Characterization of Fyn-mediated Tyrosine Phosphorylation Sites on GluRepsilon 2 (NR2B) Subunit of the N-Methyl-D-aspartate Receptor*

Takanobu NakazawaDagger §, Shoji Komai, Tohru TezukaDagger , Chihiro HisatsuneDagger , Hisashi UmemoriDagger , Kentaro Semba||, Masayoshi Mishina**, Toshiya Manabe, and Tadashi YamamotoDagger DaggerDagger

From the Department of Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 GluRepsilon 2 (NR2B) subunit of NMDA receptors. Seven out of 25 tyrosine residues in the C-terminal cytoplasmic region of GluRepsilon 2 were phosphorylated by Fyn in vitro. Of these 7 residues, Tyr-1252, Tyr-1336, and Tyr-1472 in GluRepsilon 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 GluRepsilon 2 and showed that Tyr-1472 of GluRepsilon 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 GluRepsilon 2 is important for synaptic plasticity.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 GluRzeta 1 (NR1 in rat) and modulatory subunits GluRepsilon 1-GluRepsilon 4 (NR2A-NR2D in rat) (4-6). The GluRzeta 1 subunit is essential for the function of NMDA receptor channels, whereas GluRepsilon 1-GluRepsilon 4 subunits determine the characteristics of NMDA receptor channels by forming different heteromeric configurations with the GluRzeta 1 subunit (4-6). The physiological importance of these subunits has been demonstrated by gene targeting. Mice with a null mutation in GluRzeta 1 die neonatally (7, 8). Mutant mice in which GluRzeta 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 GluRepsilon 1 mutant mice, hippocampal LTP is reduced, and spatial learning is impaired (10). Disruption of GluRepsilon 2 results in neonatal death and impairment of hippocampal long term depression (11).

GluRepsilon 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 GluRepsilon subunits is also demonstrated by gene targeting. Phenotypic defects in mice expressing C-terminally truncated GluRepsilon 1 or GluRepsilon 2 are similar to those in mice lacking entire GluRepsilon 1 or GluRepsilon 2 (13, 14). C-terminal tails of GluRepsilon subunits are likely to participate in their synaptic localization or regulation of NMDA receptor functions. Furthermore, GluRepsilon 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.

In the NMDA receptor complex, GluRepsilon 1, GluRepsilon 2, and GluRepsilon 4 are tyrosine-phosphorylated in the brain (20-22), and Fyn significantly contributes to phosphorylation of GluRepsilon 1 and GluRepsilon 2 (15, 18, 19). Among these subunits, GluRepsilon 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 GluRepsilon 2 in the rat insular cortex, and tyrosine phosphorylation of GluRepsilon 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 GluRzeta 1-GluRepsilon 2 channels in 293 cells (28). The p85 subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma , SHP2, and brain spectrin interact with GluRepsilon 2 in a tyrosine phosphorylation-dependent manner (15, 29-31). These data suggest that GluRepsilon 2 tyrosine phosphorylation may at least in part be involved in intracellular signaling in murine brain.

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 GluRepsilon 2 by the Src-family kinases, we set out experiments in which Fyn-mediated tyrosine phosphorylation sites of GluRepsilon 2 were determined. By showing that Tyr-1472 of GluRepsilon 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

Antibodies-- Rabbit polyclonal antibodies against GluRepsilon 2 were raised against a glutathione S-transferase (GST) fusion protein with mouse GluRepsilon 2 (amino acids 1034-1119) and affinity-purified. The anti-GluRepsilon 2 antibodies did not cross-react with rat NR2A (data not shown). Rabbit polyclonal antibodies against phospho-Tyr-1472 of GluRepsilon 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 GluRepsilon 2) followed by a column conjugated to the immunogen. The antibodies did not recognize NR2A (GluRepsilon 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.

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 GluRepsilon 2 proteins, the GST-GluRepsilon 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 [gamma -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-GluRepsilon 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.

Construction of cDNAs-- For epitope-tagging of GluRepsilon 2, GluRepsilon 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-GluRepsilon 2, was subcloned to pME18S (37). The expression plasmids pME-FynY531F and PSD-95 were described previously (19, 37). Various YF mutants of GluRepsilon 2 were generated by oligonucleotide-mediated site-directed mutagenesis (38). Mutations were verified by dideoxynucleotide sequencing.

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-GluRepsilon 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.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphopeptide Mapping of the C-terminal Cytoplasmic Region of the GluRepsilon 2 Subunit in Vitro-- The GluRepsilon 2 subunit contains 25 tyrosine residues in the intracellular C-terminal region. To determine the site(s) of Fyn-mediated GluRepsilon 2 phosphorylation, we constructed GST fusion proteins containing truncated segments of the intracellular C-terminal region of the GluRepsilon 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 [gamma -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 GluRepsilon 2 in vitro.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1.   Identification of tyrosine residues of GluRepsilon 2 phosphorylated by Fyn in vitro. A, schematic diagram of GST fusion proteins containing the intracellular C-terminal region of GluRepsilon 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.

Tyr-1472 as the Principal Fyn-mediated Phosphorylation Site in HEK 293T Cells-- In vivo phosphorylation of the seven tyrosine residues was examined using GluRepsilon 2-transfected HEK 293T cells. First, 293T cells were transfected with an expression plasmid encoding HA-tagged wild-type GluRepsilon 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 GluRepsilon 2 (data not shown) as well as NR2A (GluRepsilon 1) (19). The cells were lysed, and HA-tagged GluRepsilon 2 was immunoprecipitated from cellular lysates and subjected to immunoblotting with an anti-phosphotyrosine (Tyr(P)) antibody. GluRepsilon 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 GluRepsilon 2 or one of the seven single YF mutants of GluRepsilon 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 GluRepsilon 2. There was no reduction in the tyrosine phosphorylation levels of GluRepsilon 2 in the other mutants. Moreover, phosphorylation of GluRepsilon 2 Y1252F/Y1472F and Y1336F/Y1472F double mutants was less than that of GluRepsilon 2 Y1472F mutant (Fig. 2C, lanes 2, 3, and 4), and phosphorylation of GluRepsilon 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 GluRepsilon 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.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2.   Phosphorylation of Tyr-1252, Tyr-1336, and Tyr-1472 of GluRepsilon 2 by active Fyn in HEK 293T cells. A, tyrosine phosphorylation of GluRepsilon 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 GluRepsilon 2, various GluRepsilon 2 YF mutants, PSD-95, and FynY531F. The cells were lysed in TNE buffer. GluRepsilon 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-GluRepsilon 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 GluRepsilon 2 (180 kDa), GluRepsilon 2 YF mutants (180 kDa), and FynY531F (59 kDa) are indicated by arrowheads.

Characterization of Antibodies against Tyr-1472-phosphorylated GluRepsilon 2-- Antisera that recognize Tyr-1472-phosphorylated GluRepsilon 2 were raised by immunizing rabbits with phosphotyrosine-containing synthetic peptides corresponding to the amino acid sequence of GluRepsilon 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 GluRepsilon 2, 293T cells were transfected with expression plasmids encoding FynY531F, PSD-95, and either HA-tagged wild-type GluRepsilon 2 or GluRepsilon 2 Y1472F mutant. Western blots of HA-tagged GluRepsilon 2 immunoprecipitates with anti-phospho-Tyr-1472 antibodies showed their specific reactivity with tyrosine-phosphorylated wild-type GluRepsilon 2 but not with tyrosine-phosphorylated GluRepsilon 2 Y1472F mutant or non-phosphorylated GluRepsilon 2 (Fig. 3C). The level of tyrosine phosphorylation of GluRepsilon 2 Y1472F mutant was lower than that of wild-type GluRepsilon 2 but was clearly detectable using anti-Tyr(P) antibody (Fig. 2C and data not shown). The amount of immunoprecipitated GluRepsilon 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 GluRepsilon 2. The antibodies did not recognize tyrosine-phosphorylated NR2A (GluRepsilon 1) expressed in 293T cells (data not shown).



View larger version (15K):
[in this window]
[in a new window]
 
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-GluRepsilon 2 mAb (b). C, detection of Tyr-1472-phosphorylated GluRepsilon 2 in 293T cells using the anti-phospho-Tyr-1472 antibodies. 293T cells were transfected with combinations of expression plasmids encoding GluRepsilon 2, GluRepsilon 2Y1472F, PSD-95, and FynY531F. GluRepsilon 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-GluRepsilon 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) GluRepsilon 2 and GluRepsilon 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), GluRepsilon 2 (180 kDa), GluRepsilon 2 Y1472F mutant (180 kDa), and FynY531F (59 kDa) are indicated by arrowheads.

Phosphorylation of Tyr-1472 of GluRepsilon 2 in Murine Brain-- To examine whether Tyr-1472 of GluRepsilon 2 is phosphorylated in murine brain, GluRepsilon 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 GluRepsilon 2, but this immunoreactivity was completely abolished when immunoprecipitated GluRepsilon 2 were extensively dephosphorylated with BAP (Fig. 4A). The amount of GluRepsilon 2 in each lane was similar. The results demonstrated that GluRepsilon 2 was phosphorylated at Tyr-1472 in murine brain. Importantly, the immunoreactivity of anti-phospho-Tyr-1472 antibodies for immunoprecipitated GluRepsilon 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 GluRepsilon 2 was phosphorylated in murine brain and that Fyn contributed significantly to the phosphorylation events.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4.   Fyn-mediated phosphorylation of Tyr-1472 of GluRepsilon 2 in murine brain. A, phosphorylation of Tyr-1472 of GluRepsilon 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. GluRepsilon 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-GluRepsilon 2 mAb (b). B, reduced level of Tyr-1472 phosphorylation of GluRepsilon 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. GluRepsilon 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-GluRepsilon 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 GluRepsilon 2 (180 kDa) are indicated by arrowheads.

Because expression of GluRepsilon 2 is developmentally regulated (40), the profile of Tyr-1472 phosphorylation during postnatal development was examined. GluRepsilon 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 GluRepsilon 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 GluRepsilon 2 was loaded in each lane. Results suggest that phosphorylation of Tyr-1472 of GluRepsilon 2 in murine brain is developmentally regulated.



View larger version (21K):
[in this window]
[in a new window]
 
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 GluRepsilon 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-GluRepsilon 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 GluRepsilon 2 (180 kDa) are indicated by arrowheads.

Increase in Phosphorylation of Tyr-1472 of GluRepsilon 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 GluRepsilon 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 GluRepsilon 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 GluRepsilon 2 is involved in the expression of LTP.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6.   Increased Tyr-1472 phosphorylation of GluRepsilon 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 GluRepsilon 2 immunoprecipitates from non-stimulated control or stimulated slices with anti-phospho-Tyr-1472 antibodies (a) and then with anti-GluRepsilon 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. GluRepsilon 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-GluRepsilon 2 mAb (b). Positions and sizes (kDa) of standard protein markers are indicated on the left. The positions of GluRepsilon 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

Here we showed that Tyr-1472 of GluRepsilon 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 GluRepsilon 2 were significantly phosphorylated by Fyn in vitro. Among these residues, Tyr-1252, Tyr-1336, and Tyr-1472 of GluRepsilon 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 GluRepsilon 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 GluRepsilon 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 GluRepsilon 2 in GluRepsilon 1 (4-6). To further understand how tyrosine phosphorylation regulates NMDA receptor function, determination of Fyn-mediated phosphorylation sites of GluRepsilon 1 is also important.

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 alpha -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 GluRepsilon 2 after 60 min of LTP induction. This suggests that Src-family kinases may phosphorylate GluRepsilon 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 (GluRepsilon 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 GluRepsilon 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 GluRepsilon 2 as well as GluRepsilon 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 GluRepsilon 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 GluRepsilon 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).

It is reported that Src-family kinases do not potentiate recombinant NR1-NR2B channels (GluRzeta 1-GluRepsilon 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 GluRepsilon 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 GluRepsilon 2.

In summary, we report Fyn-mediated phosphorylation sites of GluRepsilon 2 showing that Tyr-1472 of GluRepsilon 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 GluRepsilon 2, it is interesting to investigate electrophysiological and behavioral changes in GluRepsilon 2 Y1472F knock-in mice, where Y1472F mutant of GluRepsilon 2 replaces wild-type GluRepsilon 2.


    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.

Dagger Dagger 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


    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, alpha -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Collingridge, G. L., and Bliss, T. V. P. (1995) Trends Neurosci. 18, 54-56[CrossRef][Medline] [Order article via Infotrieve]
2. McDonald, J. W., and Johnston, M. V. (1990) Brain Res. Rev. 15, 41-70[Medline] [Order article via Infotrieve]
3. Choi, D. W. (1988) Trends Neurosci. 11, 465-469[CrossRef][Medline] [Order article via Infotrieve]
4. Kutsuwada, T., Kashiwabuchi, N., Mori, H., Sakimura, K., Kushiya, E., Araki, K., Meguro, H., Masaki, H., Kumanishi, T., Arakawa, M., and Mishina, M. (1992) Nature 358, 36-41[CrossRef][Medline] [Order article via Infotrieve]
5. Meguro, H., Mori, H., Araki, K., Kushiya, E., Kutsuwada, T., Yamazaki, M., Kumanishi, T., Arakawa, M., Sakimura, K., and Mishina, M. (1992) Nature 357, 70-74[CrossRef][Medline] [Order article via Infotrieve]
6. Monyer, H., Sprengel, R., Schoepfer, R., Herb, A., Higuchi, M., Lomeli, H., Burnashev, N., Sakmann, B., and Seeburg, P. H. (1992) Science 256, 1217-1221[Medline] [Order article via Infotrieve]
7. Forrest, D., Yuzaki, M., Soares, H. D., Ng, L., Luk, D. C., Sheng, M., Stewart, C. L., Morgan, J. I., Connor, J. A., and Curran, T. (1994) Neuron 13, 325-338[Medline] [Order article via Infotrieve]
8. Li, Y., Erzurumlu, R., Chen, C., Jhaveri, S., and Tonegawa, S. (1994) Cell 76, 427-437[Medline] [Order article via Infotrieve]
9. Tsien, J. Z., Huerta, P. T., and Tonegawa, S. (1996) Cell 87, 1327-1338[Medline] [Order article via Infotrieve]
10. Sakimura, K., Kutsuwada, T., Ito, I., Manabe, T., Takayama, C., Kushiya, E., Yagi, T., Aizawa, S., Inoue, Y., Sugiyama, H., and Mishina, M. (1995) Nature 373, 151-155[CrossRef][Medline] [Order article via Infotrieve]
11. Kutsuwada, T., Sakimura, K., Manabe, T., Takayama, C., Katakura, N., Kushiya, E., Natsume, R., Watanabe, M., Inoue, Y., Yagi, T., Aizawa, S., Arakawa, M., Takahashi, T., Nakamura, Y., Mori, H., and Mishina, M. (1996) Neuron 16, 333-344[Medline] [Order article via Infotrieve]
12. Sheng, M. (1996) Neuron 17, 575-578[Medline] [Order article via Infotrieve]
13. Mori, H., Manabe, T., Watanabe, M., Satoh, Y., Suzuki, N., Toki, S., Nakamura, K., Yagi, T., Kushiya, E., Takahashi, T., Inoue, Y., Sakimura, K., and Mishina, M. (1998) Neuron 21, 571-580[CrossRef][Medline] [Order article via Infotrieve]
14. Sprengel, R., Suchanek, B., Amico, C., Brusa, R., Burnashev, N., Rozov, A., Hvalby, Ø., Jensen, V., Paulsen, O., Andersen, P., Kim, J. J., Thompson, R. F., Sun, W., Webster, L. C., Grant, S. G. N., Eilers, J., McNamara, J. O., and Seeburg, P. H. (1998) Cell 92, 279-289[Medline] [Order article via Infotrieve]
15. Hisatsune, C., Umemori, H., Mishina, M., and Yamamoto, T. (1999) Genes Cells 4, 657-666[Abstract/Free Full Text]
16. Leonard, A. S., and Hell, J. W. (1997) J. Biol. Chem. 272, 12107-12115[Abstract/Free Full Text]
17. Omkumar, R. V., Kiely, M. J., Rosenstein, A. J., Min, K.-T., and Kennedy, M. B. (1996) J. Biol. Chem. 271, 31670-31678[Abstract/Free Full Text]
18. Suzuki, T., and Okumura-Noji, K. (1995) Biochem. Biophys. Res. Commun. 216, 582-588[CrossRef][Medline] [Order article via Infotrieve]
19. Tezuka, T., Umemori, H., Akiyama, T., Nakanishi, S., and Yamamoto, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 435-440[Abstract/Free Full Text]
20. Dunah, A. W., Yasuda, R. P., and Wolfe, B. B. (1998) J. Neurochem. 71, 1926-1934[Medline] [Order article via Infotrieve]
21. Moon, I.-L., Apperson, M. L., and Kennedy, M. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3954-3958[Abstract]
22. Lau, L.-F., and Huganir, R. L. (1995) J. Biol. Chem. 270, 20036-20041[Abstract/Free Full Text]
23. Wang, Y.-T., and Salter, M. W. (1994) Nature 369, 233-235[CrossRef][Medline] [Order article via Infotrieve]
24. Yu, X.-M., Askalan, R., Keil, G. J., II, and Salter, M. W. (1997) Science 275, 674-678[Abstract/Free Full Text]
25. Rosenblum, K., Berman, D. E., Hazvi, S., Lamprecht, R., and Dudai, Y. (1997) J. Neurosci. 17, 5129-5135[Abstract/Free Full Text]
26. Rosenblum, K., Dudai, Y., and Richter-Levein, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10457-10460[Abstract/Free Full Text]
27. Rostas, J. A. P., Brent, V. A., Voss, K., Errington, M. L., Bliss, T. V. P, and Gurd, J. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10452-10456[Abstract/Free Full Text]
28. Köhr, G., and Seeburg, P. H. (1996) J. Physiol. (Lond.) 492, 445-452[Abstract]
29. Gurd, J. W., and Bissoon, N. (1997) J. Neurochem. 69, 623-630[Medline] [Order article via Infotrieve]
30. Lin, S.-Y., Wu, K., Len, G.-W., Xu, J.-L., Levine, E. S., Suen, P.-C., Mount, H. T. J., and Black, I. B. (1999) Mol. Brain Res. 70, 18-25[CrossRef][Medline] [Order article via Infotrieve]
31. Wechsler, A., and Teichberg, V. I. (1998) EMBO J. 17, 3931-3939[Abstract/Free Full Text]
32. Grant, S. G. N., O'Dell, T. J., Karl, K. A., Stein, P. L., Soriano, P., and Kandel, E. R. (1992) Science 258, 1903-1910[Medline] [Order article via Infotrieve]
33. Miyakawa, T., Yagi, T., Kitazawa, H., Yasuda, M., Kawai, N., Tsuboi, K., and Niki, H. (1997) Science 278, 698-701[Abstract/Free Full Text]
34. Umemori, H., Sato, S., Yagi, T., Aizawa, S., and Yamamoto, T. (1994) Nature 367, 572-576[CrossRef][Medline] [Order article via Infotrieve]
35. Zhao, W., Cavallaro, S., Gusev, P., and Alkon, D. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8098-8103[Abstract/Free Full Text]
36. Boyle, W. J., van der Geer, P., and Hunter, T. (1991) Methods Enzymol. 201, 110-149[Medline] [Order article via Infotrieve]
37. Takeuchi, M., Kuramochi, S., Fusaki, N., Nada, S., Kawamura-Tsuzuku, J., Matsuda, S., Semba, K., Toyoshima, K., Okada, M., and Yamamoto, T. (1993) J. Biol. Chem. 268, 27413-27419[Abstract/Free Full Text]
38. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382[Medline] [Order article via Infotrieve]
39. Manabe, T., Aiba, A., Yamada, A., Ichise, T., Sakagami, H., Kondo, H., and Katsuki, M. (2000) J. Neurosci. 20, 2504-2511[Abstract/Free Full Text]
40. Okabe, S., Collin, C., Auerbach, J. M., Meiri, N., Bengzon, J., Kennedy, M. B., Segal, M., and McKay, D. G. (1998) J. Neurosci. 18, 4177-4188[Abstract/Free Full Text]
41. Collingridge, G. L., Kehl, S. J., and McLennan, H. (1983) J. Physiol. (Lond.) 334, 33-46[Abstract]
42. Nicoll, R. A., and Malenka, R. C. (1995) Nature 377, 115-118[CrossRef][Medline] [Order article via Infotrieve]
43. Lu, Y. M., Roder, J. C., Davidow, J., and Salter, M. W. (1998) Science 279, 1363-1367[Abstract/Free Full Text]
44. Barria, A., Muller, D., Derkach, V., Griffith, L. C., and Soderling, T. R. (1997) Science 276, 2042-2045[Abstract/Free Full Text]
45. Fukunaga, K., Stoppini, L., Miyamoto, E., and Muller, D. (1993) J. Biol. Chem. 268, 7863-7867[Abstract/Free Full Text]
46. Lledo, P.-M., Hjelmstad, G. O., Mukherji, S., Soderling, T. R., Malenka, R. C., and Nicoll, R. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11175-11179[Abstract]
47. McGlade-McCulloh, E., Yamamoto, H., Tan, S. E., Brickey, D. A., and Soderling, T. R. (1993) Nature 362, 640-642[CrossRef][Medline] [Order article via Infotrieve]
48. Sans, N., Petralia, R. S., Wang, Y.-X., Blahos, J., II, Hell, J. W., and Wenthold, R. J. (2000) J. Neurosci. 20, 1260-1271[Abstract/Free Full Text]
49. Bolshakov, V. Y., and Siegelbaum, S. A. (1995) Science 269, 1730-1734[Medline] [Order article via Infotrieve]
50. Liao, D., and Malinow, R. (1996) Learn. Memory 3, 138-149[Abstract]
51. Lee, H.-Y., Barbarosie, M., Kameyama, K., Bear, M. F., and Huganir, R. L. (2000) Nature 405, 955-959[CrossRef][Medline] [Order article via Infotrieve]
52. Husi, H., Ward, M. A., Choudhary, J. S., Blackstock, W. P., and Grant, S. G. N. (2000) Nat. Neurosci. 3, 661-669[CrossRef][Medline] [Order article via Infotrieve]
53. Pawson, T., and Scott, J. D. (1997) Science 278, 2075-2080[Abstract/Free Full Text]
54. Brambilla, R., Gnesutta, N., Minichiello, L., White, G., Roylance, A. J., Herron, C. E., Ramsey, M., Wolfer, D. P., Cestari, V., Rossi-Arnaud, C., Grant, S. G. N., Chapman, P. F., Lipp, H. P., Sturani, E., and Klein, R. (1997) Nature 390, 281-286[CrossRef][Medline] [Order article via Infotrieve]
55. Froehner, S. C. (1993) Annu. Rev. Neurosci. 16, 347-368[CrossRef][Medline] [Order article via Infotrieve]
56. Wallace, B. G., Qu, Z., and Huganir, R. L. (1991) Neuron 6, 869-878[Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.