From the Department of Oncology and
Cellular
and Molecular Biology, Institute of Medical Science, University of
Tokyo, Tokyo 108-8639, ¶ Department of Physiology, Kobe University
School of Medicine, Kobe 650-0017, and ** Department of Molecular
Neurobiology and Pharmacology, School of Medicine, University of Tokyo
113-0033, Japan
Received for publication, September 5, 2000, and in revised form, October 5, 2000
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ABSTRACT |
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The N-methyl-D-aspartate
(NMDA) receptors play critical roles in synaptic plasticity, neuronal
development, and excitotoxicity. Tyrosine phosphorylation of NMDA
receptors by Src-family tyrosine kinases such as Fyn is implicated in
synaptic plasticity. To precisely address the roles of NMDA receptor
tyrosine phosphorylation, we identified Fyn-mediated phosphorylation
sites on the GluR The N-methyl-D-aspartate
(NMDA)1 subtype of excitatory
glutamate receptors (GluRs) play central roles in synaptic plasticity (1), neuronal development (2), and excitotoxicity (3). The receptors
are formed by assembly of two classes of subunits, a principal subunit
GluR GluR In the NMDA receptor complex, GluR The physiological importance of Fyn in the nervous system has been
suggested by analyses of fyn mutant mice. These mice show various neural defects including defective LTP, impaired spatial memory, impaired myelination, and altered ethanol sensitivity (32-34).
In addition, up-regulation of Src is observed after spatial maze
learning, suggesting involvement of other Src-family kinases in
synaptic plasticity (35). In this paper, to precisely understand the
roles of tyrosine phosphorylation of GluR Antibodies--
Rabbit polyclonal antibodies against GluR Baculoviral Expression and Purification of
GST-Fyn--
Sf9 insect cells were maintained in Sf-900 medium
(Life Technologies, Inc.) containing 10% fetal bovine serum at
27 °C. Adherent cells were infected with recombinant baculovirus
carrying the GST-human Fyn cDNA. 72 h after infection, cells
were lysed in TNE buffer (1% (w/v) Nonidet P-40, 50 mM
Tris-HCl (pH 8.0), 120 mM NaCl, 5 mM EDTA, 0.2 mM Na3VO4 with aprotinin at 50 units/ml). GST-Fyn fusion protein was purified on glutathione-Sepharose
4B (Amersham Pharmacia Biotech) according to the supplier's instruction.
Purification of GST-C1, -C2, and -C3 Proteins--
pGEX-C1, -C2,
and -C3 were described previously (15). Fusion proteins were expressed
in Escherichia coli BL21. Purification of GST fusion
proteins was described above.
Tryptic Peptide Mapping Analysis--
To prepare the in
vitro phosphorylated GluR Construction of cDNAs--
For epitope-tagging of GluR Cell Culture and Transient Transfection--
Human embryonic
kidney (HEK) 293T cells were maintained in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum at 37 °C in 5%
CO2. Cells (1.5 × 106) were transfected
with combinations of expression plasmids (5 µg each) by the standard
calcium phosphate method. The amount of DNA transfected was adjusted in
each experiment by using a control expression vector pME18S (37). Two
days after transfection, cells were collected for protein extraction.
Preparation of Lysates, Immunoprecipitation, and
Immunoblotting--
For preparation of lysates of 293T cells, cells
were washed with phosphate-buffered saline and then lysed with 1 ml of
TNE buffer. Typically, 800 µl of lysates were used for
immunoprecipitation. For preparation of whole-cell lysates of
telencephalons, samples were homogenized in 0.2 volumes (ml/g of
tissue) of RIPA buffer (1% (w/v) Nonidet P-40, 1% (w/v) sodium
deoxycholate, 50 mM Tris-HCl (pH 8.0), 120 mM
NaCl, 5 mM EDTA, 0.2 mMNa3VO4 with aprotinin at 50 units/ml) containing 0.5% SDS. The lysates were boiled for 5 min to
dissociate the NMDA receptor complex and diluted with 4 volumes of RIPA
buffer. For immunoprecipitation, lysates were cleared by centrifugation
with an excess amount of protein G-Sepharose (Amersham Pharmacia
Biotech) and then incubated with indicated antibodies on ice for 1 h. Immune complexes were collected on protein-G Sepharose and washed
five times with lysis buffer. Immunoprecipitates or lysates were
resolved by SDS/ 7.5% polyacrylamide gel electrophoresis and
transferred to polyvinylidene difluoride membranes (Bio-Rad). Then the
membranes were blocked and probed with antibodies indicated. When
necessary, the antibodies were stripped from the membranes by
incubation in 62.5 mM Tris (pH 7.4), 2% SDS, and 0.7%
2-mercaptoethanol at 60 °C for 40 min, then the membranes were
reprobed with the antibodies indicated. For quantification, the
immunoreacted protein bands were analyzed with NIH image software.
Phosphatase Treatment--
100 µl of brain lysates from
wild-type and fyn mutant mice, which contained about 150 µg of
proteins, were immunoprecipitated with anti-GluR Electrophysiology--
Extracellular field potential recordings
were performed essentially as described (39). Hippocampal slices (400 µm thick) were prepared from 6-8-week-old mice and placed in an
interface-type holding chamber for at least 1 h. A slice was then
transferred to the recording chamber and submerged beneath continuously
perfusing artificial cerebrospinal fluid (119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 2.5 mM CaCl2, 1.0 mM
NaH2PO4, 26.2 mM
NaH2CO3, 11 mM glucose) that had
been saturated with 95% O2 and 5% CO2. All
the perfusing solutions contained 100 µM picrotoxin to
block GABAA receptor-mediated inhibitory synaptic
responses. The CA3 region was surgically separated from the CA1 region
to prevent invasion of epileptiform activity. A glass recording
electrode (containing 3 M NaCl) and a tungsten bipolar
stimulating electrode were placed in the stratum radiatum. The test
stimulation was applied to Schaffer collateral fibers at 0.1 Hz. The
stimulus strength was adjusted to get the initial excitatory
postsynaptic potential slope value of 0.10-0.15 mV/ms. To induce LTP,
we applied 4 trains of tetanic stimulation (100 Hz for 1 s) at an
interval of 10 s. Axopatch one-dimensional amplifier (Axon
Instruments) was used, and the signal was filtered at 1 kHz, digitized
at 10 kHz, and stored in an IBM-compatible computer equipped with a
TL-1 DMA analog-to-digital board (Axon Instruments). All
experiments were done at 25 °C. All data are presented as the
means ± S.E. A statistical evaluation was made by use of paired t test.
Phosphopeptide Mapping of the C-terminal Cytoplasmic Region of the
GluR Tyr-1472 as the Principal Fyn-mediated Phosphorylation Site in HEK
293T Cells--
In vivo phosphorylation of the seven
tyrosine residues was examined using GluR Characterization of Antibodies against Tyr-1472-phosphorylated
GluR Phosphorylation of Tyr-1472 of GluR
Because expression of GluR Increase in Phosphorylation of Tyr-1472 of GluR Here we showed that Tyr-1472 of GluR The best characterized form of LTP occurs in the hippocampal CA1
region. LTP is initiated by transient activation of NMDA receptors and
is expressed as a persistent increase in synaptic transmission through
It is reported that Src-family kinases do not potentiate recombinant
NR1-NR2B channels (GluR In summary, we report Fyn-mediated phosphorylation sites of GluR2 (NR2B) subunit of NMDA receptors. Seven out of 25 tyrosine residues in the C-terminal cytoplasmic region of GluR
2 were
phosphorylated by Fyn in vitro. Of these 7 residues,
Tyr-1252, Tyr-1336, and Tyr-1472 in GluR
2 were phosphorylated in
human embryonic kidney fibroblasts when co-expressed with active Fyn,
and Tyr-1472 was the major phosphorylation site in this system. We then
generated rabbit polyclonal antibodies specific to
Tyr-1472-phosphorylated GluR
2 and showed that Tyr-1472 of GluR
2
was indeed phosphorylated in murine brain using the antibodies.
Importantly, Tyr-1472 phosphorylation was greatly reduced in
fyn mutant mice. Moreover, Tyr-1472 phosphorylation became
evident when hippocampal long term potentiation started to be observed,
and its magnitude became larger in murine brain. Finally, Tyr-1472
phosphorylation was significantly enhanced after induction of long term
potentiation in the hippocampal CA1 region. These data suggest that
Tyr-1472 phosphorylation of GluR
2 is important for synaptic plasticity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (NR1 in rat) and modulatory subunits GluR
1-GluR
4
(NR2A-NR2D in rat) (4-6). The GluR
1 subunit is essential for the
function of NMDA receptor channels, whereas GluR
1-GluR
4 subunits
determine the characteristics of NMDA receptor channels by forming
different heteromeric configurations with the GluR
1 subunit (4-6).
The physiological importance of these subunits has been demonstrated by
gene targeting. Mice with a null mutation in GluR
1 die neonatally
(7, 8). Mutant mice in which GluR
1 is deleted only in hippocampal
CA1 pyramidal cells reach adulthood but lack NMDA receptor-mediated
postsynaptic currents and long term potentiation (LTP) in the
hippocampal CA1 region (9). In GluR
1 mutant mice, hippocampal LTP is
reduced, and spatial learning is impaired (10). Disruption of GluR
2
results in neonatal death and impairment of hippocampal long term
depression (11).
subunits have unusually long C-terminal tails that are extended
into the cytoplasm (4-6). Several PDZ domain-containing proteins such
as PSD-95 and chapsyn-110/PSD-93 interact with the NMDA receptor
through the C-terminal PDZ binding motif (12). Importance of the
C-terminal tails of GluR
subunits is also demonstrated by gene
targeting. Phenotypic defects in mice expressing C-terminally truncated
GluR
1 or GluR
2 are similar to those in mice lacking entire
GluR
1 or GluR
2 (13, 14). C-terminal tails of GluR
subunits are
likely to participate in their synaptic localization or regulation of
NMDA receptor functions. Furthermore, GluR
2 is phosphorylated at its
C-terminal tails by Src-family of nonreceptor protein-tyrosine kinases,
such as Fyn, cyclic AMP-dependent protein kinase, protein
kinase C, and calcium/calmodulin-dependent protein kinase
II (CaMKII) (15-19). However, the physiological roles of these
phosphorylation events remain unclear.
1, GluR
2, and GluR
4 are
tyrosine-phosphorylated in the brain (20-22), and Fyn significantly contributes to phosphorylation of GluR
1 and GluR
2 (15, 18, 19).
Among these subunits, GluR
2 is the major tyrosine-phosphorylated protein in the forebrain synapse (21). Protein tyrosine phosphorylation regulates NMDA channel receptor activity. NMDA receptor-mediated currents are potentiated by Src-family tyrosine kinases and suppressed by tyrosine phosphatases (23, 24). In addition, taste-learning increases tyrosine phosphorylation of GluR
2 in the rat insular cortex, and tyrosine phosphorylation of GluR
2 is increased after induction of LTP in the dentate gyrus of anesthetized adult rats (25-27). It is reported that Src and Fyn do not potentiate the current
through recombinant GluR
1-GluR
2 channels in 293 cells (28). The
p85 subunit of phosphatidylinositol 3-kinase, phospholipase C
, SHP2,
and brain spectrin interact with GluR
2 in a tyrosine phosphorylation-dependent manner (15, 29-31). These data
suggest that GluR
2 tyrosine phosphorylation may at least in part be
involved in intracellular signaling in murine brain.
2 by the Src-family kinases, we set out experiments in which Fyn-mediated tyrosine phosphorylation sites of GluR
2 were determined. By showing that Tyr-1472 of GluR
2 is phosphorylated in murine brain, we propose that
Tyr-1472 phosphorylation plays important roles for synaptic plasticity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
were raised against a glutathione S-transferase (GST) fusion
protein with mouse GluR
2 (amino acids 1034-1119) and
affinity-purified. The anti-GluR
2 antibodies did not cross-react
with rat NR2A (data not shown). Rabbit polyclonal antibodies against
phospho-Tyr-1472 of GluR
2 were raised using a keyhole limpet
hemocyanin-conjugated synthetic peptide with the sequence
CSNGHV(phospho-Y)EKLSSI as immunogen. The antibodies were purified from
sera of the immunized rabbits by successive affinity chromatography
using a column of N-hydroxysuccinimide-activated Sepharose
4B resin (Amersham Pharmacia Biotech) conjugated to the GST-C3 protein
(to subtract IgGs against non-phosphorylated GluR
2) followed by a
column conjugated to the immunogen. The antibodies did not recognize
NR2A (GluR
1) expressed in 293T cells together with active Fyn (data
not shown). Anti-influenza hemagglutinin (HA) monoclonal antibody (mAb)
(12CA5) was purchased from Roche Molecular Biochemicals.
Anti-phosphotyrosine mAb (RC20) and anti-NR2B mAb were from
Transduction Laboratories. Rabbit anti-Fyn (Fyn3) and rabbit anti-NR2B
antibodies were from Santa Cruz Biotechnology and Chemicon, respectively.
2 proteins, the GST-GluR
2 fusion
proteins (2 µg) were phosphorylated by 1 µg of baculovirally
expressed and purified GST-Fyn in 40 µl of kinase buffer (20 mM Hepes-NaOH (pH 7.2), 10 mM
MgCl2, 3 mM MnCl2) in the presence
of 100 µM ATP and 5 µCi of
[
-32P]ATP at 30 °C for 30 min. The reactions were
terminated by the addition of 20 µl of 3× Laemmli sample buffer and
then resolved by 10% SDS-polyacrylamide gel. 32P-Labeled
GST-GluR
2 proteins were excised from the gel and then subjected to
peptide-mapping analysis according to Boyle et al. (36).
Briefly, tryptic peptide samples were electrophoresed for 40 min at 1.0 kV in pH 1.9 buffer using the HTLE7000 apparatus (CBS Scientific); the
plates were air-dried and then placed in tanks for ascending
chromatography using phosphochromatography buffer. After ascending
chromatography, the plates were air-dried and then exposed.
2,
GluR
2 cDNA (4) was inserted with the oligonucleotides encoding
an HA epitope-containing sequence, DYPYDVPDYASLV, at XmaI
site that encoded amino acid residues 66 and 67. The resultant
cDNA, HA-GluR
2, was subcloned to pME18S (37). The expression
plasmids pME-FynY531F and PSD-95 were described previously (19, 37).
Various YF mutants of GluR
2 were generated by
oligonucleotide-mediated site-directed mutagenesis (38). Mutations were
verified by dideoxynucleotide sequencing.
2 antibodies. Immune
complexes were collected on protein G-Sepharose (Amersham Pharmacia
Biotech) and washed five times with lysis buffer followed by washing
twice with bacterial alkaline phosphatase (BAP) buffer (Tris-HCl (pH
9.0), 1 mM MgCl2). Bound proteins were
incubated in 100 µl of the BAP buffer with or without 5 units of BAP
(Takara) at 37 °C for 6 h. After the BAP reaction, the beads
were washed three times and then subjected to Western blot analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 Subunit in Vitro--
The GluR
2 subunit contains 25 tyrosine residues in the intracellular C-terminal region. To determine
the site(s) of Fyn-mediated GluR
2 phosphorylation, we constructed
GST fusion proteins containing truncated segments of the intracellular
C-terminal region of the GluR
2 subunit (termed GST-C1, -C2, and -C3
proteins) (Fig. 1A). GST-C1,
-C2, and -C3 fusion proteins were bacterially expressed, purified, and
then phosphorylated in vitro with baculovirally expressed
GST-Fyn in the presence of [
-32P]ATP. The
phosphorylated proteins were subjected to tryptic phosphopeptide mapping. As shown in Fig. 1, highly phosphorylated peptides P1-P4, P5
and P6, and P7-P9 were generated from GST-C1, GST-C2, and GST-C3 fusion proteins, respectively. To identify the tyrosine residues that
were phosphorylated in GST-C3 protein, we constructed Y1252F (conversion of Tyr-1252 to Phe-1252)-, Y1336F-, and Y1472F-GST-C3 proteins by site-directed mutagenesis. Conversion of Tyr-1472 to
Phe-1472 resulted in generation of a tryptic phosphopeptide map that
lacked phosphopeptide P9 (Fig. 1C). Similarly,
phosphopeptide P7 was absent in the phosphopeptide map of Y1252F-GST-C3
protein, and phosphopeptide P8 was absent in the phosphopeptide map of Y1336F-GST-C3 protein (data not shown). The two-dimensional tryptic phosphopeptide map of GST-C1 fusion protein displayed highly
phosphorylated peptides, P1-P4 (Fig. 1D), and the map of
GST-C2 fusion protein showed highly phosphorylated peptides P5 and P6
(Fig. 1E). Phosphopeptides corresponding to P1-P4 were
absent in two-dimensional tryptic phosphopeptide maps of GST-C1 having
Y932F, Y1039F, Y1070F, or Y1109F mutations, and phosphopeptides
corresponding to P5 and P6 were missing in maps of GST-C2 having Y1109F
or Y1252F mutations (data not shown). These results indicate that
Tyr-932, Tyr-1039, Tyr-1070, Tyr-1109, Tyr-1252, Tyr-1336, and Tyr-1472
are Fyn-mediated phosphorylation sites in GluR
2 in
vitro.
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Fig. 1.
Identification of tyrosine residues of
GluR 2 phosphorylated by Fyn in
vitro. A, schematic diagram of GST fusion
proteins containing the intracellular C-terminal region of GluR
2.
B, C, D, and E,
two-dimensional tryptic phosphopeptide maps of GST-C3 (B),
GST-C3 Y1472F mutant (C), GST-C1 (D), and GST-C2
(E). Purified GST fusion proteins were phosphorylated by
GST-Fyn in vitro. The phosphorylated proteins were separated
by SDS-polyacrylamide gel electrophoresis, excised from the gels, and
digested with trypsin. The resulting tryptic peptides were separated in
the first dimension by electrophoresis and in the second dimension by
chromatography, as indicated by the arrows. The
dot in each map shows the origin of
electrophoresis. The phosphopeptides in the individual maps are
indicated by P1-P9.
2-transfected HEK 293T
cells. First, 293T cells were transfected with an expression plasmid
encoding HA-tagged wild-type GluR
2 alone or together with plasmids
encoding Fyn Y531F, which is a constitutively active form of Fyn (37),
and PSD-95. PSD-95 promotes Fyn-mediated tyrosine phosphorylation of
GluR
2 (data not shown) as well as NR2A (GluR
1) (19). The cells
were lysed, and HA-tagged GluR
2 was immunoprecipitated from cellular
lysates and subjected to immunoblotting with an anti-phosphotyrosine
(Tyr(P)) antibody. GluR
2 was prominently tyrosine-phosphorylated only when it was co-expressed with Fyn Y531F
(Fig. 2A). Next, 293T cells
were transfected with expression plasmids for either HA-tagged
wild-type GluR
2 or one of the seven single YF mutants of GluR
2
described above together with FynY531F and PSD-95 expression plasmids.
As shown in Fig. 2B, Y1472F mutation resulted in the
significant reduction of the tyrosine phosphorylation level of
GluR
2. There was no reduction in the tyrosine phosphorylation levels
of GluR
2 in the other mutants. Moreover, phosphorylation of GluR
2
Y1252F/Y1472F and Y1336F/Y1472F double mutants was less than that of
GluR
2 Y1472F mutant (Fig. 2C, lanes 2,
3, and 4), and phosphorylation of GluR
2
Y1252F/Y1336F/Y1472F triple mutant was nearly eliminated in 293T cells
(Fig. 2C, lanes 1 and 5). These
results suggest that Tyr-1252, Tyr-1336, and Tyr-1472 of GluR
2 are
phosphorylated in 293T cells when active Fyn is co-expressed. Phosphorylation of four other residues, Tyr-932, Tyr-1039, Tyr- 1070, and Tyr-1109, was under detectable level.
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Fig. 2.
Phosphorylation of Tyr-1252, Tyr-1336, and
Tyr-1472 of GluR 2 by active Fyn in HEK 293T
cells. A, tyrosine phosphorylation of GluR
2 by
active Fyn in 293T cells. B, identification of Tyr-1472 as
the major Fyn-mediated phosphorylation site in 293T cells.
C, phosphorylation of Tyr-1252, Tyr-1336, and Tyr-1472 by
active Fyn in 293T cells. 293T cells were transfected with combinations
of expression plasmids for GluR
2, various GluR
2 YF mutants,
PSD-95, and FynY531F. The cells were lysed in TNE buffer. GluR
2
immunoprecipitates (IP) from the lysates were subjected to
immunoblotting (Blot) with the anti-Tyr(P) (PY)
antibody RC20 (A, a; B, a;
and C, a). The filter used in a was
reprobed with anti-GluR
2 antibodies (A, b;
B, b; and C, b). The
expression levels of FynY531F and PSD-95 were confirmed by
immunoblotting (A, c; B, c;
C, c and data not shown). All experiments were
performed more than three times. Positions and sizes (kDa) of standard
protein markers are indicated on the left. The positions of GluR
2
(180 kDa), GluR
2 YF mutants (180 kDa), and FynY531F (59 kDa) are
indicated by arrowheads.
2--
Antisera that recognize Tyr-1472-phosphorylated GluR
2
were raised by immunizing rabbits with phosphotyrosine-containing
synthetic peptides corresponding to the amino acid sequence of GluR
2
surrounding Tyr-1472 (Fig.
3A). Antisera were extensively
preabsorbed with non-phosphorylated GST-C3 fusion protein that contains
Tyr-1472 and then affinity-purified. The purified antibodies, termed
anti-phospho-Tyr-1472 antibodies, showed selective immunoreactivity
with GST-C3 protein phosphorylated by Fyn in vitro but not
to non-phosphorylated GST-C3 protein and phosphorylated GST-C3 proteins
treated with BAP (Fig. 3B). Immunoreactivity was blocked by
preincubation of the antibodies with the antigen (data not shown). To
examine whether the purified antibodies recognize
Tyr-1472-phosphorylated GluR
2, 293T cells were transfected with
expression plasmids encoding FynY531F, PSD-95, and either HA-tagged
wild-type GluR
2 or GluR
2 Y1472F mutant. Western blots of
HA-tagged GluR
2 immunoprecipitates with anti-phospho-Tyr-1472 antibodies showed their specific reactivity with
tyrosine-phosphorylated wild-type GluR
2 but not with
tyrosine-phosphorylated GluR
2 Y1472F mutant or non-phosphorylated
GluR
2 (Fig. 3C). The level of tyrosine phosphorylation of
GluR
2 Y1472F mutant was lower than that of wild-type GluR
2 but
was clearly detectable using anti-Tyr(P) antibody (Fig. 2C
and data not shown). The amount of immunoprecipitated GluR
2 and the
expression levels of Fyn and PSD-95 were similar in each experiment
(Fig. 3C and data not shown). Thus, the
anti-phospho-Tyr-1472 antibodies specifically recognized
Tyr-1472-phosphorylated GluR
2. The antibodies did not recognize
tyrosine-phosphorylated NR2A (GluR
1) expressed in 293T cells (data
not shown).
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Fig. 3.
Characterization of rabbit
anti-phospho-Tyr-1472 polyclonal antibodies. A, the
synthetic peptide containing phospho-Tyr-1472, used for immunogen.
B, detection of the in vitro phosphorylated
GST-C3 protein with the anti-phospho-Tyr-1472 antibodies. GST-C3 fusion
protein was phosphorylated by GST-Fyn (lanes 2 and
3) and followed by treatment with BAP (lane 3).
The proteins were subjected to immunoblotting (Blot) with
anti-phospho-Tyr-1472 antibodies (a). The filter used in
a was reprobed with anti-GluR 2 mAb (b).
C, detection of Tyr-1472-phosphorylated GluR
2 in 293T
cells using the anti-phospho-Tyr-1472 antibodies. 293T cells were
transfected with combinations of expression plasmids encoding GluR
2,
GluR
2Y1472F, PSD-95, and FynY531F. GluR
2 immunoprecipitates
(IP) from the lysates were subjected to immunoblotting
(Blot) with anti-phospho-Tyr-1472 antibodies (a).
The filter used in a was reprobed with anti-GluR
2 mAb
(b). Expression levels of FynY531F and PSD-95 were confirmed
by immunoblotting (c and data not shown). Although the data
are not presented, immunoblotting of the filter used in a
with anti-Tyr(P) antibody showed basically the same pattern of
immunoreactive signals as shown in Fig. 2C, a
with respect to wild-type (WT) GluR
2 and GluR
2 Y1472F
mutant. All experiments were performed more than three times. Positions
and sizes (kDa) of standard protein markers are indicated on the left.
The positions of GST-C3 (51 kDa), GluR
2 (180 kDa), GluR
2 Y1472F
mutant (180 kDa), and FynY531F (59 kDa) are indicated by
arrowheads.
2 in Murine Brain--
To
examine whether Tyr-1472 of GluR
2 is phosphorylated in murine brain,
GluR
2 was immunoprecipitated from telencephalons, which had been
boiled to dissociate the NMDA receptor complex, and subjected to
immunoblotting with anti-phospho-Tyr-1472 antibodies. The antibodies
reacted with a protein corresponding to Tyr-1472-phosphorylated GluR
2, but this immunoreactivity was completely abolished when immunoprecipitated GluR
2 were extensively dephosphorylated with BAP
(Fig. 4A). The amount of
GluR
2 in each lane was similar. The results demonstrated that
GluR
2 was phosphorylated at Tyr-1472 in murine brain. Importantly,
the immunoreactivity of anti-phospho-Tyr-1472 antibodies for
immunoprecipitated GluR
2 from telencephalon of fyn mutant
mice was much less than that from wild-type mice (Fig. 4B).
Therefore, we concluded that Tyr-1472 of GluR
2 was phosphorylated in
murine brain and that Fyn contributed significantly to the phosphorylation events.
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Fig. 4.
Fyn-mediated phosphorylation of Tyr-1472 of
GluR 2 in murine brain. A,
phosphorylation of Tyr-1472 of GluR
2 in murine brain. Telencephalons
from wild-type mice were homogenized in RIPA, 0.5% SDS buffer, boiled,
and then diluted with 4 volumes of RIPA buffer. GluR
2
immunoprecipitates (IP) from the lysates were treated with
(lane 2) or without BAP (lane 1) and were
subjected to immunoblotting (Blot) with
anti-phospho-Tyr-1472 antibodies (a). The filter used in
a was reprobed with anti-GluR
2 mAb (b).
B, reduced level of Tyr-1472 phosphorylation of GluR
2 in
fyn mutant mice. Telencephalons from wild-type mice and
fyn mutant mice were homogenized in RIPA/0.5% SDS buffer,
boiled, and then diluted with 4 volumes of RIPA buffer. GluR
2
immunoprecipitates (IP) from the lysates of wild-type and
fyn mutant (fyn
/
) mice were subjected to
immunoblotting (Blot) with anti-phospho-Tyr-1472 antibodies
(a). The filter used in a was reprobed with
anti-GluR
2 mAb (b). All experiments were performed more
than three times. Positions and sizes (kDa) of standard protein markers
are indicated on the left. The positions of GluR
2 (180 kDa) are
indicated by arrowheads.
2 is developmentally regulated (40), the
profile of Tyr-1472 phosphorylation during postnatal development was
examined. GluR
2 immunoprecipitates from lysates of
telencephalons of mice at postnatal (P) days 3, 7, 16, 28, and 56 were
subjected to immunoblotting with anti-phospho-Tyr-1472 antibodies and
anti-phosphotyrosine antibody. The level of Tyr-1472 phosphorylation
was low at P3 and P7 but gradually increased at P16, P28, and P56 (Fig.
5). Overall tyrosine phosphorylation of GluR
2 during the same developmental period, which was determined by
blotting with an anti-Tyr(P) antibody, was similar to Tyr-1472 phosphorylation. A similar amount of immunoprecipitated GluR
2 was
loaded in each lane. Results suggest that phosphorylation of Tyr-1472
of GluR
2 in murine brain is developmentally regulated.
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Fig. 5.
Developmental change of Tyr-1472
phosphorylation in murine brain. Telencephalons from P3 to P56
wild-type mice were homogenized in RIPA/0.5% SDS buffer, boiled, and
then diluted with 4 volumes of RIPA buffer. The GluR 2
immunoprecipitates (IP) from the lysates were subjected to
immunoblotting (Blot) with anti-phospho-Tyr-1472 antibodies
(a). The filter used in a was reprobed with the
anti-Tyr(P) antibody (b) and anti-GluR
2 mAb
(c). All experiments were performed more than three times.
Positions and sizes (kDa) of standard protein markers are indicated on
the left. The positions of GluR
2 (180 kDa) are indicated by
arrowheads.
2 after LTP
Induction in the Hippocampal CA1 Region--
LTP in the CA1 region of
the hippocampus, which is a cellular model for learning and memory, is
induced by activation of postsynaptic NMDA receptors (41, 42). Because
tyrosine phosphorylation of postsynaptic NMDA receptors is implicated
in the expression of LTP at the Schaffer collateral-commissural-CA1
synapse (43), alteration in the level of Tyr-1472 phosphorylation after
induction of LTP was examined. Excitatory postsynaptic potentials were
recorded in the hippocampal CA1 region by extracellular field potential recording techniques. Tetanic stimulation of the afferent fibers (100 Hz for 1 s, repeated 4 times at 10-s intervals) gave rise to LTP
of excitatory synaptic transmission in C57BL/6 mice (184.4 ± 8.8% of base line, n = 10) (Fig.
6A). Sixty minutes after
tetanic stimulation, the ratio of the Tyr-1472 phosphorylation of
GluR
2 in the stimulated slices to that in the non-stimulated slices was 1.50 ± 0.13 (p < 0.002) (Fig. 6C;
representative data are shown in Fig. 6B). Five minutes
after tetanic stimulation, the level of Tyr-1472 phosphorylation of
GluR
2 in the stimulated slices was almost the same as that in the
control slices (data not shown). The data suggest that Tyr-1472
phosphorylation of GluR
2 is involved in the expression of LTP.
View larger version (13K):
[in a new window]
Fig. 6.
Increased Tyr-1472 phosphorylation of
GluR 2 in the hippocampal CA1 region after
induction of LTP. A, representative recording of LTP.
In the inset, sample traces (average of 10 consecutive
excitatory postsynaptic potentials (EPSP)) are shown, which
were recorded at the times indicated by the numbers in the figure. A
tungsten bipolar stimulating electrode was placed in the stratum
radiatum, and Schaffer collateral-commissural fibers were stimulated 4 times at 100 Hz for 1 s at an interval of 10 s to induce LTP.
B, representative blot of the GluR
2 immunoprecipitates
from non-stimulated control or stimulated slices with
anti-phospho-Tyr-1472 antibodies (a) and then with
anti-GluR
2 mAb (b). Hippocampal CA1 regions from
non-stimulated control (Ctrl) or stimulated (LTP)
slices were homogenized in RIPA/ 0.5% SDS buffer, boiled, and then
diluted with 4 volumes of RIPA buffer. GluR
2 immunoprecipitates
(IP) from the lysates were subjected to immunoblotting
(Blot) with anti-phospho-Tyr-1472 antibodies (a).
The filter used in a was reprobed with anti-GluR
2 mAb
(b). Positions and sizes (kDa) of standard protein markers
are indicated on the left. The positions of GluR
2 (180 kDa) are
indicated by arrowheads. C, quantification of
Tyr-1472 phosphorylation in non-stimulated control and stimulated
slices (n = 10 from each of 4 mice). The open
bar indicates the level of Tyr-1472 phosphorylation in
non-stimulated slices (Ctrl). The closed bar
indicates the level of Tyr-1472 phosphorylation in stimulated slices 60 min after the induction of LTP. An asterisk indicates
statistically significant difference from control (p < 0.002).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2, the most prominently
phosphorylated site in 293T cells, was phosphorylated in murine brain
and that Tyr-1472 phosphorylation was significantly reduced in
fyn mutant mice. We also showed that the level of Tyr-1472 phosphorylation was developmentally regulated and enhanced after induction of LTP. We found that 7 out of 25 tyrosine residues in the
intracellular C-terminal region of GluR
2 were significantly phosphorylated by Fyn in vitro. Among these residues,
Tyr-1252, Tyr-1336, and Tyr-1472 of GluR
2 were phosphorylated in HEK
293T cells when active Fyn was co-expressed. Tyr-1472 was the most prominently phosphorylated site in this system. Moreover, using anti-phospho-Tyr-1472 antibodies, we showed that Tyr-1472 of GluR
2 was phosphorylated in murine brain. When examined in vitro,
the level of Tyr-1472 phosphorylation was similar, relative to that of
other major Fyn-mediated phosphorylation sites. However in 293T cells,
active Fyn mainly phosphorylated Tyr-1472 in comparison with the other
six tyrosine residues identified in vitro. This difference
may be due in part to an excess phosphorylation reaction in
vitro or the presence of protein-tyrosine phosphatases in 293T cells. To our knowledge, Tyr-1472 of GluR
2 is a residue identified first as a tyrosine phosphorylation site of NMDA receptors. There are
cognate sites to Tyr-1252, Tyr-1336, and Tyr-1472 of GluR
2 in
GluR
1 (4-6). To further understand how tyrosine phosphorylation regulates NMDA receptor function, determination of Fyn-mediated phosphorylation sites of GluR
1 is also important.
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)
receptors (41, 42). Induction of LTP produces rapid activation of Src
within 1-5 min, and prior application of Src-specific inhibitors
prevents induction of LTP in this region (43). We observed enhancement
of Tyr-1472 phosphorylation of GluR
2 after 60 min of LTP induction.
This suggests that Src-family kinases may phosphorylate GluR
2
slowly, similar to the phosphorylation of the GluR1 subunit of AMPA
receptors by CaMKII (44). Although CaMKII is activated within 1 min
after induction of LTP (45), the potentiation of AMPA receptor-mediated
currents by CaMKII reaches a maximum 15-30 min after induction of LTP
(44, 46, 47). Similar changes are seen in the overall NR2B (GluR
2)
tyrosine phosphorylation, which is enhanced 15 min after induction of
LTP in the dentate gyrus of anesthetized adult rats (26, 27). Moreover,
we observed that the level of Tyr-1472 phosphorylation as well as
overall tyrosine phosphorylation of GluR
2 was low during embryonic
(data not shown) and early developmental stages (P3 and P7). This may
be partly due to the low expression of PSD-95, which promotes
Fyn-mediated phosphorylation of GluR
2 as well as GluR
1 (NR2A)
(19), during the early developmental stages (48). The low level of
Tyr-1472 phosphorylation in P3 and P7 mice may cause small LTP in the
early developmental stages (49, 50). The level of Tyr-1472
phosphorylation was significantly reduced in fyn mutant
mice, which show impaired hippocampal LTP and spatial learning (32),
suggesting that reduced Tyr-1472 phosphorylation of GluR
2 may partly
explain the defects in LTP and spatial learning in fyn
mutant mice. These observations suggest that Tyr-1472 phosphorylation
may be required for the expression of LTP and is, therefore, important
for synaptic plasticity in the hippocampus. Since NMDA receptors are
phosphorylated not only by Src-family kinases but also serine/threonine
kinases such as protein kinase C, protein kinase A, and CaMKII
(15-19), phosphorylation of GluR
2 by various kinases may be
involved in multiple forms of synaptic plasticity as in the case of
GluR1, in which Ser-831 phosphorylation of CaMKII and Ser-845
phosphorylation by protein kinase A differentially contribute to
hippocampal synaptic plasticity (51).
1-GluR
2 channels in mice) expressed in HEK
293 cells (28). However, since NMDA receptor protein complexes from
murine brain are composed of a variety of postsynaptic proteins (52),
some of which are missing in HEK 293 cells, contribution of Tyr-1472
phosphorylation of GluR
2 to the channel activity of NMDA receptor
in vivo should be examined. Because protein tyrosine phosphorylation regulates protein-protein interactions (53), Tyr-1472
phosphorylation by Src-family kinases may induce intracellular signal
transduction pathways by recruiting Src homology 2-containing proteins
(15). This might contribute to the biochemical changes required for
modulation of synaptic transmission and synaptic plasticity. Indeed,
many signaling pathways regulated by tyrosine kinases, such as the Ras
mitogen-activated protein kinase pathway, are involved in modulation of
synaptic transmission and long term memory (54). In addition,
clustering of receptors at the postsynaptic membrane is crucial for
rapid and efficient synaptic signaling (55). Acetylcholine receptor
clustering at neuromuscular junctions is regulated by tyrosine
phosphorylation of the receptor (56). Similarly, NMDA receptor
clustering at synapses may be regulated by Tyr-1472 phosphorylation of
GluR
2.
2
showing that Tyr-1472 of GluR
2 is a major phosphorylation site. Our
data suggest that Tyr-1472 phosphorylation may modulate hippocampal
synaptic plasticity. To further establish the physiological importance
of Tyr-1472 phosphorylation of GluR
2, it is interesting to
investigate electrophysiological and behavioral changes in GluR
2
Y1472F knock-in mice, where Y1472F mutant of GluR
2 replaces wild-type GluR
2.
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ACKNOWLEDGEMENTS |
---|
We thank S. Aizawa for fyn mutant mice. We also thank S. Nakanishi for the NR2A cDNA.
![]() |
FOOTNOTES |
---|
* This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan.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.
§ This author was supported in part by Japan Society for the Promotion of Science fellowships for Japanese Junior Scientists.
To whom correspondence should be addressed: Dept. of Oncology,
Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan. Tel.: 81-3-5449-5301; Fax: 81-3-5449-5413; E-mail: tyamamot@ims.u-tokyo.ac.jp.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M008085200
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ABBREVIATIONS |
---|
The abbreviations used are:
NMDA, N-methyl-D-aspartate;
GluR, glutamate receptor;
NR1, NMDA receptor subunit 1;
LTP, long term potentiation;
BAP, bacterial alkaline phosphatase;
Tyr(P), phosphotyrosine;
HA, influenza
hemagglutinin;
GST, glutathione-S-transferase;
CaMKII, calcium/calmodulin-dependent protein kinase II;
mAb, monoclonal antibody;
RIPA, radioimmune precipitation buffer;
HEK cells, human embryonic kidney cells;
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid.
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