The postsynaptic density protein PSD-95 differentially regulates insulin- and Src-mediated current modulation of mouse NMDA receptors expressed in Xenopus oocytes

The NMDA subtype of glutamate receptor is physically associated with the postsynaptic density protein PSD-95 at glutamatergic synapses. The channel activity of NMDA receptors is regulated by different signaling molecules, including protein tyrosine kinases. Because previous results have suggested a role for protein kinase C (PKC) in insulin potentiation of NMDA currents in oocytes, the effects of coexpression of PSD-95 on insulin and PKC potentiation of NMDA currents from these receptors were compared. Another primary objective was to determine if PSD-95 could enable Src to potentiate currents from NR2A/NR1 and NR2B/NR1 receptors expressed in XENOPUS: oocytes. The results show opposite effects of PSD-95 coexpression on Src and insulin modulation of NR2A/NR1 receptor currents. Src potentiation of mouse NR2A/NR1 currents required PSD-95 coexpression. In contrast, PSD-95 coexpression eliminated insulin-mediated potentiation of NR2A/NR1 receptor currents. PSD-95 coexpression also eliminated PKC potentiation of NR2A/NR1 receptor currents. PSD-95 may therefore play a key role in controlling kinase modulation of NR2A/NR1 receptor currents at glutamatergic synapses.     

PSD-95 eliminates Src-induced potentiation of NR1/NR2A-subtype NMDA receptor channels and reduces high-affinity zinc inhibition

The channel activity of NMDA receptors is regulated by phosphorylation by protein kinases and by interaction with other proteins. Recombinant NR1/NR2A subtype NMDA receptor channels are potentiated by the protein tyrosine kinase Src, an effect which is mediated by a reduction in the high-affinity, voltage-independent Zn(2+) inhibition. However, it has been reported that Src-induced potentiation of NMDA receptor currents in hippocampus neurons is not mediated by a reduction in Zn(2+) inhibition. The post-synaptic density protein PSD-95 interacts with the C-terminus of NR2 subunits of the NMDA receptor. Here we demonstrate that PSD-95 eliminates the Src-induced potentiation of NR1/NR2A channels expressed in oocytes and reduces the sensitivity of the channels to Zn(2+). Our results reveal that the absence of Src-induced potentiation of PSD-95-coupled NR1/NR2A channels is not to due to the reduced sensitivity of these channels to Zn(2+). These results indicate that PSD-95 functionally modulates NR1/NR2A channels and explain why Src-induced potentiation of NMDA receptor currents in hippocampus neurons is not mediated by a reduction in Zn(2+) inhibition.     

Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor

The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non-receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini-slices with 500 nM phorbol 12-myristate 13-acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti-NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 +/- 11% above control) and NR2B (41 +/- 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B.     

The effects of NR2 subunit-dependent NMDA receptor kinetics on synaptic transmission and CaMKII activation

N-Methyl-D-aspartic acid (NMDA) receptors are widely expressed in the brain and are critical for many forms of synaptic plasticity. Subtypes of the NMDA receptor NR2 subunit are differentially expressed during development; in the forebrain, the NR2B receptor is dominant early in development, and later both NR2A and NR2B are expressed. In heterologous expression systems, NR2A-containing receptors open more reliably and show much faster opening and closing kinetics than do NR2B-containing receptors. However, conflicting data, showing similar open probabilities, exist for receptors expressed in neurons. Similarly, studies of synaptic plasticity have produced divergent results, with some showing that only NR2A-containing receptors can drive long-term potentiation and others showing that either subtype is capable of driving potentiation. In order to address these conflicting results as well as open questions about the number and location of functional receptors in the synapse, we constructed a Monte Carlo model of glutamate release, diffusion, and binding to NMDA receptors and of receptor opening and closing as well as a model of the activation of calcium-calmodulin kinase II, an enzyme critical for induction of synaptic plasticity, by NMDA receptor-mediated calcium influx. Our results suggest that the conflicting data concerning receptor open probabilities can be resolved, with NR2A- and NR2B-containing receptors having very different opening probabilities. They also support the conclusion that receptors containing either subtype can drive long-term potentiation. We also are able to estimate the number of functional receptors at a synapse from experimental data. Finally, in our models, the opening of NR2B-containing receptors is highly dependent on the location of the receptor relative to the site of glutamate release whereas the opening of NR2A-containing receptors is not. These results help to clarify the previous findings and suggest future experiments to address open questions concerning NMDA receptor function.     

Insulin causes a transient tyrosine phosphorylation of NR2A and NR2B NMDA receptor subunits in rat hippocampus

NMDA receptors play a critical role in various aspects of CNS function. Hence, it is important to identify mechanisms that regulate NMDA receptor activity. We have shown previously that insulin rapidly potentiates NMDA receptor activity in both native and recombinant expression systems. Here we report that insulin causes a transient phosphorylation of NR2A and NR2B NMDA receptor subunits on tyrosine residues. Rat hippocampal slices were exposed to 1 microM insulin for 20 and 60 min and then solubilized. NR2A and NR2B subunits were immunoprecipitated and probed for tyrosine phosphorylation. Insulin incubation of hippocampal slices for 20 min elicited an increase in tyrosine phosphorylation to 176 +/- 16% (NR2A) and 203 +/- 15% (NR2B) of control levels. In contrast, 60 min of insulin incubation did not alter NR2 tyrosine phosphorylation levels (NR2A: 85 +/- 13% of control; NR2B: 93 +/- 10% of control). Although the consequence of insulin-stimulated tyrosine phosphorylation is unknown, it is possible that this site(s) is responsible for insulin potentiation of NMDA receptor activity. This possibility is consistent with our earlier finding that insulin potentiates hippocampal NMDA receptor activity after 20 min, but not after 60 min, of insulin exposure.     

Inhibition of protein kinase C reduces ischemia-induced tyrosine phosphorylation of the N-methyl-d-aspartate receptor

The role of protein kinase C (PKC) in tyrosine phosphorylation of the N‐methyl‐d ‐aspartate receptor (NMDAR) following transient cerebral ischemia was investigated. Transient (15 min) cerebral ischemia was produced in adult rats by four‐vessel occlusion and animals allowed to recover for 15 or 45 min. Following ischemia, tyrosine phosphorylation of NR2A and NR2B and activated Src‐family kinases (SFKs) and Pyk2 were increased in post‐synaptic densities (PSDs). Phosphorylation of NR2B on Y1472 by PSDs isolated from post‐ischemic forebrains was inhibited by the SFK specific inhibitor PP2, and by the PKC inhibitors GF109203X (GF), Gö6976 and calphostin C. Intravenous injection of GF immediately following the ischemic challenge resulted in decreased phosphorylation of NR1 on PKC phosphorylation sites and reduced ischemia‐induced increases in tyrosine phosphorylation of NR2A and NR2B without affecting the increase in total tyrosine phosphorylation of hippocampal proteins. Ischemia‐induced increases in activated Pyk2 and SFKs in PSDs, but not the translocation of PKC, Pyk2 or Src to the PSD, were also inhibited by GF. The inactive homologue of GF, bisindolylmaleimide V, had no effect on these parameters. The results are consistent with a role for PKC in the ischemia‐induced increase in tyrosine phosphorylation of the NMDAR, via a pathway involving Pyk2 and Src‐family kinases.     

Src-protein tyrosine kinases are required for cocaine-induced increase in the expression and function of the NMDA receptor in the ventral tegmental area

Cocaine-induced long-term potentiation of glutamatergic synapses in the ventral tegmental area (VTA) has been proposed as a key process that contributes to the development of addictive behaviors. In particular, the activation of ionotrophic glutamate NMDA receptor (NMDAR) in the VTA is critical for the initiation of cocaine sensitization. Here we show that application of cocaine both in slices and in vivo induced an increase in tyrosine phosphorylation of the NR2A, but not the NR2B subunit of the NMDAR in juvenile rats. Cocaine induced an increase in the activity of both Fyn and Src kinases, and the Src-protein tyrosine kinase (Src-PTKs) inhibitor, 4-amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo[3,4-d]pyrimidine (PP2), abolished both cocaine-induced increase in tyrosine phosphorylation of the NR2A subunit and the increase in the expression of NR1, NR2A, and NR2B in the VTA. Moreover, cocaine-induced enhancement in NMDAR-mediated excitatory post-synaptic currents was completely abolished by PP2. Taken together, these results suggest that acute cocaine induced an increase in the expression of NMDAR subunits and enhanced tyrosine phosphorylation of NR2A-containing NMDAR through members of the Src-PTKs. This in turn, increased NMDAR-mediated currents in VTA dopamine neurons. These results provide a potential cellular mechanism by which cocaine triggers NMDAR-dependent synaptic plasticity of VTA neurons that may underlie the development of behavioral sensitization.     

Roles of NMDA NR2B subtype receptor in prefrontal long-term potentiation and contextual fear memory

Cortical plasticity is thought to be important for the establishment, consolidation, and retrieval of permanent memory. Hippocampal long-term potentiation (LTP), a cellular mechanism of learning and memory, requires the activation of glutamate N-methyl-D-aspartate (NMDA) receptors. In particular, it has been suggested that NR2A-containing NMDA receptors are involved in LTP induction, whereas NR2B-containing receptors are involved in LTD induction in the hippocampus. However, LTP in the prefrontal cortex is less well characterized than in the hippocampus. Here we report that the activation of the NR2B and NR2A subunits of the NMDA receptor is critical for the induction of cingulate LTP, regardless of the induction protocol. Furthermore, pharmacological or genetic blockade of the NR2B subunit in the cingulate cortex impaired the formation of early contextual fear memory. Our results demonstrate that the NR2B subunit of the NMDA receptor in the prefrontal cortex is critically involved in both LTP and contextual memory.     

Reduced NMDA receptor tyrosine phosphorylation in PTPalpha-deficient mouse synaptosomes is accompanied by inhibition of four src family kinases and Pyk2: an upstream role for PTPalpha in NMDA receptor regulation

Mice lacking protein tyrosine phosphatase alpha (PTPalpha) exhibited defects in NMDA receptor (NMDAR)-associated processes such as learning and memory, hippocampal neuron migration, and CA1 hippocampal long-term potentiation (LTP). In vivo molecular effectors linking PTPalpha and the NMDAR have not been reported. Thus the involvement of PTPalpha as an upstream regulator of NMDAR tyrosine phosphorylation was investigated in synaptosomes of wild-type and PTPalpha-null mice. Tyrosine phosphorylation of the NMDAR NR2A and NR2B subunits was reduced upon PTPalpha ablation, indicating a positive effect of this phosphatase on NMDAR phosphorylation via intermediate molecules. The NMDAR is a substrate of src family tyrosine kinases, and reduced activity of src, fyn, yes and lck, but not lyn, was apparent in the absence of PTPalpha. In addition, autophosphorylation of proline-rich tyrosine kinase 2 (Pyk2), a tyrosine kinase linked to NMDAR signaling, was also reduced in PTPalpha-deficient synaptosomes. Altered protein tyrosine phosphorylation was not accompanied by altered expression of the NMDAR or the above tyrosine kinases at any stage of PTPalpha-null mouse development examined. In a human embryonic kidney (HEK) 293 cell expression system, PTPalpha enhanced fyn-mediated NR2A and NR2B tyrosine phosphorylation by several-fold. Together, these findings provide evidence that aberrant NMDAR-associated functions in PTPalpha-null mice are due to impaired NMDAR tyrosine phosphorylation resulting from the reduced activity of probably more than one of the src family kinases src, fyn, yes and lck. Defective NMDAR activity in these mice may also be linked to the loss of PTPalpha as an upstream regulator of Pyk2.     

Fyn tyrosine kinase reduces the ethanol inhibition of recombinant NR1/NR2A but not NR1/NR2B NMDA receptors expressed in HEK 293 cells

NMDA receptors are potentiated by phosphorylation in a subunit- and kinase-specific manner. Both native and recombinant NMDA receptors are inhibited by behaviorally relevant concentrations of ethanol. Whether the phosphorylation state of individual subunits modulates the ethanol sensitivity of these receptors is not known. In this study, the effects of Fyn tyrosine kinase on the ethanol sensitivity of specific recombinant NMDA receptors expressed in HEK 293 cells were investigated. Whole-cell mode patch clamp and ratiometric calcium imaging demonstrated that the degree of ethanol inhibition of NR1/NR2B receptors was unaffected by Fyn tyrosine kinase. In contrast, the inhibition of NR1/NR2A receptors by ethanol (100 mM) was significantly reduced under conditions of enhanced Fyn-mediated tyrosine phosphorylation of the NR2A subunit. This effect was not observed at lower concentrations of ethanol (< or = 50 mM). These results suggest that tyrosine phosphorylation of specific NMDA receptors by Fyn tyrosine kinase may regulate the sensitivity of these receptors to the sedative/hypnotic concentrations of ethanol.     

Opposing Contributions of NR1 and NR2 to Protein Kinase C Modulation of NMDA Receptors

Abstract: N -Methyl- d -aspartate (NMDA) receptors mediate increases in intracellular calcium that can be modulated by protein kinase C (PKC). As PKC modulation of NMDA receptors in neurons is complex, we studied the effects of PKC activation on recombinant NMDA receptor-mediated calcium rises in a nonneuronal mammalian cell line, human embryonic kidney 293 (HEK-293). Phorbol 12-myristate 13-acetate (PMA) pretreatment of HEK-293 cells enhanced or suppressed NMDA receptor-mediated calcium rises based on the NMDA receptor subunit composition. NR2A or NR2B, in combination with NR1₀₁₁, conveyed enhancement whereas NR2C and NR2D conveyed suppression. The PKC inhibitor bisindolylmaleimide blocked each of these effects. The region on NR2A that conveyed enhancement localized to a discrete segment of the C terminus distal to the portion of NR2C that is homologous to NR2A. Calcium-45 accumulation, but not intracellular calcium store depletion, matched PMA effects on NMDA receptor-mediated calcium changes, suggesting that these effects were not due to effects on intracellular calcium stores. The suppression of intracellular calcium transients seen with NR2C was eliminated when combined with NR1 splice variants lacking C-terminal cassette 1. Thus, the intracellular calcium effects of PMA were distinguishable based on both the NR1 splice variant and the NR2 subunit type that were expressed. Such differential effects resemble the diversity of PKC effects on NMDA receptors in neurons.     

NR2A and NR2B subunits differentially mediate MAP kinase signaling and mitochondrial morphology following excitotoxic insult

NMDA receptors are essential for neurotransmission and key mediators of synaptic signaling, but they can also trigger deleterious degenerative processes that lead to cell death. Growing evidence suggests that selective blockade of the heterogeneous subunits that comprise the NMDA receptor may enable better control of pharmacotherapies for treating neurological diseases and injuries. We investigated the relationship between NMDAR activation, MAPK signaling, and mitochondrial shape following an excitotoxic insult. NR2A- and NR2B-containing NMDARs differentially mediated acute changes in cytosolic calcium, alterations in mitochondrial morphology, and phosphorylation of the MAPKs ERK and JNK. Activation of NR2A-containing NMDARs was associated with JNK phosphorylation that was neuroprotective in neuronal cultures subjected to excitotoxicity. In contrast, activation of NR2B-containing NMDARs triggered calcium accumulation in mitochondria that was strongly associated with mitochondrial swelling and neuronal cell death. Indeed, while blockade of NR2B-containing receptors was neuroprotective, this protection was lost when NR2A-initiated JNK phosphorylation was inhibited. Given the modest selectivity of the NR2A inhibitor, NVP-AAM077, the results highlight the significance of the relative, rather than absolute, activation of these two NMDA subtypes in modulating cell death pathways. Therefore, the balance between concurrent activation of NR2B-containing and NR2A-containing NMDARs dictates neuronal fate following excitotoxicity.     

NMDA receptor subunit-dependent modulation by conantokin-G and Ala7-conantokin-G

The modulation of recombinant NMDA receptors by conantokin-G (con-G) and Ala7-conantokin-G (Ala7-Con-G) was investigated in Xenopus oocytes injected with capped RNA coding for NR1 splice variants and NR2 subunits using the two-electrode voltage clamp technique. Glutamate exhibited a marginally higher apparent affinity for NR2A-containing receptors than NR2B-containing receptors, regardless of the NR1 subunit present. Conantokins were bath applied to give cumulative concentration responses in the presence of 3 and 30 mum glutamate. Both contantokins exhibited biphasic concentration-response relationships at NR2A-containing NMDA receptors, producing potentiation at low conantokin concentrations and inhibition at high concentrations. These effects were stronger with glutamate concentrations near its EC50, and less marked at saturating concentrations. In contrast, the conantokin concentration-response relation was monophasic and inhibitory at NR2B-containing receptors. We conclude that the combinations of subunits that comprise the NMDA receptor complex influence conantokin and glutamate affinities and the nature of the responses to conantokins.     

Identification of mouse NMDA receptor subunit NR2A C-terminal tyrosine sites phosphorylated by coexpression with v-Src

The protein tyrosine kinase Src is known to regulate NMDA receptors in native neurons. While NR2A, NR2B and NR2D are known to be phosphorylated on tyrosine residues, the exact sites have remained unidentified. Immunoprecipitation of NMDA receptor subunits followed by western blotting was used to analyze the state of tyrosine phosphorylation of recombinant NMDA receptor subunits expressed in HEK293 cells. Using antiphosphotyrosine antibody PY20, we find that on expression in HEK cells, v-Src and Fyn cause detectable tyrosine phosphorylation only of NR2A. Because a stronger signal was produced by the constitutively active v-Src, the general region of v-Src phosphorylation was delimited by expression of a series of truncation mutants of NR2A. Site-directed mutagenesis on candidate sites within the likely region allowed identification of three sites, Y1292, Y1325, and Y1387 that account for a significant fraction of the total PY20 signal. Two of these sites, Y1292 and Y1387, were suggested to control current modulation by Src in previous studies of HEK cells expressing NR1/NR2A. One of these sites, Y1325, has not yet been evaluated for effects on receptor current. A unique tyrosine site, Y1267, was shown not to be a site of detectable phosphorylation, in accordance with its Src-independent regulation of receptor currents.     

Differential modulation of NR1-NR2A and NR1-NR2B subtypes of NMDA receptor by PDZ domain-containing proteins

The PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins MALS and PSD-95 localize to post-synaptic densities and bind the COOH-termini of NR2 subunits of the NMDA receptor. The effects of MALS-2 and PSD-95 on the channel activity of NMDA receptors were compared using the Xenopus oocyte expression system. Both MALS-2 and PSD-95 increased the current response of the NR1-NR2B receptor to l-glutamate. In contrast, the current response of the NR1-NR2A receptor was increased by PSD-95 but not by MALS-2. MALS-2 had no effect either on the potentiation of NR1-NR2A or NR1-NR2B channel activity by protein kinase C, or on Src-mediated potentiation of NR1-NR2A activity, whereas PSD-95 almost completely inhibited the effects of these protein kinases. Construction of chimeras of MALS-2 and PSD-95 revealed that the first two PDZ domains and two NH(2)-terminal cysteine residues are essential for the inhibitory effects of PSD-95 on protein kinase C-mediated potentiation of NR1-NR2A and NR1-NR2B channel activity, respectively. The second of the three PDZ domains of PSD-95 was required for its inhibition of Src-mediated potentiation of NR1-NR2A activity. These results indicate that the NR1-NR2A and NR1-NR2B receptors are modulated differentially by MALS-2 and PSD-95, and that similar regulatory effects of PSD-95 on these receptors are achieved by distinct mechanisms.     

Prostacyclin-induced rundown of N-methyl-D-aspartate receptor currents in HEK293 cells is protein kinase A-dependent and NR2 subunit-selective

Previous studies in neurons have demonstrated a rapid decrease in NMDA receptor currents following tyrosine kinase inhibition or exposure to platelet-derived growth factor (PDGF). Inhibitors of protein kinase A (PKA) block the PDGF-induced rundown suggesting a multistep pathway that leads to decreased amplitudes of NMDA-activated currents. In this study, HEK293 cells expressing different NMDA receptor subunits were used to study the effects of prostacyclin receptor-mediated PKA activation on the magnitude of glutamate-activated currents. The prostacyclin agonist iloprost induced a rapid and time-dependent depression of otherwise stable glutamate-activated currents in cells expressing NR1-2a/2A or NR1-2a/2D receptors but not NR1-2a/2B or NR1-2a/2C receptors. This rundown was prevented by treatment of cells with the PKA inhibitor H89. The iloprost effect persisted in cells coexpressing NR1-2a/2A receptors and either wild-type or mutant Src kinase (SrcS17A). Co-expression of PSD-95 with NR1-2a/2A receptors reduced but did not eliminate the extent of rundown. Iloprost also produced current rundown in cells expressing NR1-2a and a C-terminal truncated NR2A subunit (NR2A1050stop) but not in those transfected with an NR2A tyrosine mutant (Y842F). The iloprost-induced rundown of wild-type NR1-2a/2A receptors was prevented by prior exposure of cells to hypertonic sucrose. These results suggest that PKA influences the functional activity of NMDA receptors in an NR2 subunit-selective fashion.     

Dopamine receptors regulate NMDA receptor surface expression in prefrontal cortex neurons

Interactions between dopamine (DA) and glutamate systems in the prefrontal cortex (PFC) are important in addiction and other psychiatric disorders. Here, we examined DA receptor regulation of NMDA receptor surface expression in postnatal rat PFC neuronal cultures. Immunocytochemical analysis demonstrated that surface expression (synaptic and non-synaptic) of NR1 and NR2B on PFC pyramidal neurons was increased by the D1 receptor agonist SKF 81297 (1 microM, 5 min). Activation of protein kinase A (PKA) did not alter NR1 distribution, indicating that PKA does not mediate the effect of D1 receptor stimulation. However, the tyrosine kinase inhibitor genistein (50 microM, 30 min) completely blocked the effect of SKF 81297 on NR1 and NR2B surface expression. Protein cross-linking studies confirmed that SKF 81297 (1 microM, 5 min) increased NR1 and NR2B surface expression, and further showed that NR2A surface expression was not affected. Genistein blocked the effect of SKF 81297 on NR1 and NR2B. Surface-expressed immunoreactivity detected with a phospho-specific antibody to tyrosine 1472 of NR2B also increased after D1 agonist treatment. Our results show that tyrosine phosphorylation plays an important role in the trafficking of NR2B-containing NMDA receptors in PFC neurons and the regulation of their trafficking by DA receptors.     

Long-Term Potentiation in the CA1 Hippocampus Induced by NR2A Subunit-Containing NMDA Glutamate Receptors Is Mediated by Ras-GRF2/Erk Map Kinase Signaling

Background

NMDA-type glutamate receptors (NMDARs) are major contributors to long-term potentiation (LTP), a form of synaptic plasticity implicated in the process of learning and memory. These receptors consist of calcium-permeating NR1 and multiple regulatory NR2 subunits. A majority of studies show that both NR2A and NR2B-containing NMDARs can contribute to LTP, but their unique contributions to this form of synaptic plasticity remain poorly understood.

Methodology/Principal Findings

In this study, we show that NR2A and NR2B-containing receptors promote LTP differently in the CA1 hippocampus of 1-month old mice, with the NR2A receptors functioning through Ras-GRF2 and its downstream effector, Erk Map kinase, and NR2B receptors functioning independently of these signaling molecules.

Conclusions/Significance

This study demonstrates that NR2A-, but not NR2B, containing NMDA receptors induce LTP in pyramidal neurons of the CA1 hippocamus from 1 month old mice through Ras-GRF2 and Erk. This difference add new significance to the observation that the relative levels of these NMDAR subtypes is regulated in neurons, such that NR2A-containing receptors become more prominent late in postnatal development, after sensory experience and synaptic activity.     

Regulation of NR1/NR2C N-methyl-D-aspartate (NMDA) receptors by phosphorylation

NR2C-containing N-methyl-D-aspartate (NMDA) receptors are highly expressed in cerebellar granule cells where they mediate the majority of current in the adult. NMDA receptors composed of NR1/NR2C exhibit a low conductance and reduced sensitivity to Mg(2+), compared with the more commonly studied NR2A- and NR2B-containing receptors. Despite these interesting features, very little is known about the regulation of NR2C function. Here we investigate the role of phosphorylation of NR2C in regulating NMDA receptor trafficking and ion channel properties. We identify a phosphorylation site, serine 1244 (Ser(1244)), near the extreme COOH terminus of NR2C, which is phosphorylated by both cAMP-dependent protein kinase and protein kinase C. This residue is located adjacent to the consensus PDZ ligand, a region that regulates protein-protein interactions and receptor trafficking in NR2A and NR2B. We show that Ser(1244) on NR2C is phosphorylated in vitro, in heterologous cells, and in neurons. Moreover, we demonstrate for the first time that NR2C interacts with the PSD-95 family of PDZ domain-containing proteins but that phosphorylation of Ser(1244) does not influence this PDZ interaction. Furthermore, Ser(1244) phosphorylation does not regulate surface expression of NR1/NR2C receptors. However, we find that this site does regulate the kinetics of the ion channel: a phosphomimetic mutation at Ser(1244) accelerates both the rise and decay of NMDA-evoked currents in excised patches from HEK-293 cells. Therefore, phosphorylation of Ser(1244) does not regulate trafficking but unexpectedly affects ion channel function, suggesting that phosphorylation of Ser(1244) on NR2C may be important in defining the functional properties of NMDA receptor-mediated currents in the cerebellum.     

Protein Tyrosine Kinase-Mediated Potentiation of Currents from Cloned NMDA Receptors

Abstract: Although serine/threonine phosphorylation has been more commonly recognized as a mechanism to modulate the function of ion channels and receptors, tyrosine phosphorylation is under increasing scrutiny. An important subtype of glutamate receptor, the NMDA receptor, is shown to be regulated by insulin via protein tyrosine kinase (PTK). NMDA currents through cloned receptors are potentiated by insulin in a subunit-specific manner. The insulin-mediated potentiation of NMDA current is diminished by inhibitors of PTKs. At least one exogenous cytosolic PTK, pp60^{c- src} , is also able to potentiate NMDA current. Because later application of PTK inhibitors can reverse the seemingly stable insulin-mediated potentiation of NMDA current, it appears that tyrosine residues responsible for potentiation are continually rephosphorylated by some long-term PTK activity that was induced via insulin treatment.