We previously identified Neuregulin1 (NRG1) as a gene contributing to the risk of developing schizophrenia. Furthermore, we showed
treatment of schizophrenia. We now present evidence that ErbB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4), the
tyrosine kinase 2). NRG1 stimulation of cells expressing ErbB4 and Fyn leads to the association of Fyn with ErbB4 and consequent
on the NR2B subunit of the NMDA receptor (NMDAR), a key regulatory site that modulates channel properties. NR2B Y1472 is hypo-
phosphorylated in NRG1?/?mutant mice, and this defect can be reversed by clozapine at a dose that reverses their behavioral abnor-
malities. We also demonstrate that short-term synaptic plasticity is altered and theta-burst long-term potentiation is impaired in
NRG1?/?mutant mice, and incubation of hippocampal slices from these mice with NRG1 reversed those effects. Attenuated NRG1
Schizophrenia is a heritable, highly debilitating psychotic disor-
der that affects 0.5–1% of the general population (Task Force on
identified Neuregulin 1 (NRG1) as a gene conferring susceptibil-
ity to schizophrenia (Stefansson et al., 2002). This finding has
on populations outside of Iceland, although the exact markers
and/or haplotypes showing the most significant association to
schizophrenia vary (for review, see Harrison and Weinberger,
gene, postmortem transcript analysis has shown that several at-
risk single nucleotide polymorphisms are associated with altered
ratios of NRG1 mRNA isoforms in brains of schizophrenics
(Hasimoto et al., 2004; Law et al., 2006). In addition, the mRNA
for ErbB3 (v-erb-a erythroblastic leukemia viral oncogene ho-
molog 3), a tyrosine kinase receptor for NRG1, is significantly
underexpressed in prefrontal cortex of schizophrenics (Hakak et
al., 2001; Aston et al., 2004), and a genetic interaction has been
reported between NRG1 and ErbB4, another receptor for NRG1
(Norton et al., 2006). With the identification of NRG1 and a
number of other susceptibility genes in the past several years, a
molecular understanding of schizophrenia has begun to emerge
(for review, see Harrison and Owen, 2003; Corfas et al., 2004).
Interestingly, several of these candidate genes appear to play a
role in glutamatergic neurotransmission through the NMDA re-
ceptor (NMDAR), and mutations in these genes may confer sus-
ceptibility to schizophrenia by impairing synaptic function
(Konradi and Heckers, 2003).
crucial role in the development of many organs, including the
brain (Buonanno and Fischbach, 2001; Falls, 2003), and modu-
et al., 2000; Roysommuti et al., 2003; Gu et al., 2005; Kwon et al.,
2005; Chen et al., 2006).
NRG1 signals through the ErbB family of receptor tyrosine
has been shown to interact with PDZ [postsynaptic density-95
Correspondence should be addressed to Mark Gurney, deCODE Genetics, Sturlugata 8, 101 Reykjavik, Iceland.
TheJournalofNeuroscience,April25,2007 • 27(17):4519–4529 • 4519
(PSD-95)/Discs large/zona occludens-1] domain-containing
(Garcia et al., 2000; Huang et al., 2000). PSD-95 also interacts
with the NMDAR complex, and this interaction appears to be
critical for the phosphorylation of the NMDAR by the Src family
kinase (SFK) Fyn (Tezuka et al., 1999; Nakazawa et al., 2001).
NMDARs are ligand and voltage-gated ion channels that are
et al., 1997; Salter and Kalia, 2004). Clozapine, an antipsychotic
drug, increases NMDA EPSCs in the nucleus accumbens and in
pyramidal cells of the rat prefrontal cortex. This increase is de-
nase II, and SFKs. In the nucleus accumbens, clozapine only af-
fects NR2B subunit-containing NMDARs (Ninan et al., 2003;
Wittmann et al., 2005). Subtle misregulation of membrane po-
tential, ligand binding, or tyrosine phosphorylation may have
profound effects on NMDAR opening, thus influencing behav-
iors modulated by this channel (Moghaddam, 2003).
Although dopamine D2receptor antagonists are widely used
clinically to control the positive symptoms of schizophrenia
(Freedman, 2003), dissociative anesthetics such as phencyclidine
and ketamine that produce schizophrenia-like disorders target
the NMDAR rather than the dopamine pathway. Whereas ma-
posure to dissociative anesthetics acutely reproduces the more
clinically challenging negative and cognitive symptoms of the
tion in schizophrenia has also been suggested on pharmacologi-
Here we demonstrate that NRG1 signaling stimulates NR2B
Y1472 phosphorylation through the activation of the SFKs Fyn
and Pyk2 (proline-rich tyrosine kinase 2). Furthermore, we
present evidence that NR2B Y1472 is hypophosphorylated in
NRG1?/?and ErbB4?/?mutant mice and that this can be cor-
rected by treatment with clozapine at doses that reverse behav-
ioral abnormalities. Last, NRG1?/?mice show altered hip-
pocampal synaptic plasticity. We propose that attenuated NRG1
signaling may contribute to the pathophysiology of schizophre-
nia through dysfunction of NMDAR modulation.
Reagents. All chemicals were purchased from Sigma (St. Louis, MO)
Invitrogen (Carlsbad, CA). Recombinant human NRG1?2 epidermal
(RefSeq) accession number NP_039258] (catalog #296-HR-050; R & D
Systems, Minneapolis, MN) was used for all biochemical assays. NRG1
stimulation was performed in serum-free medium for 0–15 min. For all
domain (ECD) of NRG1?1 (amino acids 1–241; RefSeq accession num-
ber NP_039250) (catalog #RP-318-PA; LabVision, Fremont, CA). Al-
though both forms of NRG1 gave similar results in a reporter gene assay
(described below), the ? form of the protein predominates in brain.
However, during the time these studies were being performed, the
NRG1?1 protein was in short supply and was often backordered for
months from the sole supplier. For this reason, we reserved it for the
electrophysiological studies in which we felt the cellular environment
warranted the use of the more relevant form of the protein (Ozaki et al.,
Yeast two-hybrid screening. A random-primed human hippocampus
ing kit (Stratagene, La Jolla, CA) and transformed into yeast. The ErbB4
yeast cells were created by mating 5 ? 109bait cells (AH109) with 1 ?
108pretransformed library cells (Y187) and plated on minimum media,
106clones was screened. After incubation for 5–10 d at 30°C, colonies
were picked and grown for 48 h at 30°C in ?LT media before gridding
onto ?LTH agar supplemented with 5 mM 3-amino-triazole (3AT).
kit (Pierce, Rockford, IL) according to the instructions of the manufac-
turer. Prey plasmids were isolated using a 96-well MultiScreen system
(Millipore, Billerica, MA) and insert sequences amplified by PCR (for-
ward primer, 5?-TTGGAATCACTACAGGGATGTTTAATAC; reverse
primer, 5?-CTCTGCAGTAATACGACTCACTATAGGG) before se-
the appropriate plasmids using a Frozen EZ Yeast Transformation II kit
bait or with simian virus 40 prey plasmids (Clontech).
Plasmids and transfection. Human ErbB4 in pcDNA3.1 (Invitrogen)
was kindly provided by Prof. Kermit L. Carraway III (University of Cal-
ifornia Davis Cancer Center, Davis, CA). Plasmids containing the ErbB4
D843A and K751A mutations were prepared using a Quick Change XL
Site-Directed Mutagenesis kit (Stratagene). A human Fyn plasmid was
purchased from Invitrogen (clone identification number RG000030),
and the C-terminal V5 epitope tag was added by subcloning. The serum
response element (SRE)–Luc plasmid was obtained from Stratagene.
Transfections were performed using Fugene6 (Roche, Indianapolis, IN).
Mammalian tissue culture. All cells coexpressing ErbB4 and
p59Fyn–V5 were generated by double transfection, using the ErbB4 and
mycin (G418) and Zeocin both from Invitrogen]. Protein expression in
Leica (Bannockburn, IL) deconvolution microscope.
COS-7 and HEK293 cells were grown in DMEM supplemented with
10% (v/v) fetal calf serum, 100 IU/ml penicillin, and 100 ?M streptomy-
cin, and CHO-K1 and BE(2)-M17 (European Collection of Cell Cul-
tures, Wiltshire, UK) were grown in DMEM/Nutri-mix F-12 supple-
mented with 10% (v/v) fetal bovine serum (FBS) and 100 IU/ml
penicillin and 100 ?M streptomycin. All lines were maintained at 37°C,
5% CO2. Treatment of cells with the tyrosine kinase inhibitors PP2
or PP3 (4-amino-7-phenylpyrazol[3,4-d]pyrimidine) (25 nM each; Cal-
biochem, San Diego, CA) were done overnight in serum-free media.
BE(2)-M17 cells were differentiated by incubation with 10 ?M retinoic
acid (RA) for 3 d. The BE(2)-M17 cells were stimulated with 14.4 nM
and were therefore chosen to explore the effect of NRG1 stimulation on
Colocalization assay. Transiently transfected COS-7 cells were grown
on glass slides to 70% confluency, treated with 7.2 nM NRG1?2 for 10
min, fixed in 4% paraformaldehyde, and permeabilized with 100%
methanol for 1 min. Nonspecific binding sites were blocked with 10%
Biotechnology, Santa Cruz, CA) and anti-V5 (1:500; Invitrogen)] were
added overnight at 4°C. Secondary antibodies goat anti-rabbit-Alexa
Fluor 488 and goat anti-mouse-Alexa Fluor 594 (Invitrogen), each at
1:500, were added for 4 h at 4°C. Cells were mounted in DL-2-amino-5-
phosphonovaleric acid/1,4-diazabicyclo(2.2.2)octane (Millipore) and
examined with a Leica deconvolution CTR MIC Microscope using Leica
QFluoro Image Manager software.
Immunoprecipitation from mammalian tissue culture cell lysates.
radioimmunoprecipitation assay buffer [20 mM 3-(N-morpholino)-
Inhibitor Cocktail (Roche) and Phosphatase Inhibitor Cocktail Set II
4520 • J.Neurosci.,April25,2007 • 27(17):4519–4529 Bjarnadottiretal.•NRG1ModulationofNMDARPhosphorylation
(Calbiochem). Twenty-five units of Benzonase Nuclease (Novagen,
[anti-glutamate (Glu) (deCODE Biostructures, Seattle, WA), anti-
Biotechnology)]. Immunocomplexes were isolated using Protein G
boiling for 7 min in 1? SDS-PAGE sample buffer.
Mouse hippocampus, immunoprecipitation, and Western blot. Animals
NR2B Y1472 phosphorylation. Hippocampus tissue was isolated by
manual dissection and homogenized in DOC buffer (20 ?M ZnCl2, 50
mM Tris, pH 9.0, and 1% DOC) (1 ml/50 mg of tissue) containing pro-
tease and phosphatase inhibitors as above. Twenty-five units of Benzo-
nase Nuclease per 1 ml of lysate were added to each sample during a 60
concentration was determined using a BCA Protein Assay Reagent kit
(Pierce), adjusted to 2 ?g/?l in 1? SDS-PAGE sample buffer, and then
20–25 ?g of protein was loaded per gel lane. For immunoprecipitation,
DOC buffer. Lysates were incubated with the appropriate antibodies (1
?g of antibody/250 ?g of total protein) overnight at 4°C (anti-Glu, anti-
ErbB4, anti-HA, and anti-PSD-95). Immunocomplexes were isolated
in 1? SDS-PAGE sample buffer. Samples were subjected to one-
mass spectrometric analysis, or by Western blotting.
Western blotting. Samples were separated by SDS-PAGE on 4–20%
Tris-HCl Ready Gels (Bio-Rad, Hercules, CA), blotted onto nitrocellu-
lose membranes (Schleicher and Schuell, Keene, NH) and blocked in
TBST (10 mM Tris Base, pH 7.6, 150 mM NaCl, and 0.06% Tween 20)
Reykjavı ´k, Iceland). Blots were incubated with the following primary
antibodies: anti-V5 (1:1000; Invitrogen); anti-Pyk2 (1:500; Santa Cruz
Biotechnology); anti-ErbB4 (1:1000; Santa Cruz Biotechnology); anti-
phosphotyrosine 4G10 (1:2000; Upstate Biotechnology, Lake Placid,
NY); anti-phospho-p44/42 mitogen-activated protein kinase (MAPK)
Thr202/Tyr204 (1:500; Cell Signaling Technology, Danvers, MA); anti-
gen); and anti-NMDA NR2B phospho-specific-Y1472 (1:250 or 1:500;
secondary antibodies (1:2000; DakoCytomation, Carpinteria, CA; or
1:20000; Jackson Immunoresearch, West Grove, PA) for 1 h at room
temperature. Blots were incubated with ECL Plus Western Blotting De-
tection System (GE Healthcare) or SuperSignal West Femto Maximum
Sensitivity Substrate (Pierce) according to the instructions of the manu-
facturer, and signal was detected using Hyperfilm ECL (GE Healthcare).
Multiple exposures of film were made to ensure that the dynamic range
of the film had not been exceeded.
Mass spectrometry. Bands selected from silver-stained SDS-PAGE gels
were excised and destained using destainer solutions contained in the
to in-gel digestion according to a modified version of the method by
Rosenfeld et al. (1992), as described by Edmondson et al. (2002). Liquid
chromatography mass spectrometry/mass spectrometry (MS/MS) was
used for the analysis of in-gel digests.
Nanoscale reversed-phase chromatography was performed using an
Ultimate system with FAMOS autosampler (LC Packings, Amsterdam,
The Netherlands). At a flow rate of 200 nl/min, proteolytic digests were
loaded from a 10 ?l sample loop onto a 75 ?m inner diameter ? 15 cm
column packed with PepMap C18 material (LC Packings) connected
directly to the spray needle of the microelectrospray spray ionization
source (to which 3 kV was applied) of a Micromass Q-Tof-2 mass spec-
trometer (Waters, Milford, MA). Typical gradient conditions used to
elute peptides from the column were 0–50% B (where A ? 98:2 H2O/
acetonitrile incorporating 0.3% formic acid, and B ? 9:1 H2O/acetoni-
trile incorporating 0.3% formic acid) in 10 min, 50–100% B in 2 min,
100% B for 2 min, and 100–2% B in 30 s. The Q-Tof-2 electrospray
mode such that continuous cycles of one MS survey scan followed by
Mascot protein database.
In vitro kinase assay. CHO-K1 cells stably expressing ErbB4 or ErbB4/
Fyn–V5 were treated with 7.2 nM NRG1?2 and lysed in 500 ?l PTK
extraction buffer (SignaTECT Protein Tyrosine Kinase Assay System;
Promega, Madison, WI) containing Complete Protease Inhibitor Cock-
well as 25 U of Benzonase Nuclease. ErbB4 and Fyn–V5 were immuno-
precipitated from cell lysates using ?-ErbB4 or ?-V5 antibodies, respec-
tively. In vitro kinase assays with the immunoprecipitates were per-
formed using a SignaTECT Protein Tyrosine Kinase Assay System
1 was used for ErbB4 and substrate 2 for Fyn.
Luciferase reporter assay. CHO-K1 cells carrying pSRE-Luc (PathDe-
tect; Stratagene) and stably expressing ErbB4 or ErbB4/Fyn–V5 were
plated in serum-free media (96-well format) and treated for 4 h with
0–57.6 nM NRG1?2. Assay was performed using the Dual-Luciferase
manufacturer. Plates were read with a TR 717 Microplate Luminometer
(Applied Biosystems, Foster City, CA). In this assay, the NRG1?2 EGF
had an EC50of 0.7 nM (see Fig. 2 and data not shown).
Knock-out mice. NRG1(?TM)?/?and ErbB4?/?mutant mice were
obtained from Prof. Richard Harvey (The Victor Chang Cardiac Re-
search Institute, Sydney, New South Wales, Australia) and from Prof.
Greg Lemke (The Salk Institute for Biological Studies, La Jolla, CA),
respectively, as acknowledged previously (Stefansson et al., 2002). The
NRG1(?EGF)?/?mice (Erickson et al., 1997) were obtained from Ge-
nentech (South San Francisco, CA). The Fyn?/?mutant mice were ob-
tained from The Jackson Laboratory (Bar Harbor, ME). All lines were
maintained by mating to wild-type C57BL/6. Mice were housed under a
standard 12 h light/dark cycle with access to food and water ad libitum.
All animal work was approved by the respective Institutional Animal
Care and Use Committees and conducted in accordance with appropri-
ate national regulations concerning animal welfare.
Clozapine treatment. Clozapine was administered by intraperitoneal
injection as described previously (Stefansson et al., 2002).
Slice preparation. NRG1(?EGF)?/?mice (4–16 weeks of age) and
age-matched controls were used for all electrophysiology experiments.
Experiments and subsequent analysis were performed by investigators
blinded to the genotype of the mice. Slices were prepared as described
previously (Misner and Sullivan, 1999). Briefly, mice were anesthetized
with halothane, the brains were removed, and coronal slices (350 ?m)
were cut in ice-cold solution (in mM: 120 NaCl, 3.5 KCl, 0.7 CaCl2, 4.0
MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose) bubbled with 95%
O2/5% CO2. The hippocampus was dissected and placed in a chamber
containing recording buffer (in mM: 120 NaCl, 3.5 KCl, 2.6 CaCl2, 1.3
MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose) perfused with 95%
O2/5% CO2. Slices were incubated at room temperature at least 1 h
Electrophysiology. All experiments were performed as described previ-
ously (Misner and Sullivan, 1999). Individual slices were placed in a
submerged recording chamber and were perfused with recording solu-
stimulated every 20 s unless otherwise noted. Stimulus intensity was
field EPSPs were measured for field potential recordings. Data are ex-
pressed as mean ? SEM.
at varying interpulse intervals (20–200 ms). The slope of the response to
the second pulse (P2) was averaged over 5–10 trials and divided by the
a ratio (P2/P1).
Population spikes were recorded in the CA1 pyramidal cell layer, and
the amplitude of the spike from the positive to negative peak was mea-
Bjarnadottiretal.•NRG1ModulationofNMDARPhosphorylationJ.Neurosci.,April25,2007 • 27(17):4519–4529 • 4521
plying pairs of stimuli at an interpulse interval of
10 ms. The paired-pulse ratio (P2/P1) was calcu-
Long-term potentiation (LTP) was induced
first field potential to normalize the data. Te-
tanic stimulation consisted of five trains of 100
Hz stimulation lasting 200 ms at an intertrain
interval of 10 s. Synaptic fatigue was studied by
measuring the synaptic responses during the first
high-frequency train. Theta-burst stimulation
Recombinant NRG1 comprising the entire
some slices at concentrations between 0.1 and
10.0 nM after recording of a stable baseline.
Control slices were treated with vehicle (0.01%
To characterize components of the NRG1
signaling pathway downstream of ErbB4,
(ErbB4i) was used as a bait in a yeast two-
hybrid interaction screen. This bait is ca-
pable of autophosphorylation on tyrosine
in yeast cells. Tyrosine phosphorylation is
absent on ErbB4 baits in which mutations
site, K751A, or the catalytic site, D843A
(data not shown). Screening of a human
hippocampus cDNA library yielded a par-
tial cDNA clone comprising the Src ho-
mology 2 (SH2) and SH3 domains of Fyn (amino acids 60–252,
RefSeq accession number NM_002037). No Fyn clones were iso-
lated when the same cDNA library was screened using ErbB4
K751A as bait, nor did the ErbB4 K751A or D843A bait interact
with Fyn in the yeast two-hybrid growth assay (Fig. 1A). These
results suggest that Fyn and ErbB4 can interact without the need
is dependent on tyrosine phosphorylation of ErbB4, and that the
binding likely involves the Fyn SH2 domain.
a mammalian cell, full-length ErbB4 and Fyn–V5 were tran-
siently coexpressed in COS7 cells. As shown in Figure 1B, these
proteins colocalized to the plasma membrane. This colocaliza-
tion was independent of ErbB4 receptor activation by NRG1,
ylation in the transfected cells (data not shown).
To demonstrate that ErbB4 and Fyn exist in a complex, we
performed coimmunoprecipitations from CHO-K1 cells stably
munoprecipitated using anti-V5 (recognizing Fyn) but not by
control anti-Glu antibodies (Fig. 1C). Fivefold more ErbB4 was
coimmunoprecipitated with Fyn from cells stimulated with
NRG1, suggesting that activation of the receptor facilitates the
recruitment of Fyn into the ErbB4 protein complex. In addition,
immunoprecipitation of ErbB4 coprecipitates Fyn, further con-
firming the association of these two proteins (Fig. 1C). These
results demonstrate that ErbB4 and Fyn can exist in a protein
complex in mammalian cells and that the formation of the com-
plex is facilitated by ErbB4 activation and consequent tyrosine
ErbB4 has been shown to be a component of the postsynaptic
tein kinases, including Fyn and other SFKs, the NMDAR, and
interaction of PSD-95 and ErbB4 has been demonstrated previ-
characterize its binding partners in brain, ErbB4 was immuno-
precipitated from mouse hippocampal lysates, and associated
proteins were analyzed using in-gel tryptic digestion and mass
fragmentation. One associated protein contained a peptide with
modulation of NMDAR function (Huang et al., 2001), also asso-
ciates with ErbB4. This was confirmed by anti-Pyk2 immuno-
blotting of anti-ErbB4 coimmunoprecipitates (Fig. 1D). Pyk2
precipitation of Pyk2 was observed when anti-HA antibodies
were used for immunoprecipitation (Fig. 1D, middle lane).
(arrow) when transiently expressed in COS7 cells and visualized by deconvolution microscopy (n ? 3) and magnification at
1000?. C, Full-length Fyn–V5 and ErbB4 were stably coexpressed in CHO-K1 cells. Fyn–V5 coimmunoprecipitates with anti-
ErbB4 antibodies, as shown by Western blot using anti-V5 antibodies for detection. Stimulation with 7.2 nM NRG1 for 10 min
antibodies for detection, and the interaction is enhanced by NRG1 stimulation of the cells as before (middle). No interaction
4522 • J.Neurosci.,April25,2007 • 27(17):4519–4529 Bjarnadottiretal.•NRG1ModulationofNMDARPhosphorylation
The finding that Fyn and ErbB4 are found in the same protein
complex suggests that Fyn may be a downstream effector of
NRG1. The initial step of NRG1 signaling is activation of ErbB4
kinase and autophosphorylation on tyrosine residues. An in-
crease in ErbB4 tyrosine phosphorylation and associated kinase
exposed to NRG1. After immunoprecipitation of ErbB4, kinase
activity was measured using a peptide substrate. Maximum
ErbB4 kinase activity was detected within 1 min of NRG1 expo-
To determine whether Fyn was activated by NRG1 signaling,
in Fyn phosphorylation. Fyn kinase is inhibited in the basal state
through an intramolecular SH2 interaction with phosphorylated
Y531 and can be activated directly by a second kinase through
phosphorylation on Y420 (Salter and Kalia, 2004). Alternatively,
Fyn can be activated by dephosphorylation of Y531 or through
displacement of the SH2 domain by a second tyrosine-
phosphorylated binding partner, thereby allowing autophos-
phorylation of Y420. NRG1 treatment of HEK293 cells stably
phosphorylation of ErbB4 and activation of the downstream ef-
fector Erk1/2 (p44/42) as evident by elevation of the T202/Y204
phosphorylation (Fig. 3A). To monitor the effect of NRG1 stim-
ulation on Fyn, the changes in phosphorylation levels on Y531
and Y420 were evaluated. Densiometric scanning of the phos-
phorylation bands were made, and a ratio was obtained by nor-
increased in CHO-K1 cells expressing ErbB4 (f) or coexpressing ErbB4 and Fyn–V5 (Œ) in
ErbB4 using a substrate with higher affinity for Fyn. Maximum ErbB4 activity was observed
to NRG1 (n ? 3). B, Fyn potentiates NRG1-induced signal transduction activity as measured
NRG1 activation of ErbB4 regulates Fyn phosphorylation in HEK293 cells coex-
Bjarnadottiretal.•NRG1ModulationofNMDARPhosphorylationJ.Neurosci.,April25,2007 • 27(17):4519–4529 • 4523
ever, NRG1 stimulation leads to a 2.5-fold increase in PY420
phosphorylation. This NRG1-stimulated increase in Fyn Y420
phosphorylation could be attributable to stimulation of Fyn au-
tophosphorylation, to phosphorylation of Fyn by ErbB4, or to
phosphorylation of Fyn by an ErbB4-activated tyrosine kinase
other than Fyn.
These alternatives could be distinguished by using PP2, a rel-
atively selective SFK inhibitor, to block Fyn activity, whereas
ErbB4 signaling was stimulated with NRG1 (Fig. 3B). Fyn inhi-
bition is highly sensitive to PP2 with an IC50of 5 nM. We used as
a control PP3, a protein kinase inhibitor related to PP2 that has
no effect on Fyn or other SFKs. In HEK293 cells coexpressing
ErbB4 and Fyn, Fyn was phosphorylated at a low level on Y420
when treated with PP3. This could be reduced by preincubation
for 16 h with 25 nM PP2, suggesting that Fyn Y420 phosphoryla-
phosphorylation was seen, even under conditions in which PP2
was used to inhibit Fyn autophosphorylation. The control com-
pound PP3 had no effect on Fyn phosphorylation. Thus, ErbB4
or an ErbB4-activated tyrosine kinase can phosphorylate Fyn on
Y420 in response to NRG1 stimulation and activate this kinase
To test whether the observed increase in Fyn Y420 phosphor-
ylation was associated with enhanced catalytic activity, we mea-
sured Fyn kinase activity after NRG1 stimulation (Fig. 2A). Fyn
kinase activity was increased within 5 min of NRG1 stimulation,
lation levels within 15 min. These results confirm that, in mam-
malian cells, Fyn kinase is activated in response to NRG1 signal-
ing through ErbB4 either directly or indirectly through Y420
We observed greater tyrosine phosphorylation of ErbB4 in the
presence of Fyn. This suggested that Fyn might amplify NRG1
signaling by increasing ErbB4 tyrosine phosphorylation. To test
ErbB4 under conditions in which Fyn and related Src-family ki-
nases were inhibited by PP2. PP2 greatly reduced NRG1-
stimulated ErbB4 tyrosine phosphorylation (Fig. 3C), whereas
in ErbB4 phosphorylation was observed after PP2 or PP3 incu-
bation in the absence of NRG1, nor on NRG1-induced tyrosine
phosphorylation in HEK293 cells expressing only ErbB4 (data
not shown). Our results suggest that Fyn is activated in response
to NRG1 signaling and that activated Fyn contributes to ErbB4
sult in the creation of additional SH2 domain docking sites, thus
leading to further activation of Fyn as part of a positive feedback
mechanism involving NRG1, ErbB4, and SFKs.
Because Fyn kinase activity appears to contribute to NRG1-
induced tyrosine phosphorylation of ErbB4, we speculated that
NRG1-dependent signal transduction activity would be en-
we generated a CHO-K1 line expressing only ErbB4 and a sub-
blot. Both lines contained a pSRE–Luc reporter gene sensitive to
MAPK pathway activation. Expression of the luciferase reporter
and Fyn compared with cells expressing only ErbB4 ( p ? 0.05).
No significant differences in baseline luciferase activity or in the
EC50for NRG1 stimulation of reporter gene expression (?1 nM)
were found between the two cell lines. Thus, our results suggest
that Fyn is a downstream amplifier of NRG1 signaling through
NR2A and NR2B subunits of the NMDAR (Tezuka et al., 1999;
Nakazawa et al., 2001; Heidinger et al., 2002). It also has been
reported that NR2B Y1472 phosphorylation is increased after
induction of LTP in the hippocampal CA1 region (Nakazawa et
2003). As described above, we find that the SFKs Fyn and Pyk2
both associate with ErbB4. We therefore tested whether NRG1
signaling, through activation of SFKs, results in changes in the
phosphorylation state of regulatory tyrosine residues on NR2B.
For convenience, we conducted these experiments using
BE(2)-M17 human neuroblastoma cells. These cells have a
neuron-like appearance after differentiation for 72 h in the pres-
blot that BE(2)-M17 express ErbB4, Fyn, Src, Pyk2, PSD-95, and
NR1 (Fig. 4 and data not shown). We further found that RA
express an NMDAR complex containing NR1 and NR2B (Fig. 4
and data not shown). After RA stimulation, BE(2)-M17 cells be-
come sensitive to glutamate-induced excitotoxicity, implying
Retinoic acid-differentiated BE(2)-M17 cells thus provided a
model system in which to examine the coupling of NRG1 activa-
tion of ErbB4 to NMDAR phosphorylation. When differentiated
BE(2)-M17 cells were stimulated with NRG1 for 10 min, we
found increased ErbB4 tyrosine phosphorylation (Fig. 4), in-
4524 • J.Neurosci.,April25,2007 • 27(17):4519–4529 Bjarnadottiretal.•NRG1ModulationofNMDARPhosphorylation
creased Fyn/Src Y420 phosphorylation (indicative of increased
SFK activity), and increased Pyk2 Y402 phosphorylation. Pyk2
modulation of NMDAR function (Huang et al., 2001). Pyk2
might be phosphorylated directly by ErbB4 or Fyn or indirectly
through MAPK pathway activation attributable to NRG1 stimu-
lation. Importantly, we observed a coincident increase in NR2B
Y1472 phosphorylation that is associated with Fyn and Pyk2 ac-
tivation. Thus, NRG1 signaling through ErbB4 modulates NR2B
tyrosine phosphorylation in neuroblastoma cells. We lack the
reagents to distinguish between Fyn and Pyk2 as proximal effec-
tors in BE(2)-M17 cells. Furthermore, because we do not have
information about the expression of other ErbB family members
phosphorylation of NR2B in these cells.
cellular model, we hypothesized that the abnormal behavioral
phenotype seen in NRG1?/?and ErbB4?/?mutant mice (Ste-
fansson et al., 2002) might be related to altered NR2B Y1472
phosphorylation. Indeed, we found that Y1472 was hypophos-
NRG1(?TM)?/?or ErbB4?/?mutant mice compared with age-
and sex-matched controls (Fig. 5A). There was no apparent dif-
ference in total NR2B protein between the mutant and control
mice. As reported by others, NR2B Y1472 phosphorylation was
reduced in Fyn?/?null mice (Nakazawa et al., 2001). Interest-
ingly, Y1472 phosphorylation in both NRG1(?TM)?/?and
ErbB4?/?mice is reduced to the level seen in Fyn?/?null mice,
suggesting that NRG1 is the major regulator of Fyn-mediated
NR2B tyrosine phosphorylation in the hippocampus. Fyn/Src
pared with the controls, suggesting attenuated SFK activity in an
environment of altered NRG1 signaling. Although the anti-Fyn/
Src PY420 antibody reagent used for Western blotting does not
distinguish between Fyn and Src, the signal is virtually absent in
Fyn?/?null mice, suggesting that Fyn is the predominant kinase
in the hippocampal lysate. Although we could not distinguish
between Fyn and Pyk2 as effectors of NR2B Y1472 phosphoryla-
that Fyn plays a dominant role in the hippocampus. Our results
suggest that, in animals deficient in components of the NRG1
signaling pathway, altered regulation of NR2B Y1472 phosphor-
ylation may cause defects in NMDAR function.
We reported previously that the behavioral abnormality of
NRG1(?TM)?/?mutant mice in the open-field test was revers-
ible within 25 min of treatment with clozapine at a intraperito-
neal dose of 1 mg/kg (Stefansson et al., 2002). This dose had no
obvious effect on wild-type controls, although higher doses were
to wild-type and NRG1?/?mice and prepared hippocampal ly-
sates 25 min later. NR2B Y1472 phosphorylation was increased
2.5- to 3-fold when normalized to total NR2B (Fig. 5B, compare
lanes 1, 2). NR2B Y1472 phosphorylation in clozapine-treated
NRG1(?TM)?/?mice was indistinguishable from wild-type
control lysates, regardless of whether the wild-type animals re-
ceived clozapine (Fig. 5B, lanes 2–4). Thus, a dose of clozapine
that reverses the abnormal behavior in NRG1(?TM)?/?mice
restores NR2B Y1472 phosphorylation to normal levels.
Postsynaptic excitatory responses and short-term plasticity were
examined to determine the effects of NRG1 gene deletion on
synaptic transmission. In these experiments, a second NRG1
them, these mice exhibit the same phenotype as the
NRG1(?TM)?/?line, including homozygous lethality, hyperac-
Western blot. Fyn/Src Y420 phosphorylation also was reduced in ErbB4?/?and
loaded per lane. B, Clozapine reverses NR2B hypophosphorylation in NRG1(?TM)?/?mice.
NR2B Y1472 phosphorylation (top) when administered to age- and sex-matched wild-type
Bjarnadottiretal.•NRG1ModulationofNMDARPhosphorylationJ.Neurosci.,April25,2007 • 27(17):4519–4529 • 4525
tivity in the open-field assay, and de-
creased NMDAR expression (Gerlai et al.,
2000 and data not shown). Basal synaptic
responses to increasing stimulation inten-
sities, was not altered in NRG1(?EGF)?/?
mice (data not shown). PPF, a form of
short-term plasticity believed to reflect
presynaptic function (Zucker, 1989), was
measured in both wild-type and heterozy-
gous slices (Fig. 6A). Over all interpulse
intervals tested (10–200 ms), PPF was en-
hanced in slices from NRG1(?EGF)?/?
mice compared with wild type ( p ? 0.05,
two-sample t test). Moreover, these effects
with 1 nM NRG1?1 (Fig. 6B) ( p ? 0.05, one-sample t test). The
rate of synaptic fatigue during high-frequency stimulation, an
additional measure of presynaptic function, was similar between
mission mediated by GABAAreceptors, was tested by evoking
of the first spike. Paired-pulse inhibition was decreased in
NRG1(?EGF)?/?slices (64% compared with 52%) but was not
statistically significant from wild type ( p ? 0.05, two-sample t
test; data not shown). Together, these results suggest that NRG1
gene deletion selectively and reversibly alters paired-pulse facili-
tation, without effects on additional measures of basal
Because previous studies demonstrated that bath application of
NRG1 to hippocampal slices from wild type rats impaired
tetanus-induced LTP (Huang et al., 2000), we next examined the
stimulation (Fig. 7A). Although the mean potentiation 45 min
wild-type slices (123.9 ? 3.4 vs 131.8 ? 4.7%), this was not
LTP induced by tetanic stimulation was not altered in
NRG1(?EGF)?/?slices. Interestingly, LTP induced by theta-
burst stimulation (Fig. 7B), which was also reversed by NRG1 in
compared with controls (116.0 ? 2.8 vs 130.4 ? 4.3%; p ? 0.05,
two-sample t test). These results suggest that NRG1 may differ-
entially regulate synaptic plasticity, depending on the type of
Finally, we examined the effects of acute application of the solu-
ble extracellular domain of NRG1?1 on LTP induced by both
high-frequency and theta-burst stimulation in wild-type and
had no effect on basal synaptic strength at any concentration
tested, in agreement with previous reports (data not shown).
Thirty minute pretreatment with NRG1?1 dose dependently in-
hibited tetanus-induced LTP of wild-type slices (Fig. 8A), with
maximal inhibition at 1 nM (130.1 ? 4.6% for untreated slices vs
102.3 ? 2.1% for treated slices). Similarly, NRG1?1 pretreat-
with complete inhibition at 1 nM (130.4 ? 4.2 vs 106.4 ? 4.6%).
Surprisingly, acute treatment with NRG1?1 had very different
results in NRG1(?EGF)?/?slices. Compared with untreated
slices, tetanus-induced LTP was enhanced in the presence of 0.1
nM NRG1?1 treated) (Fig. 8B). This effect was reversed when
higher concentrations were applied (128.1 ? 10.8% at 1 nM and
112.1 ? 4.3% at 10 nM). Finally, the impairment of theta-burst-
acute treatment of lower concentrations of NRG1?1 but not at
the highest concentration tested (Fig. 8D). The maximal effect
was observed at 1 nM (116.0 ? 2.6% for untreated vs 135.5 ?
7.5% for treated), the same concentration that reversed the ef-
fects on paired-pulse facilitation. NRG1?1 at 0.1 nM slightly en-
gest that LTP can be impaired by either too little or too much
for synaptic plasticity.
are associated with ErbB4, the predominant neuronal receptor
kinase, as well as phosphorylation of Pyk2 on Y402, a site associ-
ated with SFK recruitment. Both kinases have been implicated
previously in the regulation of NMDAR function through ty-
(open circles; n ? 67) compared with wild-type slices (filled squares; n ? 76). B, PPF was measured in untreated
resulting rates were similar between wild-type (filled squares; 5.5 stimuli; n ? 24) and NRG1?/?(open circles; 6.2 stimuli;
not significantly different between wild-type (filled squares; n ? 24) and NRG1(?EGF)?/?
4526 • J.Neurosci.,April25,2007 • 27(17):4519–4529 Bjarnadottiretal.•NRG1ModulationofNMDARPhosphorylation
of SFKs increases NMDAR channel open probability and mean
open time (Yu et al., 1997). The NMDAR plays a key role in the
composition (Liu et al., 2004). Fyn has been implicated in vivo in
the regulation of induction of LTP in mouse hippocampus
(Grant et al., 1992; Kojima et al., 1997; Nakazawa et al., 2001).
own data identifying NRG1 as a gene playing a role in the patho-
test for NRG1-dependent phosphorylation of an important reg-
ulatory site on NR2B, Y1472. Phosphorylation of brain NR2B
Y1472 has been demonstrated previously to increase with induc-
tion of LTP (Nakazawa et al., 2001). Here we show that NRG1
signaling results in phosphorylation of NR2B Y1472 in BE(2)-
M17 human neuroblastoma cells. Furthermore, NR2B Y1472
mutant mice, and synaptic plasticity in the NRG1?/?was altered
with impaired theta-burst LTP and enhanced LTP in response to
NRG1. Both the NRG1?/?and ErbB4?/?strains exhibit modest
behavioral abnormalities (Stefansson et al., 2002), and we
showed previously that the hyperactivity of NRG1?/?mutant
mice in the open-field test was reversible by the atypical antipsy-
ical efficacy of clozapine is attributable in part to its combined
activity on numerous neurotransmitter receptors (Miyamoto et
Y1472 phosphorylation seen in untreated NRG1?/?mice. Thus,
reversal of the behavioral abnormality is associated with restora-
tion of NR2B Y1472 phosphorylation. Interestingly, clozapine
has also been shown to facilitate potentiation of synaptic trans-
mission through increased, NMDAR-mediated, EPSCs (Gem-
perle et al., 2003).
Together, these data suggest to us that NRG1-associated sus-
ceptibility to schizophrenia is at least partly associated with hy-
pofunction of NRG1 signaling through ErbB4, Fyn, and other
associated kinases such as Pyk2, that phosphorylate regulatory
sites on NMDAR subunits, resulting in abnormal modulation of
excitatory glutamatergic neurotransmission.
Modulation of LTP by NRG1 in normal rodents has been
Kwon et al., 2005). All of these investigators found that NRG1
inhibited tetanic LTP in hippocampal slices. In agreement with
theta-burst-induced LTP in wild-type slices. Comparing
NRG1?/?slices and wild-type controls, we do not find any dif-
ference in tetanus-induced LTP but do find a deficit in theta-
burst LTP. The deficits in theta-burst LTP may be in part attrib-
utable to decreased release probability in NRG1?/?slices
indicated by the enhancement of paired-pulse facilitation, such
at 1 nM. Furthermore, NRG treatment decreased paired-pulse
facilitation within the same slices after 20 min (data not shown),
suggesting an increase in release probability similar to wild-type
controls. In NRG1?/?slices, NRG1 dramatically increases
tetanus-induced LTP, demonstrating a clear and significant dif-
ference in synaptic function between wild-type and NRG1?/?
animals. Results in the NRG1?/?mice are consistent with the
hypothesis that hypofunction in the NRG1 signaling pathway
causes a decrement in LTP that can be reversed by restoration of
the NRG1/ErbB4 signaling pathway.
ing, through activation of Fyn and Pyk2, to the NMDAR com-
plex, we are able to incorporate a number of recently published
schizophrenia susceptibility genes into a unified model for this
flux, but not calcium release from other sources, has been shown
to activate the Ca2?-dependent phosphatase calcineurin
(PPP3CC). The gene encoding PPP3CC has been associated pre-
viously with schizophrenia (Gerber et al., 2003). In a negative
feedback loop, a calcium-induced increase in calcineurin phos-
[striatum enriched protein tyrosine phosphatase (PTPN5)]
(Nguyen et al., 2002; Paul et al., 2003). STEP depresses NMDAR
activity in CA1 hippocampal neurons, and its overexpression
blocks induction of NMDAR-dependent LTP, whereas inhibi-
tion of STEP enhances NMDAR-mediated, excitatory neuro-
transmission (Pelkey et al., 2002). Functional activation of the
NMDAR requires the binding of a coagonist, either glycine or
D-serine. D-Serine levels in brain are regulated by D-amino acid
oxidase (DAAO). DAAO, as well as its activator G72, have also
been implicated as genetic risk factors for schizophrenia (Chu-
makov et al., 2002). Our results demonstrating that attenuation
of NRG1 signaling leads to changes in NMDAR-mediated neu-
rotransmission is consistent with genetic data in schizophrenia
that implicates several genes involved in glutamatergic signaling.
The basis for disease susceptibility may be altered adaptability of
with NRG1?1 dose-dependently reversed deficits in theta-burst-induced LTP in
Bjarnadottiretal.•NRG1ModulationofNMDARPhosphorylationJ.Neurosci.,April25,2007 • 27(17):4519–4529 • 4527
the brain to experience and the changes in synaptic efficacy that
underlie such adaptation, attributable to misregulation of multi-
plex. The NRG1?/?mutant mice provide a novel genetic model
of schizophrenia in which a biochemical deficit correlated to
NMDAR channel modulation can be corrected by an atypical
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