Metabotropic Glutamate Receptor 1-Induced Upregulation of
NMDA Receptor Current: Mediation through the Pyk2/Src-Family
Kinase Pathway in Cortical Neurons
Vale ´rie Heidinger, Pat Manzerra, Xue Qing Wang, Uta Strasser, Shan-Ping Yu, Dennis W. Choi, and
M. Margarita Behrens
Department of Neurology and Center for the Study of the Nervous System Injury, Washington University School of
Medicine, St. Louis, Missouri 63110
The mechanism underlying the upregulation of NMDA receptor
(mGluRs), including mGluR1 and 5, is not known. Here we show
that in cortical neurons, brief selective activation of group I
mGluRs with (S)-3,5-dihydroxy-phenylglycine (DHPG) induced
a Ca2?–calmodulin-dependent activation of Pyk2/CAK? and
the Src-family kinases Src and Fyn that was independent of
protein kinase C (PKC). Activation of Pyk2 and Src/Fyn kinases
led to increased tyrosine phosphorylation of NMDA receptor
subunits 2A and B (NR2A/B) and was blocked by a selective
mGluR1 antagonist, 7-(hydroxyamino)cyclopropa[b]chromen-
1a-carboxylate ethyl ester, but not an mGluR5 antagonist, 2-methyl-
6-(phenylethynyl)pyridine. Functional linkage between mGluR1 acti-
also demonstrated after expression of these elements in human
embryonic kidney 293 cells. Supporting functional consequences,
selective activation of mGluR1 by DHPG induced a potentiation of
NMDA receptor-mediated currents that was blocked by inhibiting
levels of Pyk2 and Src, suggesting that mGluR1 may control the
basal activity of these kinases and thus the tyrosine phosphorylation
levels of NMDA receptors.
Key words: NMDA; metabotropic; Pyk2/CAK?; Src/Fyn; cal-
Cumulative evidence suggests that Src-family activation may play
a role in regulating NMDA receptor function and synaptic plas-
ticity. NMDA receptor subunit 2B (NR2B) is the major tyrosine
phosphorylated protein in the postsynaptic density fraction
(Moon et al., 1994), and its phosphorylation increases during
long-term potentiation (LTP) (Rosenblum et al., 1996; Rostas et
al., 1996). Src is associated with NMDA receptors, and phosphor-
ylation by Src can upregulate NMDA receptor current (X. M. Yu
et al., 1997; Yu and Salter, 1999). Bath application or postsynaptic
injection of Src kinase inhibitors blocks the induction of long-
term potentiation in CA1 hippocampal neurons (O’Dell et al.,
1991; Huang and Hsu, 1999). Furthermore, targeted disruption of
the gene coding for Fyn kinase, a member of the Src-family,
impairs the induction of LTP (Grant et al., 1992; Kojima et al.,
1997). Activated forms of both tyrosine kinases, Src-and Fyn,
phosphorylate recombinantly expressed NR2 subunits and up-
regulate NMDA receptor currents (Kohr and Seeburg, 1996;
Zheng et al., 1998). Deletion of the C-terminal domain of NR2A
eliminates the potentiation of NR1/NR2A-receptor currents by
Src (Kohr and Seeburg, 1996) and impairs synaptic plasticity and
contextual memory in mice (Sprengel et al., 1998).
Few studies to date have addressed the signaling mechanisms
controlling Src-family kinase activation during glutamatergic neu-
rotransmission. One possible initial signal is the calcium-mediated
activation of the proline-rich tyrosine kinase 2 (Pyk2/CAK?). In
neurons, this kinase is stimulated by increased intracellular calcium
and also by protein kinase C (PKC) activation (Lev et al., 1995).
Activated Pyk2 binds and activates Src-family kinases (Dikic et al.,
1996), thus linking increases in intracellular calcium and PKC
activity to tyrosine phosphorylation. Both PKC and Src have been
implicated in the potentiation of NMDA-mediated currents by
G-protein-coupled muscarinic or lysophosphatidic acid receptors
in hippocampal neurons (Lu et al., 1999).
Another influence on NMDA receptor function and synaptic
plasticity is the G-protein-coupled metabotropic glutamate recep-
tor (mGluR) system. There are at least eight mGluRs (mGluR
1–8), which can be divided into groups I, II, and III on the basis of
sequence homology, signal transduction mechanisms, and pharma-
cological properties (Pin and Duvoisin, 1995). Group I mGluRs,
specifically mGluR1? and 5, localize to the periphery of the
postsynaptic region (Baude et al., 1993; Lujan et al., 1997) and are
coupled to Gq-proteins, mediating increases in inositol phosphates
and the subsequent release of calcium from intracellular stores.
Activation of group I mGluRs induces pro-excitatory effects, in-
cluding increased glutamate release from cortical neurons (Strasser
et al., 1998), increased neuronal excitability (Anik-sztejn et al.,
1991), and upregulation of NMDA-mediated currents in hip-
pocampal and striatal cultures (Fitzjohn et al., 1996). In CA1
pyramidal neurons, activation of group I mGluRs induces several
excitatory responses through the activation of Ca2?-dependent
Received Nov. 28, 2001; revised April 19, 2002; accepted April 22, 2002.
This work was supported by National Institute of Neurological Disorders and
Stroke Grant NS 30337 (D.W.C.), National Science Foundation Grant IBN-9817151
(S.P.Y.), and Fondation Simone et Cino del Duca (V.H.). We thank Dr. D. Turetsky
for preparation of the ND-10 cell line, and Drs. H. Monyer and H. S. Earp for the
NR2A and PyK2 plasmids. We especially thank Dr. K. Fish and Olympus for the
analysis of the confocal images, and Dr. T. Bartfai for his support in the conclusion
of this study.
Correspondence should be addressed to Dennis W. Choi, Department of Neu-
rology and Center for the Study of the Nervous System Injury, Washington Uni-
versity School of Medicine, St. Louis, MO 63110, or M. M. Behrens, Neurophar-
macology Department, The Scripps Research Institute, 10550 North Torrey Pines
Road SR307, La Jolla, CA 92037. E-mail: email@example.com.
Copyright © 2002 Society for Neuroscience 0270-6474/02/225452-10$15.00/0
The Journal of Neuroscience, July 1, 2002, 22(13):5452–5461
and independent cationic conductances (Crepel et al., 1994; Gueri-
neau et al., 1995) and inhibition of K?currents (Charpak et al.,
1990; Guerineau et al., 1994; Luthi et al., 1996). Furthermore,
targeted disruption of the genes coding for either mGluR1 or
mGluR5 reduces hippocampal LTP and associative learning in
mice (Aiba et al., 1994; Conquet et al., 1994; Lu et al., 1997).
We have shown previously that cultured cortical neurons ex-
press both mGluR1? and mGluR5 and that selective activation of
these mGluRs increased glutamate release and potentiated
NMDA-induced neuronal death (Strasser et al., 1998). In this
study, we set out to test the hypothesis that the ability of group I
mGluRs to upregulate NMDA receptor function might be medi-
ated through Pyk2/CAK? and Src-family kinases.
Parts of this paper have been published previously in abstract
form (Behrens et al., 1999a, 2000).
MATERIALS AND METHODS
Cortical cell cultures
Mixed cultures. Mixed cortical cultures were prepared from Swiss Web-
ster mouse cortices as described previously (Rose et al., 1993). Briefly,
astrocyte cultures were prepared from postnatal (day 1–3) Swiss Webster
mice and plated at a density of 0.6 hemispheres per plate in 24-well
culture plates (Primaria, Falcon) in Eagle’s Minimal Essential Media
(MEM with Earle’s salts, glutamine free; Invitrogen, Gaithersburg, MD)
supplemented with 10% fetal bovine serum, 10% horse serum, 20 mM
glucose, and 2 mM glutamine. After the astrocyte cultures reached
confluency [14–21 d in vitro (DIV)], dissociated cortices obtained from
fetal mice at 14–16 d gestation were plated onto the previously estab-
lished glial monolayer. Cultures were kept in MEM supplemented with
5% fetal bovine serum, 5% horse serum, glucose, and glutamine as
described above. At 5 DIV, non-neuronal cell division was halted by a 2 d
exposure to 10 ?M cytosine arabinoside. Mixed cultures were then fed
every 3 d with MEM containing 10% horse serum, glucose, and glu-
tamine. Cultures were maintained in a 37°C humidified incubator in a 5%
CO2atmosphere and used at 14–16 DIV for analysis of the tyrosine
phosphorylation of NMDA receptors.
Near-pure neuronal cultures. Dissociated cortical cells were obtained
from fetal mice as above but plated in 24-well plates (four to six cortices
per plate) coated with poly-D-lysine and laminin. After 3 DIV, cytosine
arabinoside was added (10 ?M) to halt the growth of non-neuronal cells.
Under these conditions ?1% of total cells were astrocytes. Cells were
used at 9–12 DIV for the study of intracellular signaling cascades.
Dissociated neuronal cultures. Dissociated cortical neurons were pre-
pared as above but plated on poly-D-lysine/laminin-coated coverslips as
described (Goslin et al., 1998). After cell attachment, coverslips were
placed on top of a preformed cortical astrocyte monolayer that contained
N2.1 media with the addition of 5 ?M cytosine arabinoside. Cultures
were fed every 5 d by replacing one-third of the media with fresh N2.1.
Coverslips were used for confocal imaging 14 d after plating (14 DIV).
Human embryonic kidney 293 cells. ND-10 cells were grown in MEM
20 mM glucose, 2 mM glutamine, and 10% fetal calf serum. Transfection
experiments were performed at 30–50% confluency.
Immunocytochemistry and confocal imaging
For immunocytochemistry, coverslips containing neurons were lifted
from the astrocyte monolayer, washed by immersion in PBS, and fixed in
ice-cold 4% paraformaldehyde for 30 min. Coverslips were then incu-
bated for 10 min at room temperature in PBS that contained 0.25%
Triton X-100. Nonspecific sites were blocked by incubation in PBS
containing 10% normal goat serum. For double immunostaining, the
coverslips were incubated in 2% normal goat serum containing a 1:1000
dilution of a mouse monoclonal antibody against mGluR1? (Phar-
Mingen) and a 1:500 dilution of a rabbit polyclonal antibody against
mGluR5 (Chemicon) for 30 min at 37°C. Specific binding was detected
using secondary antibodies conjugated to AlexaFluor dyes (594, red, for
mGluR5; 488, green, for mGluR1?) (Molecular Probes). Images were
collected on a Delta Vision Optical Sectioning Microscope consisting of
an Olympus IX-70 microscope equipped with a mercury arc lamp. A
photometrics CH 350 cooled CCD camera and a high-precision motor-
ized XYZ stage were used to acquire multiple consecutive optical
sections at a 0.2 ?m interval for each of the fluorescent probes. A
UPAPLO 60? objective was used to collect the images.
Human embryonic kidney (HEK) 293 stably expressing NR1 subunits
(ND-10) were prepared by cotransfection with pRK5-NR1 (kind gift
from H. Monyer, University Hospital for Neurology, Heidelberg, Ger-
many) and pRc/CMV (Invitrogen, Carlsbad, CA) using Lipofectace
(Invitrogen). Selection was performed in 500 ?g/ml G418, and expres-
sion screening was performed by immunological detection of NR1 using
specific antibodies (PharMingen, La Jolla, CA). Functional assays were
performed by transient transfections with NR2A and detection of
NMDA-mediated intracellular calcium rise using fura-2 AM videomi-
croscopy or electrophysiology. The plasmids used for transient transfec-
tions were as follows: pRK5-NR2A (kind gift from Dr. H. Monyer),
pcDNA3-mGluR1?, pcDNA3-CADTK (Pyk2, wild type) (kind gift from
Dr. H. S. Earp, University of North Carolina, Chapel Hill, NC),
pUSEamp-Src (wild type), and pUSEamp-SrcDN (dominant negative)
(Upstate Biotechnology, Lake Placid, NY). Transient cotransfections of
ND-10 cells were performed using Lipofectamine (12 ?l; Invitrogen) and
2 ?g of total DNA in a final volume of 1 ml for 2 hr in OPTIMEM
(Invitrogen), as recommended by the manufacturer. After transfection,
cells were returned to growth media with the addition of L(?)-2-amino-
5-phosphonopentanoic acid (L-AP5; 500 ?M) and (2)-?-methyl-4-
carboxyphenylglycine (MCPG; 1 mM) to prevent activation of NMDA
and mGluR1 receptors during the expression period. After allowing
protein expression for 24 hr, cells were washed three times in HEPES
controlled salt solution (HCSS) containing (in mM): NaCl 120, KCl 5.4,
MgCl2 0.8, CaCl2 1.8, glucose 15, HEPES 20, pH 7.4, treated in the
absence or presence of (S)-3,5-dihydroxy-phenylglycine (DHPG) for 5
min, and immediately processed for protein extraction.
Cell treatment and protein extraction
For the study of intracellular signaling cascades, near-pure neuronal
cultures were washed three times in HCSS and incubated for 30–45 min
at 37°C in the absence or presence of the different drugs and then
exposed to DHPG or NMDA. After treatment, cells extracts were
prepared as described (Behrens et al., 1999b) in lysis buffer [1% NP-40,
20 mM Tris-Cl, pH 7.5, 10 mM EGTA, 40 mM ?-glycerophosphate, 2.5
mM MgCl2, 2 mM orthovanadate, 1 mM dithiothreitol, 1 mM phenyl-
methyl-sulfonylfluoride (PMSF), 20 ?g/ml aprotinin, 20 ?g/ml leupeptin]
and used for the detection of activated forms of Src, Fyn, and Pyk2 using
phosphospecific antibodies (Biosource International, Camarillo, CA).
For the study of NMDA receptor tyrosine phosphorylation, cell extracts
were prepared from mixed cortical cultures as described previously (Lin
et al., 1999). Briefly, cells were treated as above, and treatments were
stopped by addition of 50 ?l of SDS buffer (1% SDS, 2 mM sodium
orthovanadate, 10 ?g/ml leupeptin, 10 ?g/ml aprotinin, 1 mM PMSF).
The resulting extracts were heated at 90°C for 5 min followed by addition
of 9 vol of dilution buffer (1% NP40, 1% CHAPS, 20 mM Tris-HCl, pH
7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovana-
date, 10 ?g/ml leupeptin, 10 ?g/ml aprotinin, and 1 mM PMSF). Insol-
uble material was removed by centrifugation at 25,000 ? g for 30 min,
and supernatants were retained for immunoprecipitation with specific
antibodies [anti-NR2A and anti-NR2B from either Chemicon (Teme-
cula, CA) or Santa Cruz Biotechnology (Santa Cruz, CA)].
Protein concentration was determined by the bicinchoninic acid method
(Pierce, Rockford, IL) using bovine serum albumin as a standard.
Each protein sample (800–1000 ?g) was incubated overnight at 4°C with
anti-NR2A or anti-NR2B antibodies (3 ?l or 2 ?g, depending on the
antibody source). For immunoprecipitation of Fyn and Src, 50 ?g of total
cell lysate obtained as described above was incubated with 1 ?g of
monoclonal antibodies (anti-Fyn: BD Transduction Laboratories, La
Jolla, CA; anti-Src: Upstate Biotechnology). Immunocomplexes were
recovered with the aid of either protein G-plus or protein A-agarose.
Agarose beads were pelleted by centrifugation and washed three times in
1% NP-40/2 mM orthovanadate in PBS and once in PBS. The immuno-
complexes were resuspended in Laemmli’s buffer and heated at 90°C for
Heidinger et al. • Enhancement of NMDA Receptor Function by mGluR1 J. Neurosci., July 1, 2002, 22(13):5452–5461 5453
Western blot analysis
For the study of PyK2 and Src-family, 20 ?g of protein samples were
fractionated on 8% SDS-PAGE and transferred to nitrocellulose mem-
branes (Micron Separations, Westboro, MA) using a semidry electro-
transfer system (Novablot, Amersham Biosciences, Piscataway, NJ).
Membranes were blocked with 5% milk in TBS-T buffer (20 mM Tris, pH
7.5, 150 mM NaCl, 0.05% Tween 20) and were then incubated with
phosphospecific antibodies directed to the phosphorylated forms of PyK2
and Src-family (anti-PyK2
For detection of phosphotyrosine, proteins were immunoprecipitated using
specific antibodies, separated in 6% SDS gels, and transferred as above.
Phosphotyrosine was detected using biotinylated anti-phosphotyrosine an-
tibodies (4G10; Upstate Biotechnology). Protein bands were visualized by
chemiluminescence using SuperSignal (Pierce). For immunoprecipitation
experiments, blots were subsequently reprobed with specific antibodies
directed to Pyk2, Src, Fyn, NR2A, or NR2B (Santa Cruz; 1:1000 for Pyk2,
Fyn, and Src, and 1:400 for NR2A or NR2B).
y402, anti-Srcy418; Biosource International).
The intensity of immunoreactive bands obtained in autoradiographic
films was measured with an imaging densitometer (Bio-Rad, Hercules,
CA). Phosphorylated levels per protein unit ratios were obtained by
dividing the phosphoimmunoreactive densitometry values by those ob-
tained for the respective protein redetection blots.
Electrophysiological recordings of NMDA receptor currents
The glass-bottom 35 mm culture dish containing cortical cultures was
placed on the stage of an inverted microscope (Nikon, Tokyo, Japan),
and membrane currents were recorded by whole-cell recording using an
EPC-9 amplifier (List-Electronic, Germany). Near-spherical cells were
chosen for the recording. Recording electrodes of 5–8 M? (fire-polished;
inner diameter ? ?1–1.5 ?m) were pulled from Corning Kovar Sealing
#7052 glass pipettes (PG52151–4, World Precision Instruments) by a
Flaming-Brown micropipette puller (P-80/PC, Sutter Instrument Co.).
The offset potential of the recording pipette was routinely corrected to 0
mV after the tip was immersed in the bath medium. This potential was
also checked at the end of experiments and corrected if necessary
(usually 0–2 mV). Recordings with potential draft of ?3 mV were
discarded. A gigaseal of 10–50 G? was formed before the whole-cell or
perforated-patch recording mode was established. For perforated
patches, gramicidin D (Sigma, St. Louis, MO) was dissolved in DMSO
(10 mg/ml) and freshly diluted to a final concentration of 50 ?g/ml in the
pipette solution. After gigaohm seal formation, whole-cell configuration
was established by application of additional suction; for perforated patch,
brief voltage steps of ?10 mV were applied to monitor the changes in
input resistance and capacitance for 15–20 min before the formation of
the perforated patch (Kyrozis and Reichling, 1995; Akaike, 1996). Series
resistance compensation was routinely applied during recordings.
NMDA current was triggered at a holding potential of ?70 mV by 100
?M NMDA plus 0.1 ?M glycine delivered by the DAD-12 drug perfusion
system (Adams and List). Current signals were digitally sampled at 100
?sec (10 kHz) and filtered by a 3 kHz, three-pole Bessel filter. Current
and voltage traces were displayed and stored on a computer using the
data acquisition/analysis package, PULSE (HEKA Electronik).
The external solution contained (in mM): NaCl 120, KCl 3, CaCl22,
HEPES 10, glucose 10, and TTX 0.5 ?M. The electrode internal solution
contained (in mM): KCl 120, Na2-ATP 2, BAPTA 0.5, and HEPES 10.
Recordings were performed at room temperature (21–22°C) and at pH
7.3, under continuous bath perfusion at ?0.2 ml/min.
Unless stated otherwise, all reagents were from Sigma. Metabotropic
agonists and antagonists were from Tocris Cookson (Ballwin, MO).
DHPG-mediated activation of group I mGluRs in
cortical neuronal cultures increased the tyrosine
phosphorylation of Pyk2, Src, and Fyn kinases
Consistent with results obtained for Gq-protein-coupled recep-
tors in non-neuronal cells (Luttrell et al., 1999), selective activa-
tion of group I mGluRs by DHPG increased the phosphorylation
of the tyrosine kinases Pyk2, Src, and Fyn. Exposure of near-pure
cortical neuronal cultures to 100 ?M DHPG for 2–10 min in-
creased the phosphorylation of the autocatalytic site of Pyk2 and
the Src-family of kinases, tyrosines 402 and 416 (423 in mouse),
respectively, as detected using phosphospecific antibodies (Fig.
1a,b). An increase in the phosphorylation of the autocatalytic site
of these kinases is expected to increase its activity (Cooper and
MacAuley, 1988; Li et al., 1999). The sequence surrounding the
autocatalytic site is highly conserved in the Src-family members,
Yes and Fyn; thus the antibody is expected to recognize also the
activation of these kinases. Among these three kinases, Src and
Fyn are highly expressed in neurons and have been implicated in
mechanisms of neuronal plasticity (Grant et al., 1992; Lu et al.,
1999). To determine which Src-family member was being acti-
vated, we performed immunoprecipitation experiments using an-
tibodies specific for either Src or Fyn and determined by Western
blot the phosphorylation state of each kinase using the same
phosphospecific antibody as above (pSrcY416). Five minute expo-
sure to 100 ?M DHPG increased the phosphorylation of both Src
and Fyn in cortical neurons (Fig. 1b).
Activation of group I mGluRs induced the tyrosine
phosphorylation of NMDA receptor subunits NR2A/B
The increased phosphorylation of the PyK2, Src, and Fyn kinases
prompted us to test the hypothesis that DHPG activation of
Fyn kinases in cortical neurons. a, Time course. After 12 DIV, near-pure
neuronal cultures were exposed to 100 ?M DHPG for 2–10 min. The
phosphotyrosine content of Pyk2 and the Src-family kinases was deter-
mined by Western blot using phosphospecific antibodies against the active
forms of the kinases (p-Pyk2: pPyk2(pY402), top panels; p-Src: pSrc(pY418),
bottom panels). Fifteen micrograms of total cell extract were run per lane
of 8% SDS-PAGE gels, and the total amount of each specific protein was
determined in redetection blots using anti-Pyk2 and anti-Src antibodies
(Pyk2 and Src in each blot). b, Phosphorylation of Src and Fyn. To analyze
whether both Src and Fyn kinases were being activated by DHPG,
near-pure cortical neuronal cultures were exposed to 100 ?M DHPG for
5 min and immediately processed for protein extraction. Fifty micrograms
of total cell lysates were immunoprecipitated using anti-Src or anti-Fyn
specific antibodies as described in Materials and Methods. Immunocom-
plexes were resolved as in a, and the phosphorylation content of each
kinase was determined by Western blots using the phosphospecific anti-
body pSrc(pY418). The relative amount of normalized phosphorylation
(phosphorylation per protein unit, obtained as the ratio of phosphopro-
tein/total immunoprecipitated protein) was expressed as a percentage of
the control (basal, sham wash). Values represent the mean ? SEM
obtained for four independent experiments. * indicates statistical differ-
ence as compared with basal at p ? 0.05 by ANOVA.
DHPG induced tyrosine phosphorylation of Pyk2, Src, and
5454 J. Neurosci., July 1, 2002, 22(13):5452–5461Heidinger et al. • Enhancement of NMDA Receptor Function by mGluR1
group I mGluRs would increase the tyrosine phosphorylation of
NMDA receptors. Cortical neuronal cultures express the NMDA
receptor subunits NR1 and NR2A/B (Zhong et al., 1994).
DHPG exposure induced a rapid increase in the tyrosine phos-
phorylation content of both NR2A and 2B subunits (Fig. 2a,b).
The increase in tyrosine phosphorylation of NR2 subunits was
blocked by preincubating the cells in the presence of the Src-
kinase family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl
pyrazolo[3,4-d]pyrimidine (PP2) (Hanke et al., 1996; Salazar and
Rozengurt, 1999) (1 ?M), or the phospholipase C (PLC) inhibitor
U73122 (20 ?M) (Fig. 2). Similar results were obtained when
using the tyrosine kinase inhibitor genistein (100 ?M) or the
specific Src-family inhibitor PP1 (data not shown).
DHPG-mediated activation of the Pyk2/Src-family
pathway and phosphorylation of NR2A/B occurs
through activation of mGluR 1, not mGluR 5
Cultured cortical neurons express both mGluR1 and 5 when ana-
lyzed either by Western blots of neuronal membrane fractions
(Strasser et al., 1998) or by immunocytochemistry (Fig. 3). How-
ever, the distribution pattern for each subunit in dissociated cul-
tures is different: mGluR1? shows higher expression levels in the
processes than in the soma, whereas the opposite is true for
mGluR5 (Fig. 3). DHPG stimulates both mGluR1 and mGluR5
(EC506 and 2 ?M, respectively) (Schoepp et al., 1999). To identify
which mGluR subtype was responsible for the DHPG-mediated
activation of the Pyk2/Src pathway and tyrosine phosphorylation of
receptor subunits NR2A/B through activation of the Src-kinase family
and PLC in cortical neurons. To analyze the phosphorylation of NR2
subunits, cultures containing neurons and glia were pretreated for 20 min
in the absence or presence of the Src-family inhibitor PP2 (1 ?M) or the
PLC inhibitor U73122 (U73, 20 ?M), and then exposed to 100 ?M DHPG
for 5 min in the presence or absence of the same inhibitors. After protein
extraction in 1% SDS, NR2A (a) or NR2B (b) subunits were immuno-
precipitated with specific antibodies (Chemicon) followed by redetection
with anti-phosphotyrosine antibody (Wb ?-PY: 4G10, Upstate Biotech-
nology). The immunoreactive bands were quantified by scanning densi-
tometry, and values were calculated as phosphorylation per protein unit
(as described in Fig. 1b using anti-NR2A or -NR2B antibodies from Santa
Cruz Biotechnology). Values represent the mean ? SEM of four to six
independent experiments. C, Basal, sham wash. * indicates statistical
significance as compared with basal, and ** indicates statistical signifi-
cance as compared with DHPG alone at p ? 0.05 by ANOVA.
DHPG stimulated the tyrosine phosphorylation of NMDA
Dissociated cortical neurons were grown on glass coverslips over a bed of
astrocytes. At 14 DIV, the coverslips were lifted from the bed of glia,
washed in PBS, and immediately fixed in 4% paraformaldehyde. Detec-
tion of mGluR1 and mGluR5 was performed by double immunostaining
with specific antibodies [mGluR1: 1:1000 dilution of mouse monoclonal
antibody (BD PharMingen); mGluR5: 1:500 dilution of rabbit polyclonal
antibody (Chemicon)] and AlexaFluor-conjugated secondary antibodies
(anti-mouse AlexaFluor488 and anti-rabbit AlexaFluor594).
Cultured cortical neurons express both mGluR1 and mGluR5.
Heidinger et al. • Enhancement of NMDA Receptor Function by mGluR1 J. Neurosci., July 1, 2002, 22(13):5452–5461 5455
NR2A/B, we took advantage of subtype-selective antagonists for
xylateethylester (CPCCOEt; 200 ?M) (IC5036 ?M), and for
mGluR5, 2-methyl-6-(phenylethynyl)-pyridine (MPEP; 1–2 ?M)
(IC5034 nM) (Annoura et al., 1996; Gasparini et al., 1999; Litschig
et al., 1999). Activation of both Pyk2 and Src/Fyn by DHPG was
blocked by a 20 min preincubation with CPCCOEt, but neither
MPEP nor the NMDA antagonist MK-801 had any effect (Fig.
4a,b, top left panels and bar graphs). CPCCOEt also reduced the
basal level of phosphorylation of Pyk2 and Src/Fyn (Fig. 4a,b,
bottom left panels and bar graphs). Furthermore, DHPG enhance-
ment of NR2A/B phosphorylation was also blocked by a 20 min
preincubation with CPCCOEt but not MPEP (Fig. 5a,b), whereas
no effects were observed with the NMDA receptor antagonist
MK801 (10 ?M) or the PKC inhibitor GF 109203X (5 ?M) (data
not shown). As a control for MPEP effectiveness, concentrations as
low as 0.1 ?M MPEP reduced the ability of the selective mGluR5
agonist 2-chloro-5-hydroxyphenylglycine (Doherty et al., 1997) to
raise intracellular calcium in cortical neurons, as determined by
fura-2 AM videomicroscopy (data not shown).
Involvement of mGluR1 was confirmed by the use of other selec-
tive mGluR1 antagonists, (R,S)2-methyl-4-carboxyphenylglycine
(200 ?M), and N-phenyl-7-(hydroxyamino)cyclopropa[b]chromen-
1a-carboxamide (100 ?M) (Annoura et al., 1996) (data not shown).
Lack of mGluR5 contribution was confirmed by the use of another
selective mGluR5 antagonist, 2-methyl-6-(2-phenylethynyl)pyridine
(5 ?M) (Varney et al., 1999) (data not shown).
Activation of mGluR1 increased the tyrosine
phosphorylation of NR2A in HEK293 cells
We next set out to determine whether mGluR1 activation would
increase tyrosine phosphorylation of NR2A when reconstituted
in a cell line system. HEK293 cells, while expressing Src, express
low levels of Pyk2 (Della Rocca et al., 1997) and have low
constitutive levels of tyrosine phosphorylation (Holmes et al.,
1997), making them a favorable system in which to analyze
signaling mechanisms leading to tyrosine phosphorylation. This
cortical neurons occurs through activation of mGluR1. Near-pure cortical
neuronal cultures were pretreated for 20 min in the absence (C) or
presence of the mGluR1 antagonist CPCCOEt (CPC, 200 ?M), the
mGluR5 antagonist MPEP (MP, 1 ?M), or the NMDA receptor antago-
nist MK801 (MK, 10 ?M) and then exposed to vehicle (basal) or 100 ?M
DHPG for 5 min (in the absence or presence of the inhibitors). After
treatment, cells were immediately processed for protein extraction. The
phosphotyrosine content of Pyk2 (a) and the Src-family kinases (b) was
determined by Western blot using phosphospecific antibodies against the
active forms of the kinases (p-Pyk2: pPyk2(pY402), top panels; p-Src:
pSrc(pY418), bottom panels). Fifteen micrograms of total cell extract were
run per lane of 8% SDS-PAGE gels, and the total amount of each specific
protein was determined in redetection blots using anti-Pyk2 and anti-Src
antibodies (Pyk2 and Src in each blot). Bar graphs indicate cumulative
results obtained from four to six independent experiments. * indicates
statistical significance as compared with basal, and ** indicates statistical
significance as compared with DHPG alone, at p ? 0.05 by ANOVA.
DHPG-mediated activation of the Pyk2/Src/Fyn pathway in
was mediated by activation of mGluR1 in cortical neurons. Cultures were
pretreated for 20 min in the absence or presence of the mGluR1 antag-
onist CPCCOEt (CPC, 200 ?M) or the mGluR5 antagonist MPEP (1 ?M)
and then exposed to 100 ?M DHPG for 5 min (in the absence or presence
of the inhibitors). After protein extraction in 1% SDS, NR2A (a) or
NR2B (b) subunits were immunoprecipitated with specific antibodies
followed by redetection with anti-phosphotyrosine antibody (Wb ?-PY).
The immunoreactive bands were quantified by scanning densitometry
(bottom panels) and expressed as in Figure 2. Values represent mean ?
SEM obtained for three to six independent experiments. * indicates
statistical significance as compared with basal, and ** indicates statistical
significance as compared with DHPG alone at p ? 0.05 by ANOVA.
The tyrosine phosphorylation of NR2A/B subunits by DHPG
5456 J. Neurosci., July 1, 2002, 22(13):5452–5461Heidinger et al. • Enhancement of NMDA Receptor Function by mGluR1
system was used previously in studies demonstrating an increase
in NMDA receptor function by recombinant Src or Fyn kinases
(Kohr and Seeburg, 1996; Zheng et al., 1998; Tezuka et al., 1999;
Xiong et al., 1999). In these studies, the upregulation of recom-
binant NMDA receptor function was obtained either by intro-
ducing high levels of recombinant kinases in the patch pipette or
by co-transfection with the active forms of the kinases.
We performed transient transfection experiments in HEK293
cells stably expressing the NR1 subunit of the NMDA receptor
(clone ND-10). A small increase in the tyrosine phosphorylation
content of NR2A was observed after ND-10 cells transiently
transfected with NR2A and mGluR1? expression plasmids (1 ?g
each) were exposed to 100 ?M DHPG for 15 min in the presence
of the tyrosine phosphatase inhibitor orthovanadate (Fig. 6a).
To allow detection of NR2A phosphorylation in the absence of
orthovanadate, we increased the levels of Pyk2 and Src by addi-
tional co-transfection with expression plasmids (0.3 ?g, respec-
tively). After transient co-transfection with NR2A/mGluR1?/
Pyk2/Src plasmids, exposure to DHPG induced the activation of
both Pyk2 and Src, as assessed by detection with phosphospecific
antibodies (Fig. 6b, left panels), as well as increased phosphory-
lation of NR2A (Fig. 6c). The DHPG-mediated activation of
Pyk2 and Src was observed only when cells were co-transfected
with expression plasmids for both kinases, remaining at control
levels when ND-10 cells were transfected with only one of the
kinase expression plasmids (Fig. 6b, middle and right panels). To
confirm the involvement of Pyk2 and Src activation in mediating
the phosphorylation of NR2A on stimulation of mGluR1, similar
transfection experiments were performed in which either the
kinase-deficient form of Pyk2 (Pyk2KD, 0.3 ?g) or the dominant-
negative form of Src plasmids (pUSEamp-SrcDN, 0.3 ?g) was
substituted. Under these conditions, no DHPG-dependent increase
in tyrosine phosphorylation of NR2A was obtained (Fig. 6c).
Activation of mGluR1 increased
To establish that the observed increase in NR2A/B phosphory-
lation corresponded to enhancement of NMDA receptor func-
tion in cortical neurons, we analyzed the effects of selective
mGluR1 activation on NMDA-induced currents in the whole-cell
and perforated-patch configuration.
When mGluR1 was selectively stimulated by application of
DHPG in the presence of the mGluR5 antagonist MPEP,
NMDA receptor responses were slowly increased, reaching sta-
tistical significance only after 15 min (Fig. 7). MPEP by itself had
no effect on current when co-applied with NMDA (mean NMDA
steady-state current before MPEP: 615 ? 170 pA; after 3 min
MPEP: 593 ? 180 pA; n ? 3 neurons). Selective activation of
mGluR1 affected both peak and steady-state NMDA currents
(Fig. 7a). When currents were analyzed in the whole-cell config-
uration, some, but not all, cells studied showed an increased
NMDA current response after a 2 sec application of NMDA in
the presence of 100 ?M DHPG ? 5 ?M MPEP (data not shown).
When currents were analyzed in the perforated–patch configura-
tion, this variability was still present after 6 min but slowly
developed into a statistically significant increase in NMDA cur-
rents (Fig. 7b). Fifteen minute preincubation with either the
Src-kinase inhibitor PP2 (5 ?M) or the mGluR1 antagonist CPC-
COEt (200 ?M) reduced the basal NMDA currents (mean
NMDA steady-state current: 531.1 ? 244, n ? 10; after 15 min
preincubation with PP2: 196.4 ? 164, n ? 5; after 15 min prein-
cubation with CPCCOEt: 217 ? 38, n ? 4) and completely
prevented a DHPG-mediated increase in NMDA currents (Fig.
7). Furthermore, as occurred with the activation of the Pyk2/Src/
Fyn pathway and tyrosine phosphorylation of NR2 A/B, pro-
longed exposure to either CPCCOEt or PP2 reduced NMDA-
NR2A through activation of Pyk2 and Src kinases in HEK293 cells.
HEK293 cells stably expressing NR1 (clone ND-10) were transiently
transfected with either NR2A or mGluR1? and treated in the absence or
presence of DHPG for the indicated times in the presence of orthovana-
date (a) or transfected with NR2A/mGluR1?/Pyk2/Src and treated in the
absence or presence of DHPG for 5 min (b, c). After treatment, cultures
were immediately processed for protein extraction and Western blotting
(b) or immunoprecipitation with specific NR2A antibodies, and phospho-
tyrosine levels were detected with anti-pTyr antibody (4G10) (a, c). a,
Western blotting using anti-phosphotyrosine antibodies (?PY: 4G10, top
panel) or NR2A-specific antibody (bottom panel). b, Extracts from
ND-10 cells transfected with NR2A/mGluR1?/Pyk2/Src (?Pyk2/Src),
NR2A/mGluR1?/Pyk2 (?Pyk2), or NR2A/mGluR1?/Src (?Src) were
processed for Western blotting using either anti-Pyk2 antibodies (either
phospho-Pyk2: p-Pyk2, or anti-Pyk2) or anti-Src (either phospho-Src:
p-Src, or anti-Src). Expression levels of mGluR1? and NR1 are shown as
mGluR1a and NR1. c, Bar graph depicting the percentage increase in
tyrosine phosphorylation of NR2A after treatment in the absence or
presence of DHPG in ND-10 cells transfected with NR2A/mGluR1?/
Pyk2/Src (Pyk2/Src), with NR2A/mGluR1?/Src and the kinase deficient
form of Pyk2 (Pyk2KD/Src), or with NR2A/mGluR1?/Pyk2 and the
dominant negative form of Src (Pyk2/SrcDN). Results plotted in c are
cumulative of three independent experiments. * indicates statistical sig-
nificance as compared with control at p ? 0.05 by ANOVA.
Activation of mGluR 1 induces the tyrosine phosphorylation of
Heidinger et al. • Enhancement of NMDA Receptor Function by mGluR1J. Neurosci., July 1, 2002, 22(13):5452–5461 5457
mediated currents below their initial level (Fig. 7b). CPCCOEt by
itself had no direct effect when co-applied with NMDA (mean
NMDA steady-state current before CPCCOEt: 240 ? 74 pA;
after 3 min CPCCOEt: 280 ? 87 pA; n ? 5 neurons).
Mechanism of activation of the Pyk2/Src pathway in
Activation of Pyk2 in neurons was shown to depend indirectly on
increases in intracellular calcium, possibly through PKC activation
(Lev et al., 1995; Lu et al., 1999). However, in non-neuronal
systems, activation of Pyk2 can also occur through a Ca2?–
calmodulin-dependent pathway (Della Rocca et al., 1997). The
lack of effects of the PKC inhibitor GF109203X on tyrosine phos-
phorylation of NR2A/B prompted us to study the possibility of
calmodulin dependence in the activation of the Pyk2/Src/Fyn
pathway by DHPG in cortical neurons. Although exposure of
near-pure neuronal cultures to the PKC activator phorbol-12-
myristate-13-acetate (PMA, 1 ?M) induced a marked increase in
Pyk2 phosphorylation that was inhibited by the specific PKC inhib-
itor GF109203X (Pyk2 phosphorylation after PMA was 250 ? 10%
of control; after PMA ? GF109203X it was 100 ? 13% of control),
neither this inhibitor nor the myristoylated inhibitor peptide (19–
27) had any effect on either the basal phosphorylation level or the
DHPG-mediated phosphorylation of Pyk2 or Src/Fyn (Fig. 8a,b).
On the other hand, pre-exposure to the calmodulin antagonists
calmidazolium chloride (30 ?M), W7 (100 ?M), or ophiobolin (30
?M) (data not shown) prevented the effects of DHPG on Pyk2 and
Src/Fyn phosphorylation (Fig. 8a,b). As occurred with the
mGluR1 antagonist CPCCOEt (Fig. 4), calmodulin antagonists
were able to decrease the basal phosphorylation levels of Pyk2 and
Src/Fyn kinases (Fig. 8a,b, bottom panels).
The main findings of the present study are that (1) selective
activation of group I mGluRs stimulated tyrosine phosphoryla-
tion of NR2A/B subunits in cortical neurons; (2) this stimulation
was mediated specifically by mGluR1 and an intracellular cascade
involving PLC, calmodulin, Pyk2, and the Src-family kinases
(Src/Fyn); (3) the mGluR1-mediated tyrosine phosphorylation of
NMDA receptors had functional consequences, increasing
NMDA receptor-mediated current; and (4) the tonic activity of
mGluR1 regulated the basal activity of Pyk2 and Src/Fyn and
thus may control the basal tyrosine phosphorylation of NMDA
receptors in cortical neurons.
current in cortical neurons. NMDA-induced currents in cortical neurons
were recorded using the perforated-patch configuration. After obtaining
stable recording of NMDA currents, DHPG (100 ?M) plus the mGluR5
antagonist MPEP (2 ?M) was applied, and NMDA currents were re-
corded for the next 20 min. a, Raw traces depicting the effect of selective
activation of mGluR1 before and after 15 min exposure to DHPG/MPEP
in the absence (top traces) or presence of the Src-family inhibitor PP2 (5
?M) (middle traces) or the mGluR1 antagonist CPCCOEt (200 ?M)
(bottom traces). b, Cumulative data obtained for the effects of selective
activation of mGluR1 with DHPG/MPEP on NMDA steady-state cur-
rents in the absence or presence of the Src-family inhibitor PP2 (5 ?M) or
the mGluR1 antagonist CPCCOEt (200 ?M). Symbols indicate the fol-
lowing: NMDA currents in the absence (ƒ) or presence of DHPG/MPEP
(F), in the presence of DHPG/MPEP and 200 ?M CPCCOEt (E), or in
the presence of DHPG/MPEP and 5 ?M PP2 (?). Neurons were prein-
cubated with the inhibitors for 15 min. ? and # indicate statistical
significance as compared with time 0 (before DHPG) at p ? 0.05 by
ANOVA; n ? 5–8 neurons per condition.
Selective activation of mGluR1 increased NMDA receptor
cortical neurons was blocked by calmodulin antagonists but not PKC
inhibitors. Cultures were preincubated for 20 min in the absence or
presence of PKC inhibitors, GF109203X (GF, 5 ?M) or the myristoylated
inhibitor peptide (19–27) (PKCi, 25 ?M); calmodulin antagonists, calmi-
dazolium chloride (CZ, 30 ?M) or W7 (100 ?M), and then exposed to
either vehicle (basal) or 100 ?M DHPG for 5 min in the presence or
absence of the inhibitors. Detection and analysis of p-Pyk2 (a) and
p-Src/Fyn (b) was performed as in Figure 4. Bar graphs represent cumu-
lative data of five independent experiments. * indicates statistical signif-
icance as compared with basal, and ** indicates statistical significance as
compared with DHPG alone at p ? 0.05 by ANOVA.
DHPG-mediated activation of the Pyk2/Src/Fyn pathway in
5458 J. Neurosci., July 1, 2002, 22(13):5452–5461Heidinger et al. • Enhancement of NMDA Receptor Function by mGluR1
The cross-linking of group I mGluRs and NMDA receptors
through the synaptic protein Shank provides a structural basis for
the functional coupling observed here. Shank proteins are asso-
ciated with the NMDA receptor/PSD-95 complex and appear to
be recruited to excitatory synapses by virtue of their interaction
with GKAP (Naisbitt et al., 1999), a synaptic protein that binds to
the guanylate kinase domain of PSD-95 (Kim et al., 1997; Naisbitt
et al., 1997). In addition, Shank contains domains that interact
with Homer, a neuronal protein that selectively binds to the C
terminus of group I mGluRs and IP3receptors (Tu et al., 1999).
The Homer–Shank interaction promotes clustering of group I
mGluRs and may explain the perisynaptic localization of these
metabotropic glutamate receptors (Baude et al., 1993; Lujan et
al., 1997). Thus, Shank may be a molecular bridge linking the
NMDA receptor complex with Homer and its associated proteins,
the group I mGluRs and IP3receptors, permitting biochemical
linkage between group I mGluRs and NMDA receptors (Sala et
al., 2001). Consistent with this view, mGluR1 and mGluR5 were
implicated in the regulation of the locomotor network output in
lamprey spinal cord neurons through a postsynaptic interaction
with NMDA receptors (Krieger et al., 2000).
The enhancement of NMDA receptor currents by G-protein-
coupled muscarinic receptors has been linked to the sequential
activation of PKC and Src and the consequent tyrosine phosphor-
ylation of NR2 subunits (Lu et al., 1999). In hippocampal neu-
rons, activation of mGluR5, not mGluR1, was recently shown to
be responsible for the upregulation of NMDA currents (Man-
naioni et al., 2001) by a mechanism probably mediated by PKC
(Bruno et al., 2001), and Pyk2 was shown as a key tyrosine kinase
in the induction of hippocampal LTP (Huang et al., 2001). PKC
can activate neuronal Pyk2 (Lev et al., 1995), and phorbol ester-
mediated activation of PKC can induce the tyrosine phosphory-
lation of NMDA receptors in hippocampal neurons (Grosshans
and Browning, 2001). Taken together, these results suggest that in
hippocampal neurons, activation of mGluR5, not mGluR1, may
upregulate NMDA receptor currents through a PKC-mediated
activation of the Pyk2/Src kinase pathway.
In cortical neurons, however, inhibition of PKC had no effect on
either basal or DHPG-mediated activation of the Pyk2/Src/Fyn
pathway, nor did it have an effect in NR2 phosphorylation (this
study). Thus, we conclude that if there is a PKC-mediated regula-
tion of the Pyk2/Src/Fyn pathway in mGluR1-induced phosphor-
ylation of NMDA receptors in cortical neurons, such involvement
is not via classical or novel family members (targets for the inhib-
itors used), but rather through atypical members insensitive to
these inhibitors (Toullec et al., 1991; Martiny-Baron et al., 1993).
Alternatively, other molecules besides PKC may be responsible for
the mGluR1-mediated activation of Pyk2 observed in cortical neu-
rons. The strong suppression of both basal and DHPG-mediated
phosphorylation of Pyk2 and Src/Fyn observed with calmodulin
inhibitors suggests that the mGluR1-induced activation of the Pyk2
Src/Fyn pathway in cortical neurons is mediated preferentially
mGluR1 and mGluR5 can interact with calmodulin in a calcium-
dependent manner (Minakami et al., 1997; Ishikawa et al., 1999),
and recent study of Chinese hamster ovary cells stably expressing
mGluR1? showed that stimulation of mGluR1 induces the activa-
tion of focal adhesion kinase through a calmodulin-dependent
mechanism independent of PKC (Shinohara et al., 2001).
Upregulation of NMDA-receptor function by tyrosine phos-
phorylation is well established, although the exact mechanisms
are not precisely known. Application of recombinant Src kinase
increases whole-cell currents through NMDA receptors, whereas
application of a purified protein tyrosine phosphatase decreases
these currents (Yu and Salter, 1999). The increase in NMDA
channel activity caused by tyrosine phosphorylation was sug-
gested to reflect enhanced gating of existing receptors, rather
than recruitment of new receptors (Salter, 1998), and Src-
mediated phosphorylation of recombinant NR2A/B was pro-
posed to relieve the voltage-independent Zn2?inhibition of
NMDA receptors (Zheng et al., 1998; Xiong et al., 1999; Vissel
et al., 2001), although these results were not reproduced in native
receptors (Xiong et al., 1999). However, sequence analysis shows
that there are at least 25 tyrosine residues susceptible to phos-
phorylation in NR2A and NR2B, and their individual effects on
NMDA receptor function are just beginning to be elucidated
(Zheng et al., 1998; Cheung and Gurd, 2001; Nakazawa et al.,
2001; Roche et al., 2001; Vissel et al., 2001). It is possible then
that tyrosine phosphorylation of NMDA receptors may play a
role other than direct regulation of NMDA currents. Recently, a
different mechanism of regulation of NMDA receptor function
by tyrosine phosphorylation was proposed. Tyrosine phosphory-
lation of NR2 subunits prevented the downregulation of recom-
binant NR1/2A receptor (Vissel et al., 2001) and prevented the
calpain-mediated truncation of the C-terminal domains of
NMDA receptors in synaptic membranes (Bi et al., 2000). Taken
together, the above results suggest that tyrosine phosphorylation
of NR2 subunits may not only induce the direct upregulation of
NMDA receptor current but may also control receptor recycling,
thus supporting a major role for the Pyk2/Src/Fyn pathway in the
stability and function of postsynaptic NMDA receptors. Recent
findings showing that activation of mGluR1? causes a rapid
increase in the number of functional NMDA receptors when
expressed in Xenopus oocytes (Lan et al., 2001), and present
results showing that mGluR1 antagonists reduce the basal activity
of the Pyk2/Src/Fyn pathway and bring the tyrosine phosphory-
lation levels and currents of NMDA receptors below their initial
level, suggest the interesting possibility that mGluR1, by control-
ling the basal tyrosine phosphorylation levels of NR2 subunits,
may also regulate receptor recycling as described by Vissel and
collaborators (2001) and thus may increase the number of func-
tional NMDA receptors at the synapse in cortical neurons.
Postsynaptic mGluR1 and mGluR5 may have different effects
on NMDA receptor function. As noted above, a general pro-
excitatory role of group I mGluRs is well accepted, although
some opposite effects have been observed (for review, see Nico-
letti et al., 1999). In particular, activation of postsynaptic group I
mGluRs induced an immediate, membrane-delimited downregu-
lation of NMDA-mediated currents in cortical neurons (S. P. Yu
et al., 1997) that appears to be preferentially mediated by
mGluR5 (S.-P. Yu, M. M. Behrens, and D. W. Choi, unpublished
observations). However, this membrane-delimited effect of
mGluR5 was not observed when currents were analyzed in the
perforated-patch recording setting, suggesting the involvement of
soluble factors in the mGluR1-mediated upregulation of NMDA
currents. In the whole-cell recording setting, these soluble com-
ponents are washed out, thus allowing the observation of the
membrane-delimited modulation of NMDA currents by mGluR5.
What is then the physiological consequence of this membrane-
delimited modulation of NMDA receptors by mGluR5? One
possibility is that although both mGluR1? and mGluR5 can
localize to the perisynaptic region and thus to the vicinity of
NMDA receptors, they do not do so in the same synapse, and the
tyrosine phosphorylation effects of mGluR1 activation are ob-
Heidinger et al. • Enhancement of NMDA Receptor Function by mGluR1 J. Neurosci., July 1, 2002, 22(13):5452–5461 5459
served only in those synapses containing mGluR1? and NMDA
receptors and not in those containing mGluR5. Indeed, the dif-
ferential distribution of mGluR1? and mGluR5 observed in dis-
sociated cortical neurons (Fig. 3) would give support to this
hypothesis. An alternative possibility would be that mGluR1?
and mGluR5 do colocalize to the same synapses, with the net
effect on NMDA receptors varying as a function of factors such
as time and activity in other relevant signaling pathways affecting
Src-family kinases or NMDA receptor phosphorylation.
Recent data obtained in hippocampal CA1 pyramidal neurons
also suggest differential effects for mGluR1 and mGluR5 (Man-
naioni et al., 2001). Using an approach similar to the one used in
the present study, those investigators concluded that mGluR1?
mediated the DHPG-induced increases in intracellular calcium,
whereas mGluR5 mediated the DHPG-induced suppression of
the Ca2?-activated potassium current (IAHP) and potentiation of
NMDA receptor current. The suggestion that mGluR1? and
mGluR5 might regulate NMDA receptors differently in CA1
pyramidal neurons versus cortical neurons is intriguing and most
likely explained by differential access to signaling cascades. Both
mGluR1? and mGluR5 are able to interact with the anchoring
protein Homer, which through the protein Shank produces the
link to NMDA receptors (Sala et al., 2001). A plausible explana-
tion then to the differential effects of group I mGluRs on NMDA
receptors is that mGluR1? and mGluR5 have other partners
besides Homer that allow the biochemical coupling of either
mGluR1? or mGluR5 to the tyrosine phosphorylation of NMDA
receptors. In mass spectroscopy analysis of the NMDA receptor
complex, as many as 70 signaling proteins were associated with
NMDA receptors at postsynaptic densities, including the compo-
nents of the biochemical network described in this work, i.e.,
mGluR1, PyK2, Src, and Fyn (Husi et al., 2000; Walikonis et al.,
2000). The presence or absence of specific signaling components
at individual synapses might convey specificity to NMDA recep-
tor regulation by mGluRs, as well as to the cascades of enzymatic
reactions that carry the NMDA receptor-mediated signal into the
interior of the cell (Kennedy, 2000). Localization might also
convey regional specificity to signaling cascades; for example,
mGluR1-induced activation of intracellular signaling pathways in
cortical neurons appears primarily in dendrites (M. M. Behrens
and T. Bartfai, unpublished observations).
Aiba A, Chen C, Herrup K, Rosenmund C, Stevens CF, Tonegawa S (1994)
Reduced hippocampal long-term potentiation and context-specific deficit
in associative learning in mGluR1 mutant mice. Cell 79:365–375.
Akaike N (1996) Gramicidin perforated patch recording and intracellular
chloride activity in excitable cells. Prog Biophys Mol Biol 65:251–264.
Aniksztejn L, Bregestovski P, Ben-Ari Y (1991) Selective activation of
quisqualate metabotropic receptor potentiates NMDA but not AMPA
responses. Eur J Pharmacol 205:327–328.
Annoura M, Fukunaga A, Uesugi M, Tatsuoka T, Horikawa Y (1996) A
novel class of antagonists for metabotropic glutamate receptors,
7-(hydroxyamino)cyclopropa[b]chromen1a-carboxylates. Bioorg Med
Chem Lett 6:763–766.
Baude A, Nusser Z, Roberts JD, Mulvihill E, McIlhinney RA, Somogyi
P (1993) The metabotropic glutamate receptor (mGluR1 alpha) is
concentrated at perisynaptic membrane of neuronal subpopulations as
detected by immunogold reaction. Neuron 11:771–787.
Behrens MM, Heidinger V, Strasser U, Yu S-P, Choi DW (1999a)
3,5-DHPG-mediated activation of group I mGluRs induces NMDA
receptor tyrosine phosphorylation. Neuropharmacology 38:A5.
Behrens MM, Strasser U, Koh JY, Gwag BJ, Choi DW (1999b) Preven-
tion of neuronal apoptosis by phorbol ester-induced activation of pro-
tein kinase C: blockade of p38 mitogen-activated protein kinase. Neu-
Behrens MM, Heidinger V, Manzerra P, Ichinose T, Yu S-P, Choi DW
(2000) Neuronal mGluR1 activation enhances NMDA receptor func-
tion via Src-family kinase-mediated phosphorylation. Soc Neurosci
Bi R, Rong Y, Bernard A, Khrestchatisky M, Baudry M (2000) Src-
mediated tyrosine phosphorylation of NR2 subunits of N-methyl-D-
aspartate receptors protects from calpain-mediated truncation of their
C-terminal domains. J Biol Chem 275:26477–26483.
Bruno V, Battaglia G, Copani A, Cespedes VM, Galindo MF, Cena V,
Sanchez-Prieto J, Gasparini F, Kuhn R, Flor PJ, Nicoletti F (2001) An
activity-dependent switch from facilitation to inhibition in the control
of excitotoxicity by group I metabotropic glutamate receptors. Eur
J Neurosci 13:1469–1478.
Charpak S, Gahwiler BH, Do KQ, Knopfel T (1990) Potassium conduc-
tances in hippocampal neurons blocked by excitatory amino-acid trans-
mitters. Nature 347:765–767.
CheungHH,Gurd JW (2001) Tyrosine
N-methyl-D-aspartate receptor by exogenous and postsynaptic density-
associated Src-family kinases. J Neurochem 78:524–534.
Conquet F, Bashir ZI, Davies CH, Daniel H, Ferraguti F, Bordi F,
Franz-Bacon K, Reggiani A, Matarese V, Conde F, Collingridge GL,
Crepel F (1994) Motor deficit and impairment of synaptic plasticity in
mice lacking mGluR1. Nature 372:237–243.
Cooper JA, MacAuley A (1988) Potential positive and negative autoreg-
ulation of p60c-src by intermolecular autophosphorylation. Proc Natl
Acad Sci USA 85:4232–4236.
Crepel V, Aniksztejn L, Ben-Ari Y, Hammond C (1994) Glutamate
metabotropic receptors increase a Ca(2?)-activated nonspecific cationic
current in CA1 hippocampal neurons. J Neurophysiol 72:1561–1569.
Della Rocca GJ, van Biesen T, Daaka Y, Luttrell DK, Luttrell LM,
Lefkowitz RJ (1997) Ras-dependent mitogen-activated protein kinase
activation by G protein-coupled receptors. Convergence of Gi- and
Gq-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase.
J Biol Chem 272:19125–19132.
Dikic I, Tokiwa G, Lev S, Courtneidge SA, Schlessinger J (1996) A role
for Pyk2 and Src in linking G-protein-coupled receptors with MAP
kinase activation. Nature 383:547–550.
Doherty AJ, Palmer MJ, Henley JM, Collingridge GL, Jane DE (1997)
(RS)-2-chloro-5-hydroxyphenylglycine (CHPG) activates mGlu5, but
no mGlu1, receptors expressed in CHO cells and potentiates NMDA
responses in the hippocampus. Neuropharmacology 36:265–267.
Fitzjohn SM, Irving AJ, Palmer MJ, Harvey J, Lodge D, Collingridge GL
(1996) Activation of group I mGluRs potentiates NMDA responses in
rat hippocampal slices Neurosci Lett [Erratum (1996) 207:142]
Gasparini F, Lingenhohl K, Stoehr N, Flor PJ, Heinrich M, Vranesic I,
Biollaz M, Allgeier H, Heckendorn R, Urwyler S, Varney MA, John-
son EC, Hess SD, Rao SP, Sacaan AI, Santori EM, Velicelebi G, Kuhn
R (1999) 2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, se-
lective and systemically active mGlu5 receptor antagonist. Neurophar-
Goslin K, Asmussen H, Banker G (1998) Rat hippocampal neurons in
low-density cultures. In: Culturing nerve cells, Ed 2 (Banker G, Goslin
K, eds), pp 339–370. Cambridge, MA: MIT.
Grant SG, O’Dell TJ, Karl KA, Stein PL, Soriano P, Kandel ER (1992)
Impaired long-term potentiation, spatial learning, and hippocampal
development in fyn mutant mice. Science 258:1903–1910.
Grosshans DR, Browning MD (2001) Protein kinase C activation in-
duces tyrosine phosphorylation of the NR2A and NR2B subunits of the
NMDA receptor. J Neurochem 76:737–744.
Guerineau NC, Gahwiler BH, Gerber U (1994) Reduction of resting K?
current by metabotropic glutamate and muscarinic receptors in rat CA3
cells: mediation by G-proteins. J Physiol (Lond) 474:27–33.
Guerineau NC, Bossu JL, Gahwiler BH, Gerber U (1995) Activation of
a nonselective cationic conductance by metabotropic glutamatergic and
muscarinic agonists in CA3 pyramidal neurons of the rat hippocampus.
J Neurosci 15:4395–4407.
Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH,
Weringer EJ, Pollok BA, Connelly PA (1996) Discovery of a novel,
potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck-
and FynT-dependent T cell activation. J Biol Chem 271:695–701.
Holmes TC, Berman K, Swartz JE, Dagan D, Levitan IB (1997) Ex-
pression of voltage-gated potassium channels decreases cellular protein
tyrosine phosphorylation. J Neurosci 17:8964–8974.
Huang CC, Hsu KS (1999) Protein tyrosine kinase is required for the
induction of long-term potentiation in the rat hippocampus. J Physiol
Huang Y, Lu W, Ali DW, Pelkey KA, Pitcher GM, Lu YM, Aoto H,
Roder JC, Sasaki T, Salter MW, MacDonald JF (2001) CAKbeta/
Pyk2 kinase is a signaling link for induction of long-term potentiation
in CA1 hippocampus. Neuron 29:485–496.
Husi H, Ward MA, Choudhary JS, Blackstock WP, Grant SG (2000)
Proteomic analysis of NMDA receptor-adhesion protein signaling com-
plexes. Nat Neurosci 3:661–669.
Ishikawa K, Nash SR, Nishimune A, Neki A, Kaneko S, Nakanishi S
(1999) Competitive interaction of seven in absentia homolog-1A and
5460 J. Neurosci., July 1, 2002, 22(13):5452–5461Heidinger et al. • Enhancement of NMDA Receptor Function by mGluR1
Ca2?/calmodulin with the cytoplasmic tail of group 1 metabotropic Download full-text
glutamate receptors. Genes Cells 4:381–390.
Kennedy MB (2000) Signal-processing machines at the postsynaptic
density. Science 290:750–754.
Kim E, Naisbitt S, Hsueh YP, Rao A, Rothschild A, Craig AM, Sheng M
(1997) GKAP, a novel synaptic protein that interacts with the guany-
late kinase-like domain of the PSD-95/SAP90 family of channel clus-
tering molecules. J Cell Biol 136:669–678.
Kohr G, Seeburg PH (1996) Subtype-specific regulation of recombinant
NMDA receptor-channels by protein tyrosine kinases of the src family.
J Physiol (Lond) 492:445–452.
Kojima N, Wang J, Mansuy IM, Grant SG, Mayford M, Kandel ER (1997)
Rescuing impairment of long-term potentiation in fyn-deficient mice by
introducing Fyn transgene. Proc Natl Acad Sci USA 94:4761–4765.
Krieger P, Hellgren-Kotaleski J, Kettunen P, El Manira AJ (2000) In-
teraction between metabotropic and ionotropic glutamate receptors
regulates neuronal network activity. J Neurosci 20:5382–5391.
Kyrozis A, Reichling DB (1995) Perforated-patch recording with gram-
icidin avoids artifactual changes in intracellular chloride concentration.
J Neurosci Methods 57:27–35.
Lan JY, Skeberdis VA, Jover T, Zheng X, Bennett MV, Zukin RS
(2001) Activation of metabotropic glutamate receptor 1 accelerates
NMDA receptor trafficking. J Neurosci 21:6058–6068.
Lev S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM,
Plowman GD, Rudy B, Schlessinger J (1995) Protein tyrosine kinase
PYK2 involved in Ca(2?)-induced regulation of ion channel and MAP
kinase functions. Nature 376:737–745.
Li X, Dy RC, Cance WG, Graves LM, Earp HS (1999) Interactions
dependent tyrosine kinase and focal adhesion tyrosine kinase. J Biol
Lin SY, Wu K, Len GW, Xu JL, Levine ES, Suen PC, Mount HT, Black IB
(1999) Brain-derived neurotrophic factor enhances association of protein
tyrosine phosphatase PTP1D with the NMDA receptor subunit NR2B in
the cortical postsynaptic density. Brain Res Mol Brain Res 70:18–25.
Litschig S, Gasparini F, Rueegg D, Stoehr N, Flor PJ, Vranesic I, Prezeau
L, Pin JP, Thomsen C, Kuhn R (1999) CPCCOEt, a noncompetitive
metabotropic glutamate receptor 1 antagonist, inhibits receptor signal-
ing without affecting glutamate binding. Mol Pharmacol 55:453–461.
Lu WY, Xiong ZG, Lei S, Orser BA, Dudek E, Browning MD, Mac-
Donald JF (1999) G-protein-coupled receptors act via protein kinase
C and Src to regulate NMDA receptors. Nat Neurosci 2:331–338.
Lu YM, Jia Z, Janus C, Henderson JT, Gerlai R, Wojtowicz JM, Roder
JC (1997) Mice lacking metabotropic glutamate receptor 5 show im-
paired learning and reduced CA1 long-term potentiation (LTP) but
normal CA3 LTP. J Neurosci 17:5196–5205.
Lujan R, Roberts JD, Shigemoto R, Ohishi H, Somogyi P (1997) Dif-
ferential plasma membrane distribution of metabotropic glutamate
receptors mGluR1 alpha, mGluR2 and mGluR5, relative to neurotrans-
mitter release sites. J Chem Neuroanat 13:219–241.
Luthi A, Gahwiler BH, Gerber U (1996) A slowly inactivating potassium
current in CA3 pyramidal cells of rat hippocampus in vitro. J Neurosci
Luttrell LM, Daaka Y, Lefkowitz RJ (1999) Regulation of tyrosine
kinase cascades by G-protein-coupled receptors. Curr Opin Cell Biol
Mannaioni G, Marino MJ, Valenti O, Traynelis SF, Conn PJ (2001)
Metabotropic glutamate receptors 1 and 5 differentially regulate CA1
pyramidal cell function. J Neurosci 21:5925–5934.
Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug
H, Marme D, Schachtele C (1993) Selective inhibition of protein kinase
C isozymes by the indolocarbazole Go 6976. J Biol Chem 268:9194–9197.
Minakami R, Jinnai N, Sugiyama H (1997) Phosphorylation and calmod-
ulin binding of the metabotropic glutamate receptor subtype 5
(mGluR5) are antagonistic in vitro. J Biol Chem 272:20291–20298.
Moon IS, Apperson ML, Kennedy MB (1994) The major tyrosine-
phosphorylated protein in the postsynaptic density fraction is N-methyl-
D-aspartate receptor subunit 2B. Proc Natl Acad Sci USA 91:3954–3958.
Naisbitt S, Kim E, Weinberg RJ, Rao A, Yang FC, Craig AM, Sheng M
(1997) Characterization of guanylate kinase-associated protein, a
postsynaptic density protein at excitatory synapses that interacts di-
rectly with postsynaptic density-95/synapse-associated protein 90.
J Neurosci 17:5687–5696.
Naisbitt S, Kim E, Tu JC, Xiao B, Sala C, Valtschanoff J, Weinberg RJ,
Worley PF, Sheng M (1999) Shank, a novel family of postsynaptic
density proteins that binds to the NMDA receptor/PSD-95/GKAP
complex and cortactin. Neuron 23:569–582.
Nakazawa T, Komai S, Tezuka T, Hisatsune C, Umemori H, Semba K,
Mishina M, Manabe T, Yamamoto T (2001) Characterization of Fyn-
mediated tyrosine phosphorylation sites on GluR epsilon 2 (NR2B)
subunit of the N-methyl-D-aspartate receptor. J Biol Chem 276:693–699.
Nicoletti F, Bruno V, Catania MV, Battaglia G, Copani A, Barbagallo G,
Cena V, Sanchez-Prieto J, Spano PF, Pizzi M (1999) Group-I metabo-
tropic glutamate receptors: hypotheses to explain their dual role in
neurotoxicity and neuroprotection. Neuropharmacology 38:1477–1484.
O’Dell TJ, Kandel ER, Grant SG (1991) Long-term potentiation in the
Pin JP, Duvoisin R (1995) The metabotropic glutamate receptors: struc-
ture and functions. Neuropharmacology 34:1–26.
Roche KW, Standley S, McCallum J, Dune Ly C, Ehlers MD, Wenthold
RJ (2001) Molecular determinants of NMDA receptor internaliza-
tion. Nat Neurosci 4:794–802.
Rose K, Goldberg M, Choi D (1993) Cytotoxicity in murine cortical cell
culture. In: In vitro biological methods. Methods in toxicology (Tyson
C, Frazier J, eds), pp 46–60. San Diego: Academic.
Rosenblum K, Dudai Y, Richter-Levin G (1996) Long-term potentia-
tion increases tyrosine phosphorylation of the N-methyl-D-aspartate
receptor subunit 2B in rat dentate gyrus in vivo. Proc Natl Acad Sci
Rostas JA, Brent VA, Voss K, Errington ML, Bliss TV, Gurd JW (1996)
Enhanced tyrosine phosphorylation of the 2B subunit of the N-methyl-
D-aspartate receptor in long-term potentiation. Proc Natl Acad Sci USA
Sala C, Piech V, Wilson NR, Passafaro M, Liu G, Sheng M (2001)
Regulation of dendritic spine morphology and synaptic function by
Shank and Homer. Neuron 31:115–130.
Salazar EP, Rozengurt E (1999) Bombesin and platelet-derived growth
factor induce association of endogenous focal adhesion kinase with Src
in intact Swiss 3T3 cells. J Biol Chem 274:28371–28378.
Salter MW (1998) Src, N-methyl-D-aspartate (NMDA) receptors, and
synaptic plasticity. Biochem Pharmacol 56:789–798.
Schoepp DD, Jane DE, Monn JA (1999) Pharmacological agents acting
at subtypes of metabotropic glutamate receptors. Neuropharmacology
Shinohara Y, Nakajima Y, Nakanishi S (2001) Glutamate induces focal
adhesion kinase tyrosine phosphorylation and actin rearrangement in
heterologous mGluR1-expressing CHO cells via calcium/calmodulin
signaling. J Neurochem 78:365–373.
Sprengel R, Suchanek B, Amico C, Brusa R, Burnashev N, Rozov A,
Hvalby O, Jensen V, Paulsen O, Andersen P, Kim JJ, Thompson RF,
Sun W, Webster LC, Grant SG, Eilers J, Konnerth A, Li J, McNamara
JO, Seeburg PH (1998) Importance of the intracellular domain of
NR2 subunits for NMDA receptor function in vivo. Cell 92:279–289.
Strasser U, Lobner D, Behrens MM, Canzoniero LM, Choi DW (1998)
Antagonists for group I mGluRs attenuate excitotoxic neuronal death
in cortical cultures. Eur J Neurosci 10:2848–2855.
Tezuka T, Umemori H, Akiyama T, Nakanishi S, Yamamoto T (1999)
PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the
N-methyl-D-aspartate receptor subunit NR2A. Proc Natl Acad Sci USA
Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane
M, Baudet V, Boissin P, Boursier E, Loriolle F, Duhamel L, Charon D,
Kirilovsky J (1991) The bisindolylmaleimide GF 109203X is a potent
Tu JC, Xiao B, Naisbitt S, Yuan JP, Petralia RS, Brakeman P, Doan A,
Aakalu VK, Lanahan AA, Sheng M, Worley PF (1999) Coupling of
mGluR/Homer and PSD-95 complexes by the Shank family of postsyn-
aptic density proteins. Neuron 23:583–592.
Varney MA, Cosford ND, Jachec C, Rao SP, Sacaan A, Lin FF, Bleicher
L, Santori EM, Flor PJ, Allgeier H, Gasparini F, Kuhn R, Hess SD,
Velicelebi G, Johnson EC (1999) SIB-1757 and SIB-1893: selective,
noncompetitive antagonists of metabotropic glutamate receptor type 5.
J Pharmacol Exp Ther 290:170–181.
Vissel B, Krupp JJ, Heinemann SF, Westbrook GL (2001) A use-
dependent tyrosine dephosphorylation of NMDA receptors is indepen-
dent of ion flux. Nat Neurosci 4:587–596.
Walikonis RS, Jensen ON, Mann M, Provance Jr DW, Mercer JA,
Kennedy MB (2000) Identification of proteins in the postsynaptic den-
sity fraction by mass spectrometry. J Neurosci 20:4069–4080.
Xiong ZG, Pelkey KA, Lu WY, Lu YM, Roder JC, MacDonald JF,
Salter MW (1999) Src potentiation of NMDA receptors in hippocam-
pal and spinal neurons is not mediated by reducing zinc inhibition.
J Neurosci 19:RC37(1–6).
Yu SP, Sensi SL, Canzoniero LM, Buisson A, Choi DW (1997)
Membrane-delimited modulation of NMDA currents by metabotropic
glutamate receptor subtypes 1/5 in cultured mouse cortical neurons.
J Physiol (Lond) 499:721–732.
Yu XM, Salter MW (1999) Src, a molecular switch governing gain con-
trol of synaptic transmission mediated by N-methyl-D-aspartate recep-
tors. Proc Natl Acad Sci USA 96:7697–7704.
Yu XM, Askalan R, Keil II GJ, Salter MW (1997) NMDA channel
regulation by channel-associated protein tyrosine kinase Src. Science
Zheng F, Gingrich MB, Traynelis SF, Conn PJ (1998) Tyrosine kinase
potentiates NMDA receptor currents by reducing tonic zinc inhibition.
Nat Neurosci 1:185–191.
Zhong J, Russell SL, Pritchett DB, Molinoff PB, Williams K (1994)
Expression of mRNAs encoding subunits of the N-methyl-D-aspartate
receptor in cultured cortical neurons. Mol Pharmacol 45:846–853.
Heidinger et al. • Enhancement of NMDA Receptor Function by mGluR1 J. Neurosci., July 1, 2002, 22(13):5452–5461 5461