(Received for publication, May 4, 1995; and in revised form, June 9, 1995)
From the
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:
-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.
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, ()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.
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, 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
-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.
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.
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 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.
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 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.
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.
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.