©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Tyrosine Phosphorylation of N-Methyl-

D

-aspartate Receptor Subunits (*)

(Received for publication, May 4, 1995; and in revised form, June 9, 1995)

Lit-Fui Lau Richard L. Huganir (§)

From the Department of Neuroscience, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Protein-tyrosine phosphorylation has recently been suggested to play an important role in synaptic transmission at the neuromuscular junction. The role of tyrosine phosphorylation in the modulation of synaptic function in the central nervous system, however, is not clear. In this study, immunocytochemical staining with an anti-phosphotyrosine antibody demonstrates that there are high levels of phosphotyrosine, which co-localizes with glutamate receptors at excitatory synapses on cultured hippocampal neurons. In addition, the tyrosine phosphorylation of various subtypes of glutamate receptors were examined using subunit-specific antibodies. Glutamate receptors are the major excitatory neurotransmitter receptors in the central nervous system and are classified into three major classes: alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate, kainate, and N-methyl-D-aspartate (NMDA) receptors, based on their electrophysiological and pharmacological properties. NMDA receptors play a central role in synaptic plasticity, synaptogenesis, and excitotoxicity and are thought to be heteromeric complexes of the two types of subunits: NR1 and NR2(A-D) subunits. Immunoaffinity chromatography of detergent extracts of rat synaptic plasma membranes on anti-phosphotyrosine antibody-agarose showed that the NR2A and NR2B subunits but not the NR1 subunit are tyrosine-phosphorylated. Conversely, immunoprecipitation of the NR1, NR2A, and NR2B subunits with subunit specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies confirmed that the NR2A and NR2B subunits but not the NR1 subunit were phosphorylated on tyrosine residues. No tyrosine phosphorylation of the AMPA (GluR1-4) and kainate (GluR6/7, KA2) receptor subunits was detected. It was estimated that 2.1 ± 1.3% of the NR2A subunits and 3.6 ± 2.4% of the NR2B subunits were tyrosine-phosphorylated in vivo. In addition, endogenous protein-tyrosine kinases in synaptic plasma membranes phosphorylated the NR2A subunit in vitro, increasing its phosphorylation 6-8-fold but did not phosphorylate NR1 or NR2B. These studies demonstrate that NMDA receptor subunits are differentially tyrosine-phosphorylated and suggest that tyrosine phosphorylation of the NR2 subunits may be important for regulating NMDA receptor function.


INTRODUCTION

Protein-tyrosine phosphorylation has been shown to play major roles in the regulation of cellular growth, proliferation, and differentiation (Hunter and Cooper, 1985; Hunter, 1987). However, studies demonstrating high levels of protein-tyrosine kinases and phosphatases in the central nervous system have suggested that tyrosine phosphorylation is also involved in the regulation of neuronal processes (Cooke and Perlmutter, 1989; Wagner et al., 1991a). Moreover, high levels of protein-tyrosine kinases and phosphatases and their substrates at synapses, both presynaptically and postsynaptically, suggest that tyrosine phosphorylation may regulate synaptic transmission (Huganir et al., 1984; Maness et al., 1988; Pang set al., 1988). For example, immunocytochemical staining of muscle with anti-phosphotyrosine antibodies detects high levels of phosphotyrosine, which co-localizes with the distribution of the nicotinic acetylcholine receptor at the neuromuscular junction (Qu et al., 1990; Qu and Huganir, 1994). A significant percentage of the phosphotyrosine at the neuromuscular junction appears to be due to tyrosine phosphorylation of the nicotinic acetylcholine receptor (Huganir et al., 1984; Hopfield et al., 1988; Wagner et al., 1991b; Qu and Huganir, 1994). In addition, endogenous protein-tyrosine kinases in isolated postsynaptic membranes phosphorylate the nicotinic acetylcholine receptor in vitro and in vivo (Hopfield et al., 1988; Huganir, 1991). Tyrosine phosphorylation of the nicotinic acetylcholine receptor appears to be involved in the regulation of receptor desensitization (Hopfield et al., 1988; Huganir and Greengard, 1990) and clustering of the receptor at the neuromuscular junction (Qu et al., 1990; Wallace, 1991, 1994; Qu and Huganir, 1994).

The role of tyrosine phosphorylation in the regulation of ligand-gated ion channels in the central nervous system has been less clear. The major excitatory neurotransmitter receptors in the central nervous system are the glutamate receptors (Seeburg, 1993; Hollmann and Heinemann, 1994). These receptors can be divided into three major classes: AMPA, (^1)kainate, and NMDA receptors, based on their selective agonists and on their physiological properties (Seeburg, 1993; Hollmann and Heinemann, 1994). AMPA and kainate receptors mediate rapid excitatory transmission in the central nervous system, whereas NMDA receptors play primarily a modulatory role and are important in synaptic plasticity, neuronal development, and excitotoxicity (Seeburg, 1993; Hollmann and Heinemann, 1994). Recent molecular cloning studies have demonstrated that NMDA receptors are hetero-oligomers consisting of two types of subunits: NR1 and NR2 subunits (Moriyoshi et al., 1991; Meguro et al., 1992; Monyer et al., 1992). The NR2 family consists of four homologous subunits, namely, NR2A, NR2B, NR2C, and NR2D, which are differentially expressed in various brain regions (Meguro et al., 1992; Monyer et al., 1992, 1994).

Recent studies have provided evidence that NMDA receptors are regulated by tyrosine phosphorylation. Studies in dorsal horn neurons have demonstrated that NMDA receptors are potentiated by perfusion of protein-tyrosine kinase or protein-tyrosine phosphatase inhibitors (Wang and Salter, 1994). In addition, protein-tyrosine kinase inhibitors attenuate NMDA-receptor function (Wang and Salter, 1994). However, it is not known whether the effect of protein-tyrosine kinases is mediated through direct phosphorylation of NMDA receptors. In the present study, we examined whether the NMDA receptors are tyrosine-phosphorylated. Immunocytochemical studies of hippocampal neurons in culture demonstrated that there is a high level of phosphotyrosine at excitatory synapses that co-localized with glutamate receptors. Moreover, biochemical analysis showed that although the AMPA and kainate receptors do not appear to be tyrosine-phosphorylated, the NR2A and NR2B subunits but not the NR1 subunit are specifically phosphorylated on tyrosine residues.


EXPERIMENTAL PROCEDURES

Materials

Sodium orthovanadate, ammonium molybdate, phenylmethylsulfonyl fluoride, sodium pyrophosphate, sodium fluoride, and protein A-Sepharose were purchased from Sigma. Trasylol was obtained from Mobay Chemical, and leupeptin, chymostatin, and antipain were from Chemicon. Tween 20 and Triton X-100 were from Bio-Rad. SDS was obtained from U. S. Biochemical Corp. ECL reagent and peroxidase conjugated anti-mouse antibody were ordered from Amersham. Polyvinylidene difluoride membrane was from Millipore. Anti-phosphotyrosine antibody and agarose-conjugated anti-phosphotyrosine antibody were purchased from Upstate Biotechnology Inc. Peroxidase-conjugated donkey anti-rabbit antibody and rhodamine-conjugated anti-mouse antibody were from Jackson ImmunoResearch Laboratories, Inc. Fluorescein streptavidin and biotinylated anti-rabbit antibody were purchased from Vector Laboratories Inc. Paraformaldehyde was from Polysciences, Inc. 1,4-Diazabicyclo[2.2.2]octane was obtained from Aldrich, and PermaFluor was purchased from Immunon. The cDNA clone for the NR2A subunit was a generous gift from Dr. Shigetada Nakanishi, and that for the NR2B subunit was kindly provided by Dr. Stephen Heinemann. Both clones were in pRK5 expression vector. QT-6 fibroblasts were generously supplied by Dr. John Merlie.

Antibodies

NR1-NH(2) and NR1-COOH were antibodies raised against the N- (residues 19-38) and C-terminal (residues 919-938) peptides of NR1, respectively (Tingley et al., 1993). The NR1-TM3/4 antibody, which recognized a fusion protein encoding the intracellular domain between the proposed third and fourth transmembrane domains of NR1 (residues 660-811), was a generous gift from Dr. Stephen Heinemann, Salk Institute (Brose et al., 1993). The NR2A antibody was raised against a hexahistidine fusion protein of the 1 C-terminal domain (residues 1247-1464). The NR2B antibody recognized a C-terminal peptide (residues 1463-1482) of NR2B. All the above antibodies are affinity-purified rabbit polyclonal antibodies except NR1-TM3/4, which is a mouse monoclonal antibody.

Preparation of Synaptic Plasma Membranes

Synaptic plasma membranes were prepared essentially according to the procedures of Blackstone et al.(1992). All procedures were performed at 4 °C. All reagents used were made with tyrosine phosphatase inhibitors (1 mM sodium vanadate and 0.1 mM ammonium molybdate) and protease inhibitors (20 units/ml Trasylol, 20 µg/ml leupeptin, 20 µg/ml antipain, 20 µg/ml pepstatin A, 20 µg/ml chymostatin, and 0.1 mM phenylmethylsulfonyl fluoride). In brief, male Sprague-Dawley rats (150-175 g) were decapitated, and the cerebral cortices were immediately removed and homogenized in 10 volumes of HEPES-buffered sucrose (0.32 M sucrose and 4 mM HEPES, pH 7.4). The homogenate was centrifuged at 800 g for 10 min to remove the nuclear fraction. The supernatant was then spun at 9,000 g for 15 min to yield the crude synaptosomal fraction. This pellet was resuspended in 10 volumes of HEPES-buffered sucrose and then respun at 10,200 g for another 15 min. The resulting pellet was lysed by hypo-osmotic shock in water, rapidly adjusted to 1 mM HEPES, and mixed constantly for 30 min. The lysate was then centrifuged at 25,000 g for 20 min, and the pellet was resuspended in HEPES-buffered sucrose (0.25 M sucrose and 4 mM HEPES, pH 7.4). The resuspended membranes were then carefully layered on top of a discontinuous gradient containing 0.8 M/1.0 M/1.2 M sucrose. Synaptic plasma membranes were recovered in the layer between 1.0 and 1.2 M sucrose and resuspended in phosphate-buffered saline (pH 7.4) with inhibitors of tyrosine phosphatases and proteases as described above.

Transient Expression of the NR2 Subunits in QT-6 Fibroblasts

The cDNA encoding the NR2A and NR2B subunits were respectively transfected into QT-6 fibroblasts using calcium phosphate co-precipitation with 20 µg of DNA/10-cm culture dish (Blackstone et al., 1992). 48 hours following transfection, QT-6 fibroblasts were harvested in 50 mM phosphate-buffered saline (pH 7.4) containing 20 units/ml Trasylol, 0.1 mM phenylmethylsulfonyl fluoride, and 10 mM EDTA and then diluted directly into sample buffer (Laemmli, 1970).

Immunocytochemistry

Low density rat hippocampal neuronal culture was performed according to the method of Goslin and Banker (1991). Briefly, hippocampi from E18/19 pups were removed, digested in trypsin and DNase, and plated on circular glass coverslips for 3-4 h for attachment. The coverslips were then flipped upside down on a layer of glial cells, which supplied the necessary factors for the growth and survival of the hippocampal neurons. After 3-4 weeks hippocampal neurons were fixed, permeabilized, and then blocked by 10% normal goat serum at 37 °C for 1 h. Primary antibodies were added and incubated overnight at 4 °C. Staining with the NR2B antibody was amplified with biotinylated anti-rabbit antibody and fluorescein streptavidin. The anti-phosphotyrosine antibody was detected with rhodamine-conjugated anti-mouse antibody. Mounting of coverslips was performed with PermaFluor with 2.5% 1,4-diazabicyclo[2.2.2]octane to reduce photobleaching of the fluorescent dyes.

Immunoprecipitation

Synaptic plasma membranes (250 µg) were first solubilized in 2% SDS in solubilization buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na(2)HPO(4)bullet7H(2)O, 1.4 mM KH(2)PO 5 mM EDTA, 5 mM EGTA, 1 mM sodium vanadate, 10 mM sodium pyrophosphate, 50 mM NaF, 20 units/ml Trasylol, 20 µg/ml leupeptin, 20 µg/ml antipain, 20 µg/ml pepstatin A, 20 µg/ml chymostatin, and 0.1 mM phenylmethylsulfonyl fluoride) and then diluted with 5 volumes of cold 2% Triton X-100 in solubilization buffer. They were incubated with either 50 µl of agarose-conjugated anti-phosphotyrosine antibody or 5 µg of each polyclonal antibody against various NMDA receptor subunits and 100 µl of protein A-Sepharose for 2-3 h at 4 °C. The reaction mixture was then washed several times, and the proteins were eluted from protein A-Sepharose by Laemmli sample buffer (Laemmli, 1970).

Immunoblot

Synaptic plasma membranes proteins were resolved on SDS-polyacrylamide gels and then electrotransferred to polyvinylidene difluoride membrane. The membrane was then blocked with 0.5% milk/0.1% Tween 20 in Tris-buffered saline for 1 h followed by 90 min of incubation of primary antibody. After several washes with the blocking buffer, peroxidase-conjugated secondary antibody (either anti-rabbit or mouse) was added and incubated for 45-60 min. The membrane was washed, and enhanced chemiluminescence was carried out according to protocols provided by Amersham. Both primary and secondary antibodies were made in the blocking buffer. The signal developed by enhanced chemiluminescence on preflashed autoradiographic film was quantitated using a densitometer (Molecular Dynamics).

In Vitro Tyrosine Phosphorylation by Endogenous Kinases

Synaptic plasma membranes (250 µg) in 50 µl phosphate-buffered saline (with inhibitors of tyrosine phosphatases and proteases as described above) were mixed with 50 µl of 2 phosphorylation buffer (40 mM Tris, 0.2 mM dithiothreitol, 4 mM MnCl(2), 200 µM ATP, 40 mM MgCl(2), 2 mM EDTA, 2 mM EGTA, 2 mM sodium vanadate, and 2 mM ouabain) and incubated at 37 °C for the indicated amount of time. The reaction was stopped by the addition of 50 µl of 6% SDS and neutralized with 750 µl of 2% Triton X-100 in solubilization buffer. Immunoprecipitation by various NMDA receptor antibodies followed by immunoblot with anti-phosphotyrosine antibody was then carried out as mentioned above. The same membrane was stripped (according to protocol provided by Amersham) and reprobed with NR1-TM3/4, NR2A, or NR2B antibodies to show the actual presence of these NMDA receptor subunits.


RESULTS

Specificity of Antibodies against NMDA Receptor Subunits

Five different NMDA receptor antibodies were used in the present study. Their specificities were tested by immunoblot of synaptic plasma membranes in the absence and presence of their respective immunogens. As shown in Fig. 1A, all three NR1 antibodies, namely NR1-COOH, NR1-NH(2), and NR1-TM3/4, recognized a single protein with an apparent molecular mass of 120 kDa. In the presence of their respective immunogens, none of the antibodies recognized the 120-kDa protein. The NR2A and NR2B antibodies each detected a single protein with an apparent molecular mass of 170 and 180 kDa, respectively (Fig. 1A). Similarly, the signal obtained could be abolished when the antibodies were incubated in the presence of their immunogens.


Figure 1: Specificity of antibodies against NMDA receptor subunits. A, synaptic plasma membrane proteins (25 µg) resolved by 7.5% SDS-PAGE were transferred to polyvinylidene difluoride membrane and immunoblotted by antibodies NR1-COOH, NR1-NH(2), NR1-TM3/4, NR2A, and NR2B in the presence or the absence of their respective immunogens (3.8-50 µg/ml). B, QT-6 fibroblasts expressing beta-galactosidase as control (mock), NR2A subunit (2A), or NR2B subunit (2B) were harvested, resolved by 5% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and immunoblotted by the NR2A (left panel) and NR2B (right panel) antibodies. Synaptic plasma membrane proteins (SPM) were included for comparison. Molecular weight markers are shown by the numbers on the left.



Because the NR2A and NR2B subunits are homologous to each other and migrate closely on SDS-PAGE, we further characterized the specificity of the NR2A and NR2B antibodies by immunoblotting QT-6 fibroblasts transiently transfected with either the NR2A or the NR2B subunit. As shown in Fig. 1B, the NR2A antibody recognized a single protein band only from QT-6 fibroblasts transfected with the NR2A cDNA. On the other hand, the NR2B antibody detected a single protein band only from cells transfected with the NR2B cDNA. Therefore, both NR2 antibodies were highly specific and did not cross-react with the other NR2 subunit.

High Levels of Phosphotyrosine Are Localized to Synaptic Regions and Co-localize with the NMDA Receptor

To study the role of tyrosine phosphorylation in the regulation of synaptic transmission in the central nervous system, cultured rat hippocampal neurons were stained with anti-phosphotyrosine antibodies. Using these antibodies, phosphotyrosine was found clustered along dendritic processes and was enriched in dendritic spines (Fig. 2, A and D) similar to the distribution of glutamate receptor subunits (Craig et al., 1993, 1994; Roche and Huganir, 1995). Double labeling of these neurons with an anti-NR2B subunit antibody demonstrated high levels of punctate staining along the dendrites (Fig. 2, B and E), which largely co-localized with the phosphotyrosine staining (Fig. 2, C, D, and E). The phosphotyrosine and NR2B staining was blocked in the presence of 0.2 mM phosphotyrosine and 100 µg/ml of the NR2B immunogen, respectively (data not shown), suggesting that the staining was specific. The NR1 and NR2A antibodies did not specifically stain NMDA receptor subunits in the hippocampal cultures; therefore, no data are presented for these two antibodies. The high level of synaptic phosphotyrosine staining is similar to that seen at the neuromuscular junction (Qu et al., 1990) and suggested to us that like the nicotinic acetylcholine receptor, glutamate receptors may be tyrosine-phosphorylated.


Figure 2: Co-localization of NR2B with tyrosine-phosphorylated proteins. Rat hippocampal neurons (3-4 weeks in culture) were stained with the anti-phosphotyrosine antibody (A) and the NR2B antibody (B) and indirectly labeled with rhodamine and fluorescein, respectively. C shows the double exposure of hippocampal neurons stained with both antibodies. D and E are magnifications of regions indicated by the arrows in A and B, respectively, to show the co-localization of the NR2B subunit and phosphotyrosine.



Differential Tyrosine Phosphorylation of NMDA Receptor Subunits

To determine if glutamate receptor subunits were tyrosine-phosphorylated in the central nervous system, rat cortical synaptic plasma membranes were solubilized with 2% SDS and boiled to denature the NMDA receptor subunits, and then the extracts were diluted with 5 volumes of 2% Triton X-100 and isolated by immunoaffinity chromatography using agarose-conjugated anti-phosphotyrosine antibodies. Immunoblots using the NMDA receptor subunit-specific antibodies showed the presence of NR2A and NR2B but not NR1 (Fig. 3A), suggesting that both NR2 subunits but not the NR1 subunit were phosphorylated on tyrosine residues. Binding of both NR2 subunits to the agarose-conjugated anti-phosphotyrosine antibody appeared to be specific because it was inhibited in the presence of 0.2 mM phosphotyrosine (Fig. 3A). In contrast, no specific binding of the AMPA receptor subunits GluR1-3 or the kainate receptor subunits GluR6/7 to the anti-phosphotyrosine-agarose was observed. (^2)


Figure 3: Tyrosine phosphorylation of NMDA receptor subunits. A, synaptic plasma membrane proteins (250 µg) solubilized in 2% SDS were immunoprecipitated by 50 µl of agarose-conjugated anti-phosphotyrosine antibody in the absence(-) or the presence (+) of 0.2 mM phosphotyrosine (PY) and resolved by 5% SDS-PAGE. Immunoblotting was then performed using the NR1-TM3/4 (NR1), NR2A, or NR2B antibody. An immunoblot using the anti-phosphotyrosine antibody on total synaptic plasma membrane proteins (25 µg) was shown on the left (total). The numbers indicate positions of molecular weight markers. B, synaptic plasma membrane proteins (250 µg) solubilized in 2% SDS were immunoprecipitated in the absence of antibody (control) or in the presence of an irrelevant IgG, NR1-NH(2) plus NR1-COOH (NR1), NR2A, or NR2B antibody. The proteins were then resolved by 5% SDS-PAGE, followed by immunoblotting using anti-phosphotyrosine antibody. The tyrosine-phosphorylated proteins immunoprecipitated by the NR2A or NR2B antibody were not detected when the immunoprecipitation was done in the presence of the respective immunogens (Im) (3.8 and 10 µg/ml). Molecular weight markers are shown by the numbers on the left. C, synaptic plasma membrane proteins (250 µg) solubilized in 2% SDS were immunoprecipitated by the NR2A or NR2B antibody and separated by 5% SDS-PAGE. Immunoblot was then performed using anti-phosphotyrosine antibody in the absence (control) or presence of 0.2 mM phosphotyrosine (PY), phosphoserine (PS), or phosphothreonine (PT). The level of tyrosine phosphorylation on NR2A was enhanced by incubating the synaptic plasma membranes for 2 min under phosphorylating conditions.



The above results were confirmed by immunoprecipitation of the various glutamate receptor subunits with subunit-specific antibodies followed by anti-phosphotyrosine antibody immunoblots. As shown in Fig. 3B, immunoprecipitation using the NR2A and NR2B antibodies revealed tyrosine-phosphorylated 170- and 180-kDa proteins, respectively, the observed molecular masses of NR2A and NR2B (Fig. 1A). These two phosphotyrosine-containing proteins were not detected if the immunoprecipitation was done in the presence of the respective immunogens. In addition, no tyrosine-phosphorylated proteins were detected if the immunoprecipitation was done in the absence of an antibody (Fig. 3B) or in the presence of an irrelevant antibody, demonstrating that the tyrosine-phosphorylated proteins did not bind nonspecifically to protein A-Sepharose or antibodies in general. In contrast, immunoprecipitation using the NR1 antibodies did not yield any tyrosine-phosphorylated proteins, even though NR1 was shown to be present (see Fig. 5, bottom panels). These data confirmed that NR2A and NR2B were tyrosine-phosphorylated, whereas NR1 did not contain phosphotyrosine. In addition, no tyrosine phosphorylation of the AMPA receptor subunits GluR1-3 or the kainate receptor subunits KA2 or GluR6 was detected.^2


Figure 5: Phosphorylation of NMDA receptor subunits by endogenous protein-tyrosine kinases. Synaptic plasma membranes (250 µg) were incubated under phosphorylating conditions for the indicated time periods, immunoprecipitated with the NR1-NH(2) plus NR1-COOH (NR1), NR2A, or NR2B antibody, and resolved by 5% SDS-PAGE. Immunoblot was then performed using the anti-phosphotyrosine antibody (PY). The NR1-TM3/4, NR2A, and NR2B antibodies (NR) were also used to immunoblot the lanes under NR1, NR2A, and NR2B, respectively.



To determine the specificity of the anti-phosphotyrosine antibody, we performed immunoblot in the presence of phosphotyrosine, phosphoserine, or phosphothreonine. Only phosphotyrosine was shown to inhibit the signal detected by the anti-phosphotyrosine antibody (Fig. 3C), suggesting that the signal was due to phosphorylation of NR2A and NR2B on tyrosine but not serine or threonine residues.

Stoichiometry of Tyrosine Phosphorylation of NR2A and NR2B Subunits

We next determined the stoichiometry of tyrosine phosphorylation of the NR2A and NR2B subunits. As shown in Fig. 4, almost all the tyrosine-phosphorylated proteins were successfully adsorbed by subjecting the solubilized synaptic plasma membranes extract to five consecutive immunoprecipitations with the agarose-conjugated anti-phosphotyrosine antibody (compare lanes 1 and 7 of Fig 4). However, when the same samples were immunoblotted with the NR2A and NR2B antibodies, most of these subunits remained in the supernatant. Quantitation of the autoradiographic films by densitometry showed that 2.1 ± 1.3% (range, n = 2) of the NR2A and 3.6 ± 2.4% (range, n = 2) of the NR2B subunits were found in the immunoprecipitate and thus tyrosine-phosphorylated (Fig. 4, lanes 2-6 compared with lane 1). Similar findings were also obtained when postsynaptic density fractions were used. Therefore, it appears that both NR2A and NR2B had a low stoichiometry of tyrosine phosphorylation.


Figure 4: Stoichiometry of tyrosine phosphorylation of the NR2A and NR2B subunits. Detergent extracts of synaptic plasma membrane proteins (100 µg) were subject to five consecutive immunoprecipitations each using 20 µl of agarose-conjugated anti-phosphotyrosine antibody. Lane 1, 2.5 µg of total synaptic plasma membrane proteins before the immunoprecipitations; lanes 2-6, pellets from each immunoprecipitation; lane 7, supernatant equivalent to 2.5 µg of total synaptic plasma membrane proteins after the immunoprecipitations. The top panel shows the immunoblot by the anti-phosphotyrosine (PY) antibody. The same membrane was then stripped and reprobed by the NR2A (middle panel, 2A) and then the NR2B antibodies (bottom panel, 2B), respectively. A Bio-Rad mini-gel system was used in this particular experiment.



In Vitro Tyrosine Phosphorylation by Endogenous Kinases

Because high levels of protein-tyrosine kinases have been found in synaptic plasma membranes (Hirano et al., 1988; Cooke and Perlmutter, 1989; Wagner et al., 1991a), we tested whether they would phosphorylate the NMDA receptor subunits in vitro. Incubation of the synaptic plasma membrane preparations under phosphorylating conditions increased the tyrosine phosphorylation of the NR2A subunit 6-8-fold (Fig. 5, upper panel). In contrast, no increase in tyrosine phosphorylation of the NR1 and NR2B subunits was detected under the same conditions (Fig. 5, upper panel). The amount of NMDA receptors immunoprecipitated was shown to be similar throughout the incubation period (Fig. 5, lower panel).


DISCUSSION

The nicotinic acetylcholine receptor and other synaptic proteins have been shown to be highly tyrosine-phosphorylated at the neuromuscular junction (Qu et al., 1990; Qu and Huganir, 1994; Huganir et al., 1984; Hopfield et al., 1988; Wagner et al., 1991b, 1993). In this study, we demonstrate that excitatory synapses between hippocampal neurons in culture contain high levels of phosphotyrosine that co-localize with the NMDA receptor in dendritic spines. Using subunit-specific antibodies we have analyzed the tyrosine phosphorylation of the glutamate receptors present in the cerebral cortex. We have shown that although the NR1 subunit of the NMDA receptor does not contain phosphotyrosine, both the NR2A and NR2B subunits are tyrosine-phosphorylated. The tyrosine phosphorylation of only NR2 subunits but not NR1 is consistent with the notion that NR2 subunits are for modulation of NMDA receptor functions, whereas the NR1 subunit is critical for its basic functioning. Our results on tyrosine phosphorylation of NR2B are also consistent with the recent results of Moon et al.(1994), who, by protein sequencing, found that NR2B is the major tyrosine-phosphorylated 180-kDa glycoprotein in postsynaptic density (Gurd, 1985). We have not found any evidence for tyrosine phosphorylation of the AMPA receptor subunits GluR1-4 or the kainate receptor subunits GluR6/7 or KA2.

The low stoichiometry of tyrosine phosphorylation of the NR2 subunits may reflect only the basal phosphorylation level and allows for a large increase in signal in response to various physiological and/or pathological conditions. In fact, incubation of synaptic plasma membranes under phosphorylating conditions increased tyrosine phosphorylation of the NR2A subunit 6-8-fold by endogenous protein-tyrosine kinase(s). Alternatively, because the low stoichiometry of tyrosine phosphorylation was estimated using synaptic plasma membranes isolated from the whole cerebral cortex, it is possible that the tyrosine-phosphorylated form of the NR2A and NR2B subunits may exist in highly specific regions of the brain, giving a very high local stoichiometry. These could be specific regions of the brain, specific subpopulations of neurons within a certain region, specific synapses within one single neuron, or even regions within a single synapse. It should also be emphasized that the low stoichiometry of NR2B is not mutually exclusive with the findings by Moon et al.(1994) that the major tyrosine-phosphorylated protein in the postsynaptic density is the NR2B subunit.

Recent electrophysiological studies have shown that tyrosine phosphorylation can increase NMDA receptor function (Wang and Salter, 1994). However, it is unclear whether the effect is due to direct tyrosine phosphorylation of the NMDA receptor itself or that some other indirect mechanism is involved. Here, we provide two potential targets, namely the NR2A and NR2B subunits, on which the protein-tyrosine kinases may act to enhance the NMDA current. Tyrosine phosphorylation of the nicotinic acetylcholine receptor has been shown to affect its channel properties (Hopfield et al., 1988). Tyrosine phosphorylation of the nicotinic acetylcholine receptor may also play a role in the regulation of receptor clustering (Qu et al., 1990; Wallace, 1991, 1994), suggesting that tyrosine phosphorylation of the NR2 subunits may also serve a similar function. Finally, a variety of recent studies have shown that tyrosine phosphorylation of proteins regulates protein-protein interactions between tyrosine-phosphorylated proteins and SH2-containing proteins. It is possible that tyrosine phosphorylation of the NR2A and NR2B subunits regulates targeting of specific SH2-containing proteins to the synapse.

As mentioned earlier, our data not only demonstrate that NR2A and NR2B are basally tyrosine-phosphorylated but also show that endogenous protein-tyrosine kinases in the synaptic region are able to rapidly phosphorylate NR2A and potentially affect NMDA receptor function. Potential candidates for such protein-tyrosine kinase activity include EGF receptor (Faundez et al., 1992), IGF-1 receptor (Hanissian and Sahyoun, 1992), pp60 (Sugrue et al., 1990), and pp59 (Grant et al., 1992; Swope and Huganir, 1993), which have been shown to be present in synaptic membranes. In contrast, tyrosine phosphorylation of NR2B was not increased under the same conditions. It is possible that the protein-tyrosine kinases specific for NR2B might have been removed during preparation of synaptic plasma membranes or that the phosphorylating conditions were not optimal for the NR2B protein-tyrosine kinase.

The data presented in this paper characterize the tyrosine phosphorylation of the NMDA receptor and suggest that tyrosine phosphorylation of the NR2 subunits of the NMDA receptor may play an important role in regulating the function of the NMDA receptor. Long term potentiation, a phenomenon thought to be important for establishing learning and memory in animals (Bliss and Collingridge, 1993), is inhibited by protein-tyrosine kinase inhibitors (O'Dell et al., 1991). In addition, Grant et al.(1992) also found that mice lacking the protein-tyrosine kinase, fyn, displayed diminished long term potentiation as well as spatial learning and memory. Because of the critical role of the NMDA receptor in the induction of long term potentiation and in spatial learning and memory, it is possible that tyrosine phosphorylation of the NMDA receptor is an important modulator of synaptic plasticity.


FOOTNOTES

*
This research was supported by the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Howard Hughes Medical Inst., The Johns Hopkins University School of Medicine, Dept. of Neuroscience, 725 North Wolfe St., 900 PCTB, Baltimore, MD 21205-2185. Tel.: 410-955-4050; Fax: 410-955-0877.

(^1)
The abbreviations used are: AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate; NMDA, N-methyl-D-aspartate; PAGE, polyacrylamide gel electrophoresis.

(^2)
Roche and Huganir, unpublished data.


ACKNOWLEDGEMENTS

-We acknowledge Drs. Stephen Heinemann and Nils Brose for generosity in providing us with the NR1-TM3/4 antibody and the cDNA clone for the NR2B subunit. We also thank Dr. Shigetada Nakanishi for providing the cDNA clone for the NR2A subunit and Dr. John Merlie for providing the QT-6 fibroblasts. We also thank Katherine Roche for help in culturing hippocampal neurons, Dr. Sheridan Swope, Eric Fung, and Whittemore Tingley for their comments on the manuscript, and Cindy Finch for preparing the manuscript.


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