGE and phosphoimaging (Fig.
7A). For the FLAG-hDAT, basal phosphorylation is markedly enhanced both upon stimulation with PMA and
substance P (Fig. 7A). The phosphorylated FLAG-hDAT is seen
as a broad band with an apparent mass of ~100 kDa, which represents
the mature, fully glycosylated monomeric form of the transporter (22).
As expected, the band was not observed in the HEK293 cells solely
expressing the hNK-1 receptor (control in Fig.
7A). Notably, the phosphorylated band was also not observed upon truncation of the 22 N-terminal residues (
1-22FLAG-HA-hDAT). Parallel Western blotting analysis of the same immunoprecipitated cell
extracts verified the presence of FLAG-hDAT in quantities similar to
1-22FLAG-HA-hDAT (Fig. 7B). Taken together, these data
suggest that one or more of the serines present in the first 22 N-terminal residues of the transporter represent the major target for
phosphorylation of the hDAT.
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Fig. 7.
Phosphorylation of the
1-22FLAG-HA-hDAT is significantly reduced as
compared with the FLAG-hDAT. HEK293-hNK-1 cells transiently
transfected with either FLAG-hDAT,
1-22FLAG-HA-hDAT, or carrier
vector DNA were metabolically labeled with
[32P]orthophosphate in phosphate-free DMEM supplemented
with phosphate-free serum for 4 h at 37 °C prior to stimulation
of the cells with substance P (200 nM), PMA (1 µM), or vehicle (control) for 1 h at 37 °C. The
transporter was immunoprecipitated from the cell extracts with the M2
antibody directed against the FLAG epitope. The immunoprecipitates were
analyzed by SDS-PAGE, and phosphoproteins detected by exposure to a
PhosphorImager screen. The data shown are from a representative
experiment out of three separate experiments. B, parallel
Western blot analysis of the immunoprecipitates (150 µg of protein
from FLAG-hDAT and
1-22FLAG-HA-hDAT-expressing cells using
biotinylated M2 antibodies as described under "Experimental
Procedures").
1-22FLAG-HA-hDAT--
Despite the major difference
in phosphorylation of FLAG-hDAT and
1-22FLAG-HA-hDAT, both
constructs displayed a similar response to substance P and PMA after 30 min of stimulation (Fig. 5). However, this does not exclude the
possibility that phosphorylation of the distal N terminus could play a
role in the rate at which the response develops. Accordingly, we
performed time-course experiments of the response to substance P on
both FLAG-hDAT and
1-22FLAG-HA-hDAT. As illustrated in Fig.
8, the rate of the responses to substance P was essentially identical in the two constructs with
t1/2 values of 9 (7-16) min and 12 (10-16) min
(means of n = 3 (S.E. interval)) for FLAG-hDAT and
1-22FLAG-HA-hDAT, respectively. It should be noted that, because of
the rapidity of the response, reliable determinations at early time
points were rather difficult explaining the relatively large error bars
(Fig. 8).
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Fig. 8.
Time course of substance P-mediated
regulation of FLAG-hDAT compared with
1-22FLAG-HA-hDAT. HEK293-hNK-1 cells
transiently transfected with FLAG-hDAT (A) or
1-22FLAG-HA-hDAT (B) were incubated at 37 °C in the
presence of 200 nM substance P for the indicated amounts of
time before performing [3H]DA uptake. The data are shown
as percentage of maximum inhibition of uptake (t = 60 min) (means ± S.E. of n = 3). The
t1/2 for FLAG hDAT is 9 (7-16) min and 12 (10-16)
min for
1-22FLAG-HA-hDAT (means of n = 3 (S.E.
interval), non-linear regression analysis using one-phase exponential
association). Note that the apparent absence of error
bars for time points 30 and 60 min is a result of the fact
that the standard error is smaller than the size of the symbols.
1-22FLAG-HA-hDAT, N'+C, and ICL were verified by detection of specific [3H]DA uptake in the transfected cells. In
1-22FLAG-HA-hDAT, N'+C, and ICL, [3H]DA uptake values
were 80 ± 28, 62 ± 15, and 29 ± 6.8%, respectively (means ± S.E.; n = 3) of that observed for
FLAG-hDAT (0.14 ± 0.04 pmol/min/105 cells, mean ± S.E.; n = 3). As shown in Fig.
9, the response to both PMA and substance
P was somewhat smaller in the transfected N2A cells (~20% reduction
in uptake versus 40-60% in HEK293 cells) but still
reproducible and highly significant for the mutants tested (FLAG-hDAT,
1-22FLAG-HA-hDAT, N'+C, and ICL) (Fig. 9). The almost identical
effects of substance P and PMA in the mutants as compared with those in
the WT support the conclusion that regulation via a G protein-coupled
receptor and/or PKC is independent of alteration in Ser/Thr
phosphorylation of hDAT in a neuronally derived cell line as well as in
HEK293 cells.
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Fig. 9.
Regulation of DAT and selected Ser/Thr
mutants expressed in N2A cells by substance P and PMA. N2A-hNK-1
cells transiently expressing the indicated constructs (Table I) were
stimulated with substance P (200 nM) or PMA (1 µM) for 30 min at 37 °C before measurement of
[3H]DA uptake. The data shown are the relative
[3H]DA uptake expressed as a percentage of uptake in
untreated cells (mean ± S.E.; n = 3 experiments
performed in triplicate). The decrease in [3H]DA uptake
as compared with untreated control cells was significant for all
constructs tested (*, p < 0.05; **, p = 0.01). No effect of substance P was observed when the same
experiments were performed on N2A cells not expressing hNK-1 (data not
shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q-coupled hNK-1 (substance P) receptor, which is known
to be expressed in dopaminergic neurons (17). Like direct activation of
PKC by PMA, activation of the hNK-1 receptor by substance P caused a
marked reduction in uptake and a concomitant internalization of the
hDAT.
1-22FLAG-HA-hDAT), we performed systematic mutagenesis of multiple
serines and threonines in the predicted intracellular domains of the
transporter. None of the mutants, including one in which eight PKC
consensus sites were simultaneously mutated and another without any
serines or threonines in the entire N and C termini, displayed a
significantly altered fractional response to substance P or PMA in
either HEK293 cells or N2A cells (Figs. 4 and 5). Several of the
mutants did, however, display decreased overall uptake capacity as
compared with that of the WT (Table II), but this was a result of
lowered surface expression of the mutant transporters rather than of
altered function of the transporter molecules in the plasma membrane,
as indicated from surface biotinylation experiments of selected mutants
(Fig. 6 and data not shown). It should be noted that a few
non-consensus serines and threonines in the fourth and fifth
intracellular loops and at the predicted cytoplasmic/transmembrane
border were not mutated in the present study. Nonetheless, given the
profound effects observed, we find it very unlikely that non-detectable
phosphorylation of these few non-mutated, non-consensus site serines
and/or threonines, such as, e.g., Ser-333 and Ser-334 (Fig.
3), is the critical driving force for the internalization observed.
Hence, altogether our data provide strong evidence that phosphorylation
is not required for internalization of the transporter. Furthermore,
the data suggest that N-terminal serines are the major phosphorylation sites in the hDAT. Obviously, we cannot exclude the alternative possibility that the N terminus is not directly phosphorylated but
instead is critical for phosphorylation of other sites in the
transporter. It is therefore particularly interesting that a recent
in vivo study supports the conclusion that serines in the
N-terminal cytoplasmic tail of the DAT represent the major sites of
phosphorylation in response to PKC activation (19). In this study,
enzymatic digestion of 32PO4-labeled DAT was
carried out followed by immunoprecipitation of the resulting peptide
fragments with N- or C-terminal specific antibodies (19). Specifically,
proteolytic cleavage with endoproteinase Asp-N resulted in isolation of
phosphorylated N-terminal fragments (19). Moreover, it was found that
brief aminopeptidase treatment removed all detectable phosphorylation
from the DAT (19).
-dependent neurotransmitter
transporters have been shown to be regulated by
PKC-dependent mechanisms. These include SERT (33, 34), NET
(35, 36), GAT-1 (37), and GLYT-1 (38, 39). Activation of PKC is
generally found to inhibit transporter uptake, and for SERT, NET, and
GAT-1, PKC activation also leads to sequestration of the transporters from the cell surface to an intracellular compartment (33, 35-37). In
SERT, as in DAT, PKC activation causes direct phosphorylation (34),
which has led to the prevailing assumption that phosphorylation of the
transporter is critical for PKC-mediated down-regulation. This
assumption was further supported by a close association between the
time course of down-regulation of SERT and phosphorylation (34). In
SERT, the effect of PKC activation can be modulated by substrates (40).
The substrates serotonin and amphetamine caused retention of the
transporter on the surface upon PKC activation, whereas no effect was
observed in the presence of inhibitors such as cocaine, citalopram, and
imipramine (40). Interestingly, the effect of the substrates was
directly reflected in the level of phosphorylation; in response to
phorbol esters the level of SERT phosphorylation increased, but in the
presence of substrates this increase was abolished (40). These data are
consistent with a critical role of SERT phosphorylation in regulating
the subcellular localization of the transporter and accordingly its activity, However, no thorough mutagenesis study aimed at identifying the critical phosphorylation sites in SERT has been carried out; thus,
it is in principle possible that, at least in transfected cells, the
alteration in direct phosphorylation of SERT is secondary and is not
essential for transporter internalization. Accordingly, it is possible
that phosphorylation is dependent upon internalization and that the
suppression of phosphorylation by substrates is because of suppression
of trafficking. It is also possible that phosphorylation may have
distinct roles in different transporters. Indeed, the N and C termini,
which contain the majority of the potential phosphorylation sites, are
poorly conserved among the different transporters within the family (1,
3). SERT and DAT also clearly differ in the effect of substrate and
inhibitors on their PKC-mediated down-regulation. In contrast to the
SERT, DAT substrates such as amphetamine and dopamine have been
reported not to affect PKC-mediated internalization in transfected MDCK
cells (13). In the same cells as those used in the present study
(HEK293), however, it has recently been shown that substrates by
themselves promote internalization of the transporter with dopamine
causing a modest effect and amphetamine displaying the strongest effect
(41).
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ACKNOWLEDGEMENTS |
---|
We thank Birgitte Sander Nielsen, Dorthe Vang, and Lisbeth Sørensen for excellent technical assistance. We thank Dr. Claus Hansen for helpful technical advice on the phosphorylation experiments, Dr. Roxanne Vaughan for helpful discussions, Dr. Harald Sitte for comments on the manuscript, and Dr. Nanna Koschmieder Jorgensen for providing access to the confocal microscope donated by the Velux Foundation.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DA 11495 and MH 57324 (to J. A. J.) and by grants from the Lundbeck Foundation (to U. G.), the Danish Health Science Research Council (to U. G.), the Novo Nordic Foundation (to U. G.), and the Lebovitz Trust (to J. A. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Molecular Neuropharmacology Group, Dept. of Pharmacology 18.6, Panum Inst., University of Copenhagen, DK-2200 Copenhagen N, Denmark. Tel.: 45-3532-7548; Fax: 45-3532-7555; E-mail: gether@mfi.ku.dk.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M205058200
2 L. Norregaard, C. J. Loland, and U. Gether, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
DAT, dopamine
transporter;
NET, norepinephrine transporter;
SERT, serotonin
transporter;
hDAT, human dopamine transporter;
GAT, -aminobutyric
acid transporter;
GLYT, glycine transporter;
WT, wild type;
EGFP, enhanced green fluorescent protein;
SP, substance P;
NK, neurokinin;
HEK293, human embryonic kidney-293;
PKC, protein kinase C;
PMA, 4-
-phorbol 12-myristate 13-acetate;
HA, hemagglutinin;
PBS, phosphate-buffered saline.
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