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Introduction
The dopamine (DA) D1 receptor
(D1R) and the glutamate (Glu) NMDA receptor (NMDAR) represent two functionally
and structurally diverse receptor classes.
D1R belongs to G-protein-coupled receptor family and couples to
Gαs proteins. NMDAR are ligand-gated ion channels composed of multiple subunits (NR1, NR2A-D, and
NR3A-C)[1]. A remarkable property of NMDAR is its high permeability to
Ca2+. Alteration of NMDAR-mediated
Ca2+ influx is reported to be involved in schizophrenia and in excitotoxic neuronal death associated with brain disorders such as stroke, epilepsy, and
trauma[2].
D1R and NMDAR are co-localized in several brain structures, including caudate-putamen, nucleus accumbens,
hippocampus, and prefrontal cortex[3_5].
D1R and NMDAR are constitutively interacting in cells as revealed by
co-immunoprecipitation and fluorescence resonance energy transfer
(FRET) studies[6]. The direct physical interaction is
mediated through the C terminal of the respective
protein[7]. In addition to the direct interaction at the receptor level,
previous studies have also shown that the downstream signal
molecules mediated by D1R is also found to regulate NMDAR
function. For instance, D1R stimulated protein kinase A (PKA)
was demonstrated to phosphorylate the NR1 subunit of
NMDAR[8] and to enhance NMDA-mediated
excitability[9]. Furthermore, the activation of
D1R was also found to enhance NMDA currents via protein kinase C
(PKC)-dependent mechanisms[10]. However, the direct
D1R-NMDAR interaction was also reported to result in the inhibition of
NMDAR-mediated currents[8], indicating that the
D1-NMDAR interaction is rather a diverse and complex event.
D1R and NMDAR are presented at high concentrations
in the postsynaptic density (PSD)[6]. PSD is a highly
organized subcellular fraction in which NMDAR co-exists with
scaffolding proteins such as PSD-95 and other signaling
proteins[11]. PSD-95 comprises three primary decidual zone (PDZ)
domains, a Src-homology three domain, and a domain
homologous to guanylate kinase[12]. It is known that PSD-95
utilizes those domains for protein interaction to recruit
signaling proteins and to mediate its assembly with other
components of the PSD.
Recent information indicates that PSD-95 can regulate
membrane trafficking and intracellular signaling of a number
of neurotransmitter receptors including NMDA,
α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and
5-hydroxytryptamine 2A (5-HT2A) receptors via physical
interactions[13_15]. As D1R and NMDAR are presented at high
density in the PSD[6], it is conceivable that PSD-95 may also
regulate D1/NMDA receptor interaction. To elucidate the
role of PSD-95 in D1R-modulated functions of the NR1a/NR2B
receptor, HEK293 cells expressing D1R and NR1a/NR2B
receptors were employed to study the role of PSD-95 in the
D1R-modulated NR1a/NR2B receptor-mediated
Ca2+ influx. Our study demonstrated that PSD-95 was required in
D1R modulating NR1a/NR2B receptor function, and this
modulatory effect depended on PKA and PKC.
Materials and methods
Materials Fura-2 acetoxymethyl ester (Fura-2 AM) was
from Molecular Probe (Eugene, OR, USA).
3-Isobutyl-1-methyl-2,6 (1H, 3H)-purinedione (IBMX) and Pluronic-127,
monoclonal anti-D1 DA receptor antibody produced in rat
clone 1-1-F11 s.E6, monoclonal anti-c-myc antibody produced
in mouse clone 9E10, anti-mouse IgG (whole molecule)-TRITC
antibody produced in goats, anti-HA antibody produced in
rabbits, leupeptin, pepstatin A, aprotinin, and
phenyl-methanesulfonyl fluoride (PMSF) were purchased from Sigma
(St Louis, MO, USA). Normal mouse IgG-HRP (horseradish
peroxidase) was from Santa Cruz (Santa Cruz, CA, USA),
anti-mouse IgG (H+L)-AP was from Promega (Madison, WI,
USA), and [3H]SCH23390 was from Amersham (Cleveland,
OH, USA). l-Glutamate acid sodium salt and glycine (Gly)
were from Sino-American Biotech (Beijing, China).
(±)-SKF-38393 hydrochloride, DA, H89, 8-Br-cAMP, and chelerythrine
were from RBI (Natick, MA, USA). The cAMP assay kit was
from the Shanghai University of Traditional Chinese
Medicine (Shanghai, China). Other reagents were obtained as
indicated in the text.
Cell culture and transfection Human embryonic kidney
293 (HEK293) cells, a generous gift from Dr Gang PEI
(Institute of Biochemistry and Cell Biology, Chinese
Academy of Science, Shanghai, China), were maintained in
Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand
Island, NY, USA) supplemented with 10% newborn calf
serum (SiJiQing, Hangzhou, China), penicillin (100 U/mL), and
streptomycin (100 U/mL). 2.5×104 cells were plated on
poly-L-lysine-coated glass coverslips. Transfections were
performed while cells reached 80% confluence. NR1a, NR2B,
D1-enhanced yellow fluorescent protein (EYFP), and PSD-95
were delivered at a ratio of 1:1:1:1 by the calcium-phosphate
transfection method. Ketamine 1 mmol/L was added to the
culture dish to prevent excitotoxicity during transfection. If
not indicated specifically, the cells used for the experiments
were harvested 24 h after transfection. NR1a and NR2B cDNA
were generous gifts from Dr John WOODWARD (Medical
University of South Carolina, Charleston, USA), cDNA for
PSD-95 were from Dr Morgan SHENG(Harvard University,
Boston, MA, USA), and D1-EYFP was constructed by Dr
You HE in our Laboratory.
Assays of cAMP content After 18_24 h transfection with
plasmid-encoded D1-EYFP receptors, the cells were reseeded
into a 96-well plate (1×104 cells/well) for 12 h. The cells were
pre-incubated with 50 µL serum free DMEM containing 500
µmol/L IBMX prior to D1R agonist
R-(+)-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol
hydrochloride (SKF38393) stimulation for an additional 10 min. The
reaction was then terminated on ice by adding 100 µL 1 mol/L
trichloroacetic acid. Following the addition of 20 µL 2 mol/L
K2CO3, the sample was centrifuged for 5 min at
12 000×g. The supernatant was kept (diluted in 1:10) for determining cAMP
content. All experiments were performed in duplicate, and
each experiment was repeated at least 3 times.
Internalization assays Cells expressing
D1-EYFP were treated either with 10 µmol/L DA or vehicle for 30 min in
serum-free DMEM and then washed 3 times with ice-cold
phosphate-buffered saline (PBS). The cells were fixed in 4%
paraformaldehyde for 20 min at room temperature before 3
washes of PBS. The receptor internalization was then
observed with a Leica SP2 confocal microscope (Leica
microsystem, Heideberg, Germany). The excitation
wavelength for yellow fluorescent protein (YFP) is at 514 nm.
Calcium imaging Transfected cells were incubated with
Fura-2 AM (4 µmol/L with 0.025% Pluronic-127) for 45 min in
extracellular solution (135 mmol/L NaCl, 5.4 mmol/L KCl, 1.8
mmol/L CaCl2, 10 mmol/L glucose, and 5 mmol/L HEPES
(2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), pH
6.8). After 3 washes with Fura-free extracellular solution (pH
7.2), the dishes were mounted onto the stage of an Olympus
BX51WI upright microscope (Tokyo, Japan). The cells were
perfused continuously at a flow rate of 1.2_1.5 mL/min.
Glu/Gly were applied via a computer-controlled Y-tube (outer
j=100 µm) situated 80 µm above the cells. The cells were
exposed to alternating 340 nm and 380 nm light every 2 s
during and immediately after Glu/Gly application. The
signals were acquired via a charge coupled device (CCD)
camera (CoolSNAP HQ, Roper Scientific, Duluth, GA, USA). To
minimize UV exposure, images were taken every 15_60 s
between drug applications. Ratio images were generated with
MetaFlour software from Universal Imaging (West Chester,
PA, USA). Intracellular calcium concentrations were
determined by the ratio of the intensity of 340/380.
NMDAR-dependent increases in intracellular calcium were calculated
by subtracting the average baseline value from the peak value
obtained during Glu/Gly application.
Data analysis All data were expressed as
mean±SEM. Unless otherwise indicated, the statistical significance was
determined using the least significant difference (LSD) test
following ANCOVA with SPSS 11.0 (SPSS, Chicago,
USA). The covariate was the average baseline NMDA response
before perfusion of the antipsychotic drugs.
Results
Establishing and characterizing D1R-NR1a/NR2B
receptor co-expression in HEK293 cells The HEK293 cells
were co-transfected with D1-EYFP and the NR1a/NR2B
receptor. To verify the success of transfection, the cells
were locally perfused with Glu/Gly (100/10 µmol/L) for
3 s. This treatment induced reproducible
Ca2+ influx (Figure 1A, upper panel). The application of 1 µmol/L MK-801
significantly blocked Glu/Gly-induced
Ca2+ influx (Figure 1A, lower panel). Employing confocal microscopy, we observed the
D1-EYFP receptor location on the cell membrane (Figure 1B,
upper left). The application of 10 µmol/L DA to the cells
induced significant internalization of
D1R (Figure 1B, upper right). Moreover, SKF38393 resulted in an increase in cAMP
accumulation in a dose-dependent manner (Figure 1B, lower
panel). The mean EC50 value of SKF38393 was 0.16 µmol/L,
similar to a previous report[16]. Thus,
D1R and the NR1a/NR2B receptor were functionally expressed in HEK293 cells.
D1R activation did not affect NR1a/NR2B-mediated
Ca2+ influx in HEK293 cells that expressed
D1R and NR1a/NR2B We next tested whether
D1R activation could modulate NR1a/NR2B receptor-mediated
Ca2+ influx in HEK293 cells expressing both the
D1R and NR1a/NR2B receptor. Glu/Gly-induced
Ca2+ influx in the presence of DA (100 µmol/L) is
shown in Figure 2A, 2B. The results clearly indicated that
the activation of D1-EYFP did not alter
NR1a/NR2B-mediated Ca2+ influx in our system. To exclude the possibility
that the EYFP tag interrupts the interaction between the
D1R and the NR1a/NR2B receptor, non-tagged
D1R-NR1a/NR2B receptor co-transfected cells were tested. Again, the
activation of D1R did not alter NR1a/NR2B-mediated
Ca2+ influx as well in the cells (Figure 2A, 2B, right). Thus, it is clear that
D1R is unable to modulate NR1a/NR2B receptor function in
our in vitro co-expression system. In order to test whether
the lack of D1R modulation on NR1a/NR2B-mediated
response as described earlier was due to insufficient PKA
activation by D1R in the system, the cell permanent PKA
activator 8-Br-cAMP was used. Incubation of the cells with
10 µmol/L 8-Br-cAMP elicited no effect on the Glu/Gly-
induced Ca2+ influx in HEK293 cells expressing both the
D1R and the NR1a/NR2B receptor (Figure 3). Taken together, our
data indicate that the activation of D1R or PKA fails to
modulate NR1a/NR2B receptor-mediated
Ca2+ influx in HEK293 cells expressed with the
D1R and the NR1a/NR2B receptor.
PSD-95 is required for D1R modulation of NR1a/NR2B
receptor function Since it has been shown that the scaffold
protein PSD-95 is associated with NMDAR and is involved
in the regulation of receptor function, we wondered whether
D1R activation could modulate NR1a/NR2B receptor
function when PSD-95 is presented in our expression system.
Interestingly, after co-expression with PSD-95, activation of
D1R by DA led to an enhancement in NR1a/NR2B receptor
function in a concentration-dependent manner. DA 10
µmol/L or 100 µmol/L induced a 34.0%±5.3%
(F1,37=64.95, n=20,
P<
0.01) or a 48.6%±10.3%
(F1,97=67.58, n=50,
P<0.01) increase in Ca2+ influx, respectively (Figure 4A). As expected, the
enhanced NR1a/NR2B receptor function induced by DA was
blocked by 5 µmol/L SCH23390, a selective
D1R antagonist (DA vs DA+SCH23390,
F1,65=3.71, n=67,
P<0.01, independent-samples t-test, Figure 4B,C), indicating a
D1R-mediated event. This result demonstrated clearly that PSD-95 was required
for the modulatory effect of D1R on NR1a/NR2B receptor
function.
PKA is involved in the modulation of NR1a/NR2B
receptor function by D1R activation
The above result reveals an important role of PSD-95 involved in
D1R-modulated NR1a/NR2B receptor function. As we know,
D1R activation can lead to an increase in cAMP formation and PKA activation.
We next tested whether PKA is involved in
D1R-enhanced NR1a/NR2B receptor-mediated
Ca2+ influx in the cells which were co-expressed with PSD-95. After bath application of
PKA selective inhibitor H89 (5 µmol/L), NR1a/NR2B
receptor-mediated Ca2+ influx was
significantly attenuated; an average 38.7%±4.1% of inhibition was observed
(F1,2=66.40, n=13,
P<0.01, compared to that in the absence of H89, Figure
5), indicating that NR1a/NR2B receptor-mediated
Ca2+ influx is subjected to PKA regulation. Furthermore, H89 not only
completely abolished the DA-enhanced
Ca2+ influx mediated by NMDA agonists, but also resulted in an additional
inhibition while DA was presented. To check if H89-mediated
inhibition is reversible, H89-treated cells were washed out for 10
min prior to the reapplication of Glu/Gly. However,
NR1a/NR2B receptor-mediated Ca2+
influx was unable to recover from the inhibition (Figure 5A).
PKC is involved in the modulation of NR1a/NR2B
receptor function induced by D1R
activation As D1R may also activate PKC via a phospholipase C (PLC)-mediated
mechanism and NMDAR was reported to be phosphorylated by PKC, the role of PKC was explored. NR1a/NR2B
receptor-mediated Ca2+ influx was not altered by selective
PKC inhibitor chelerythrine (4.0%±2.4% of inhibition,
n=15, Figure 6). However, chelerythrine (5 µmol/L) indeed
attenuated the DA-enhanced NR1a/NR2B receptor-mediated
Ca2+ influx (15.8%±3.4%,
F1,27=24.89, n=15,
P<0.01) with less
potency than that of H89. Thus, it indicates that PKC also
contributed to the D1R modulation of NR1a/NR2B receptor
function.
Discussion
The present study demonstrates that in HEK293 cells
co-transfected with the D1R and NR1a/NR2B receptor,
D1R activation enhances NR1a/NR2B-mediated
Ca2+ influx only in the presence of PSD-95. Moreover, when PKA or PKC
activity was inhibited, the D1R-modulated NR1a/NR2B
receptor function was also significantly attenuated. To our
knowledge, this is the first evidence that
D1/NR1a/NR2B
receptor interaction depends on PSD-95.
PSD-95 is a scaffold protein abundant in PSD. PSD-95
can recruit signaling proteins and mediate assembly with
other components of the PSD. It is known that PSD-95 plays
an important role in mediating neurotransmitter receptor
functions such as NMDA and 5-HT2A
receptors[13_15]. The present result indicates that
D1R activation fails to modulate NR1a/NR2B receptor-mediated
Ca2+ influx unless PSD-95 is
co-expressed with the D1R and the NR1a/NR2B receptor.
Thus, it is clear that PSD-95 is required for
D1R-modulated NR1a/NR2B receptor function. Other studies also support
the important role of scaffold proteins in the regulation of
NMDAR function in transfected HEK293 cells. For instance,
the PKA-modulated NMDA current was significantly
enhanced while the cells were co-expressed with yotiao,
another scaffold protein enriched in the
PSD[17]. We here found that the physical interaction between PSD-95 and
D1R was essential for the functional expression of
D1R-modulated NMDAR function. It appears that PSD-95 acts as a
core component in recruiting D1R, NMDAR, and other
signal transduction molecules to form a multiple protein
complex that allows the interaction between
D1R and NMDAR. Indeed, it has been suggested that PSD-95 facilitates the
dynamic regulation of phosphoprotein (such as PKA or PKC)
and sequentially attaches them to the substrate (for example,
the receptors). In this manner, the signals can be efficiently
transduced from one kinase to the
next[18]. It remains
unknown how D1R and PSD-95 interact, and furthermore,
how this physical interaction regulates NR1a/NR2B
receptor function.
Both PKA and PKC are found to be involved in the
D1R modulation of NR1a/NR2B receptor function in the presence
of PSD-95 in our system. Previous findings also suggested
that an anchored pool of PKA was required for the
augmentation of NMDAR-induced currents in HEK293
cells[17]. In neurons, the activation of the
D1R has been shown to enhance the NMDA current via PKA- and PKC-dependent
mechanisms[9,10]. It has also been reported that PKA, but
not PKC, phosphorylates NMDAR or receptor-associated
proteins, thereby inducing a conformational change of the
receptor[19]. It appears that the role of PKA and PKC in
D1R-modulated NMDAR function varied according to the cell
system employed[19].
A previous report of HEK293 cells co-expressed with
D1R and NMDAR indicated that the activation of
D1R could lead to an inhibition of the NMDAR
current[7]. It is worthy to note that the PKA and PKC inhibitors were used throughout
the experiments in this study in order to exclude the
potential post-translational effect of
D1R-stimulated PKA or PKC on NMDAR function. Thus, the observed inhibitory effect
of D1R activation on the NMDA current is more likely a
result of the conformation change due to the direct physical
association between the two receptors in their system.
Indeed, it appears that there are two potential mechanisms
for D1R-modulated NMDAR function. The confirmation
change resulted from the physical interaction between the
two receptors inhibiting the NMDAR
function[7], whereas post-translational modification such as PKA or
PKC-mediated phosphorylation enhanced NMDAR
function[9,10]. The present data demonstrated that
D1R- enhanced NR1a/NR2B Ca2+ influx was observed only in the presence of PSD-95,
which was indeed subjected to the regulation of PKA or
PKC, indicating that post-translational mechanisms play an
essential role in D1R-modulated NMDAR function.
In summary, the present results demonstrate that the
D1R activation of NR1a/NR2B receptor function requires the
presence of PSD-95. The activation of
D1R enhances NR1a/NR2B receptor-mediated
Ca2+ influx and is dependent on PKA and
PKC (Figure 7). It appears that PSD-95 is a core molecule in
recruiting the two receptors to form a functional complex.
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