287

Neuregulin and ErbB receptor signaling pathways in the

nervous system
Andres Buonanno* and Gerald D Fischbach‡
The neuregulins are a complex family of factors that perform
many functions during neural development. Recent
experiments have shown that neuregulins promote neuronal
migration and differentiation, and regulate the selective
expression of neurotransmitter receptors in neurons and at the
neuromuscular junction. They also regulate glial commitment,
proliferation, survival and differentiation. At interneuronal
synapses, neuregulin ErbB receptors associate with PDZ-
domain proteins at postsynaptic densities where they can
modulate synaptic plasticity. How this combinatorial network —
comprising many neuregulin ligands that signal through distinct
combinations of dimeric ErbB receptors — elicits its multitude
of biological effects is beginning to be resolved.
Addresses
*Section on Molecular Neurobiology, Building 49, Room 5A-38,
National Institutes of Health, Bethesda, Maryland 20892-4480, USA;
e-mail: buonanno@helix.nih.gov
‡ Columbia University, College of Physicians and Surgeons,
630 West 168th Street, New York, New York 10032, USA
Current Opinion in Neurobiology 2001, 11:287–296
0959-4388/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Much of our current knowledge about NRG signaling
pathways in the nervous system originates from studies of
EGF and NRG actions in non-neural cell types. At present,
four genes encoding NRGs have been identified in verte-
brates (Figure 1). Alternate RNA splicing further enhances
the complexity of this EGF-like family. NRG-1–4 bind
preferentially to the ErbB-3 and -4 receptors, which then
form homo- or heterodimers by recruiting either ErbB-1 or
-2 co-receptors to propagate signaling (see [1]). ErbB-2 is
thought to be an important co-receptor on the basis of
in vivo and in vitro experiments.
Like other factors signaling through receptor tyrosine
kinases (RTKs), NRG binding to its receptor promotes
tyrosine cross-phosphorylation, the association of proteins
containing phosphotyrosine-binding or Src homology-2
domains with the phosphorylated residues, and the activa-
tion of intracellular effector pathways that mediate the
specific biological responses. Notably, many of the effector
molecules used by NRGs in the mammalian nervous sys-
tem are related to those used by the EGF ligands in
Drosophila and Caenorhabditis elegans.

Abbreviations
In this review, we will summarize the latest findings
AChR acetylcholine receptor regarding the spectrum of NRG actions and the signaling
Cdk5 serine/threonine cycline-dependent kinase 5 pathways used in neurons, glia and at muscle synapses. We
CRD cysteine-rich domain will discuss studies that reveal how different biological
EGF epidermal growth factor
ERBIN erbB-2 interacting protein
mechanisms have evolved to regulate the complexity and
FAK focal adhesion kinase specificity of the NRG–ErbB signaling pathway. These
GABP GA binding protein studies are beginning to address why so many NRG iso-
MAGUK membrane-associated guanylate kinase forms are generated, and how distinct isoforms elicit
MAPK mitogen-activated protein kinase
specific biological responses.
NMDA N-methyl D-aspartate
NMJ neuromuscular junction
NRG neuregulin Structural similarities between NRGs
PI3K phosphatidylinositol-3-OH kinase To date ten genes have been found to encode EGF or
PICK-1 protein kinase C interacting protein EGF-like ligands in vertebrates (see Figure 1b). The
PSD postsynaptic density
RTK receptor tyrosine kinase EGF-like domain of NRG is required for receptor binding,
TM transmembrane and on its own is sufficient to elicit ErbB receptor dimer-
ization, tyrosine phosphorylation and the activation of
Introduction downstream signaling pathways. This domain contains
The neuregulins (NRGs) are a family of proteins containing roughly 50 amino acids and is characterized by three pairs
an epidermal growth factor (EGF)-like motif that activates of cysteines that are important for its tertiary structure and
membrane-associated tyrosine kinases related to the EGF biological function. As yet, the criteria to classify an EGF-
receptor (known as ErbB-1). Initially, NRG-1 was found to like ligand as an NRG have not been clearly defined;
function in the nervous system in the proliferation of Schwann however, the NRG-1–4 subfamily of proteins share high
cells, and in the regulation of nicotinic acetylcholine receptor sequence homology in their EGF-like domain that distin-
(AChR) transcription at the neuromuscular junction (NMJ). guishes them from other EGF ligands (Figure 1b). The
More recently, the NRGs have been found to regulate early diversity of the NRG ligands is further increased by alter-
fate determination, differentiation, migration and survival of nate RNA splicing and promoter usage (reviewed in [2,3]).
satellite cells, Schwann cells and oligodendrocytes. In neu-
rons, NRGs promote neuronal migration, and selectively Although the EGF-like motif can mimic most of the
increase the expression of other neurotransmitter receptors. biological effects of the full-length protein, the other
288 Signalling mechanisms

Figure 1

Structure and homology of NRG ligands.

(a) Four genes encode NRG-1, -2, -3 and -4 in (a)
NRG-1 NRG-2 NRG-3 NRG-4
vertebrates. The diversity of NRGs is further
increased by alternate RNA splicing and
promoter usage (see [2,3]). The three types of
NRG-1 are characterized by different
extracellular domains. Type I NRG-1 has an
immunoglobulin-like (IgG-like) domain, followed
by a region high in glycosylation; Type II has a ++ ++ ++ ++
++ ++ ++ ++
GGF-specific (‘Kringle’) domain and a IgG-like
domain; and Type III has a cysteine-rich domain
(CRD). Downstream from the variable amino-
terminal region, the transmembrane (TM) forms
of NRG contain the following: an EGF-like
domain, with alternate splice variants at the
most carboxy-terminal region to give α and β
isoforms; a juxtamembrane region carrying
protease-cleavage sites; a TM domain; and a
long cytoplasmic tail. EGF-domain β isoforms
are prevalent in the nervous system, whereas α
isoforms are found in mesenchyme [85]; both Type I Type II Type III
variants display different ErbB receptor-binding
affinities. Splice variants missing the TM
IgG-like Cysteine-rich EGF-like Transmembrane
domain (which are presumably secreted
directly) have been cloned for all NRG-1 Kringle ++ Glycosylation sites EGF-α/β Protease cleavage
isoforms. Type III NRG-1 has an additional
membrane association domain which results in (b)
NRG α/β domains
a membrane-bound ligand with reverse
orientation [59,60••]. NRG-2 is most closely NRG-1α HLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCQPGFTGARCTENVPMKV
related to NRG-1; splicing also generates α NRG-1β HLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASF
and β EGF-domain isoforms. NRG-3 and -4 NRG-2α HARKCNETAKSYCVNGGVCYYIEG---INQLSCKCPNGFFGQRCLEKLPLRL
are most distantly related, and to date no splice NRG-2β HARKCNETAKSYCVNGGVCYYIEG---INQLSCKCPVGYTGDRCQQFAMVNF
variants have been found. NRG-3 has a region NRG-3 HFKPCRDKDLAYCLNDGECFVIETLT-GSHKHCRCKEGYQGVRCDQFLPKTD
rich in potential O-linked sugars, whereas NRG-4 HEQPCGPRHRSFCLNGGICYVIPT---IPSPFCRCIENYTGARCEEVFLPSS
NRG-4 has short amino- and carboxy-terminal
domains. (b) Alignment of the human EGF-like EGF SDSECPLSHDGYCLHDGVCMYIEA---LDKYACNCVVGYIGERCQYRDLKWW
domains in NRGs and other EGF-related TGF-α HFNDCPDSHTQFCFH-GTCRFLVQ---EDKPACVCHSGYVGARCEHADLLAV
ligands. HB-EGF, heparin-binding EGF; HB-EGF KRDPCLRKYKDFCIH-GECKYVKE---LRAPSCICHPGYHGERCHGLSLPVE
EPIR, epiregulin; BTC, betacellulin; EPIR QITKCSSDMDGYCLH-GQCIYLVD---MREKFCRCEVGYTGLRCEHFFLTVH
APR, amphiregulin/Schwannoma-derived BTC HFSRCPKQYKHYCIK-GRCRFVVA---EQTPSCVCDEGYIGARCERVDLFYL
growth factor. The α and β splice variants for APR KKNPCNAEFQNFCIH-GECKYIEH---LEAVTCKCQQEYFGERCGEKSMKTH
NRG-1 and -2 are boxed; variants for NRG-3
Loops A B C
and -4 have not been described. The EGF-like
domains of the EGF superfamily have three
conserved cysteine pairs (green), which form a (c) Ectodomain Transmembrane domain
three-loop (A, B, C) structure necessary for NRG-1 KHLGIEFMEAEELYQKRVLTITGICIALLVVGIMCVVAYCKTKKQRK
proper conformation. The position of amino NRG-2 LYMPDPKQKAEELYQKRVLTITGICVALLVVGIVCVVAYCKTKKQRK
acids conserved in all NRGs is marked in red; NRG-3 DHLGIEFMESEEVYQRQVLSISCIIFGIVIVGMFCAAFYFKSKKQAK
a conservative substitution is in yellow. The
spacing of the six cysteines, in combination NRG-4 CEEVFLPSSSIPESNLSAAFVVLAVLLTLTIAALCFLCRKGHLQRAS
with other conserved residues
Current Opinion in Neurobiology
(HX3CX7CXN(G/D)GXCX10–13C(K/R)CX5G
XRC), distinguishes NRGs from the other EGF
ligands that preferentially bind the ErbB-1 NRG-1 has the highest sequence homology juxtamembrane ectodomain. NRG-4 has no
receptor. (c) Alignment of NRG sequences in with NRG-2 in the 23-amino-acid TM domain significant homology in the TM or
the TM domain and adjacent ectodomain. (92% identical) and with NRG-3 in the juxtamembrane regions with NRG-1, -2 or -3.

domains can contribute to specific biological functions
(see below). Figure 1 shows schematically the four
NRGs with the conserved EGF-like motif, and the dif-
ferent structural domains that distinguish the distinct
types of NRG-1 isoforms (Types I–III). NRG-1 and -2
are closely related, whereas the sequence and predicted
structure of NRG-3 and -4 are distantly related to those
of NRG-1.
The differential regional and temporal expression of
NRGs suggests specific biological roles for each isoform.
Nrg-1, the first gene to be cloned and extensively charac-
terized, shows the broadest expression pattern during
embryonic development and in adulthood. NRG-1 is
expressed in neurons and glia, as well as in heart, liver,
stomach, lung, kidney, spleen and skin [4]. Expression of
NRG-2–4 has been analyzed mainly in adult tissues.
 Neuregulin and ErbB receptor signaling pathways Buonanno and Fischbach
NRG-2 ([5,6]; also known as Don-1 [7] and NTAK [8]) is
expressed mostly in central nervous system neurons and
heart, whereas NRG-3 [9] expression is confined to the
nervous system. In contrast, NRG-4 transcripts are detect-
ed in adult pancreas and skeletal muscle but not in neural
tissues [10•]. The distinct expression patterns of NRG-1–3
in adult brain suggests that they are involved in numerous
functions, including the regulation of neuronal plasticity.
Spectrum of NRG functions and signaling
pathways in different cell types
Signaling in neurons
Neurons express different combinations of NRG-1–3 and
ErbB receptors during development. Unfortunately, tar-
geted null mutations of Nrg-1, erbB-2 and erbB-4 result in
an embryonic-lethal cardiac phenotype at about embryon-
ic day 10 (E10), thereby impeding the study of these
factors in later neural development. Nonetheless, early in
development, NRG-1 and ErbB-2 null mice have reduced
numbers of cranial sensory neurons, and ErbB-4-deficient
mice have abnormal targeting of cranial sensory and motor
axons (see [1,3]).
Roles for NRG-1–ErbB signaling later in neural develop-
ment have been shown in mouse model studies of an
ErbB-3 null mutation [11], selective mutations of NRG-1
ectodomains [12••,13], an ErbB-2 null mutation rescued by
transgenic expression of ErbB-2 in cardiac myocytes
[14••,15••] and Cre-targeted ErbB-2 ablation in Schwann
cells [16•]. In all of these mice, peripheral motorneuron
axons defasciculate as they enter the muscle mass and fail
to form mature neuromuscular junctions. Different num-
bers of motor and sensory neurons die, probably an
indirect result of the lack of trophic support provided by
Schwann cells [17••].
A novel function of NRG-1 in regulating proliferation of
neuroepithelia and differentiation of retinal ganglion neu-
rons was revealed by circumventing early lethality using
viral delivery of a ribozyme that selectively degrades
NRG-1 mRNA [18]. These experiments emphasize the
importance of NRG-1–ErbB signaling for neuronal survival
and differentiation. Targeted mutations of NRG-2 and -3,
and conditional ErbB knock-outs in neurons, might provide
insights on other NRG functions in the developing brain.
In vitro studies have suggested additional functions of
NRG signaling in neuronal development. NRG stimulates
neurite outgrowth in PC-12 cells, dorsal root ganglia neu-
rons and cerebellar granule cells [19,20]. The migration of
cerebellar and cerebral cortical neurons along radial glia
shows a dependence on NRG-1 and the activation of ErbB
receptors [21,22].
As initially described in skeletal muscle in vitro, NRG sig-
naling regulates the expression or function of ligand- and
voltage-gated channels in central neurons. In organotypic
cerebellar slices, NRG-1 (Type I) selectively induces
289
N-methyl D-aspartate (NMDA) receptor 2C subunit
expression, whereas no effect is observed in 2B subunit
expression [23,24]. A similar NRG isoform increases the
expression of the β2 subunit of the γ-amino butyric acid
(GABA) receptor in dissociated granule cells [20]. NRG-1
Type I and Type III isoforms have different potencies in
regulating neuronal AChRs in developing chick sympa-
thetic ganglia. Both isoforms are equipotent in increasing
mRNAs encoding the α5, α7 and β4 subunits; however,
Type III more potently upregulates α3 transcripts [25].
NRG-1 may also have post-translation effects on ion chan-
nel function. In maturing ciliary ganglia neurons, NRG-1
functions synergistically with transforming growth factor-β
to increase currents elicited by calcium-activated potassi-
um channels — an effect that is not blocked by the protein
synthesis inhibitor anisomysin [26]. In all the experiments
discussed above, application of NRG-1 to cultured postsy-
naptic cells mimics the effects elicited by presynaptic
terminals, which normally express NRG-1, suggesting that
NRGs have similar functions during normal development.
At present, little is known of NRG signaling pathways in
neurons. In adult brain, ErbB-2 and -4 receptors accumu-
late in regions enriched in dendritic processes [27], and in
cultured neurons NMDA and ErbB receptors accumulate
at synaptic puncta [28••]. Moreover, ErbB receptors are
enriched in preparations of postsynaptic densities (PSDs).
A consensus binding site in the carboxy-terminal end of
ErbB-4 directly interacts with the PDZ domains of mem-
brane-associated guanylate kinases (MAGUKs) found at
PSDs, and with β2 syntrophin, which harbors a single PDZ
domain. β2 syntrophin is an extrinsic protein enriched at
postsynaptic sites at the neuromuscular junction and other
cellular junctions, and associates with the dystrophin com-
plex. The association between ErbB-4 and the MAGUK
PSD-95 occurs in vivo and in vitro, and potentiates NRG
signaling by facilitating dimerization of ErbB [28••,29••].
The colocalization of NMDA and ErbB receptors at PSDs,
where they interact with the same family of MAGUKs, may
serve to recruit src-related tyrosine kinases (see [28••]).
Recently, Tezuka et al. [30] have shown that PSD-95, which
interacts with NMDA and ErbB receptors, recruits fyn
kinase to a complex and promotes the tyrosine phosphoryla-
tion of the NR2A subunit. This kinase has been implicated
in long-term potentiation at CA1 synapses [31]. Thus, local
NRG-mediated signaling cascades at PSDs might con-
tribute to synaptic plasticity, as recent experiments suggest
[29••]. Future targeted mutations of NRGs may identify
other roles in activity-dependent synaptic plasticity.
Signaling at the NMJ synapse
Muscle nicotinic AChR genes remain the most extensively
studied targets of NRG action (see [1,3]). Developing mus-
cles and cultured myotubes express ErbB-2–4 receptors on
their surface and, like AChRs, ErbBs localize to the NMJ
by an agrin-dependent mechanism (see [32], and also
Update). But in contrast to AChRs, agrin and MuSK (the
290 Signalling mechanisms

Figure 2

Signaling pathways used in muscle and

Drosophila. Extraordinary evolutionary (a) (b)
Star Rhomboid
conservation exists in signaling pathways Spitz
used downstream of ErbB and Drosophila
EGF receptor (DER) activation by EGF-like Sevenless
ligands. (a) This NRG/ErbB signaling NRG-1 Argos
Vein
pathway in muscle was drafted from results Sevenless
obtained in cultured myotubes and in vivo. It ErbB receptors
2 3 4 2 DER receptor
depicts activation of ErbB receptors by Grb2 Shc
different NRG-1 (Type I and III) isoforms; Drk Shc Drk
association of adaptor proteins (Grb2 and
Shc) with tyrosine-phosphorylated
Ras Ras
receptors; activation of the MAPK pathway SHP2
and the SHP2 tyrosine phosphatase; Raf Csk Raf Csk
phosphorylation of the GABP Ets
transcription factors; and transcriptional MAPK MAPK
upregulation of AChR and utrophin genes.
(b) Signaling pathways used by the RTKs
Nucleus
(DER and Sevenless receptor) in P P
P P
Drosophila. Sevenless is not a member of
Pnt Pnt
the EGF-like ligand family, but this RTK uses GABP-β GABP-α
similar pathways to Vein and Spitz, which
Ets site
are both members. Spitz is synthesized as a N box E box
Yan
TM protein that requires the concerted (Ets site)
actions of the transmembrane proteins Current Opinion in Neurobiology
Rhomboid and Star for its release.
Stimulation of these RTKs recruits adaptor Corkscrew (Csk) as an inhibitory stimulates the activity of the Ets
proteins Shc and Drk (Grb2 homologue), feedback loop. The adaptor proteins factors Pointed (Pnt; activator) and
and activates the tyrosine phosphatase activate the MAPK signaling pathway, which Yan (inhibitor).

agrin receptor), which are localized to the top of the junc-
tional folds, ErbB-2, ErbB-4 and NRG-1 (Type I) are found
at the depths of the secondary junctional folds [33••]. This
remarkable segregation between the aggregating and
inducing activity of the AChR was not previously appreci-
ated, and suggests that the signaling pathways are distinct.
NRG-1 increases the transcriptional rates of AChR genes
in vitro (see [3,32]), and at synaptic nuclei in vivo [34].
Adult mice heterozygous for a mutation ablating the IgG-
like domain of Nrg-1 express roughly half the normal
number of junctional receptors and manifest smaller
miniature endplate potentials [34]. Mice with targeted
ablations of the Type III cysteine-rich domain (CRD) of
Nrg-1, erbB-2 (rescued by transgenic expression of ErbB-2
in heart) and erbB-3 express AChRs in a broadened ‘synap-
tic’ region in the central part of muscle fibers during
development, despite the extensive defasciculation and
sprouting of motorneuron axons [12••,14••,15••]. Levels of
AChR expression in the ‘synaptic’ zone may be reduced
[14••] and postsynaptic secondary folds appear stunted
[35]. These studies suggest that NRG-1 regulates different
aspects of NMJ formation, maturation and maintenance
during fetal and postnatal development.
Recently, pathways leading from NRG-1 binding to the
selective transcriptional regulation of AChR genes at the
NMJ have been identified (Figure 2a). In muscle, the acti-
vation of ErbB tyrosine phosphorylation by NRG-1 causes
association with the adaptor protein Shc [36], stimulation
of the Ras/Raf/ERK mitogen-activated protein kinase
(MAPK) pathway, and transcriptional upregulation of the
AChR δ and ε subunit promoters in cultured cells [32].
The administration of NRG-1 to cultured myocytes also
stimulates the tyrosine phosphatase SHP2 [37•], which
results in increased levels and phosphorylation of the Ets-
domain transcription factor GA-binding protein (GABP)-α,
which heterodimerizes with GABP-β, and increases tran-
scription of the δ and ε subunits of the AChR [38,39].
Several groups have identified independently a DNA ele-
ment (currently known as the ‘N box’) that is bound by Ets
transcription factors and may account for the synaptic tran-
scription of AChR δ and ε subunit genes [38–40]. Moreover,
Nichols et al. [41] have identified an N-box mutation in the
ε subunit promoter in a patient with congenital myasthenic
syndrome with reduced AChR expression. The utrophin
gene, which is expressed at the NMJ and regulated by NRG,
also requires N-box elements for its function [42•,43•].
Consistent with these findings, inhibition of GABP func-
tion in vivo results in reduced expression of the AChR ε
subunit, utrophin and acetylcholinesterase — all compo-
nents of the NMJ [44••]. These results suggest that the
regulation of Ets transcription factors by NRGs may con-
stitute a transcriptional code for muscle ‘synaptic’ genes.
Notably, the NRG signaling pathways identified in muscle
are analogous to RTK pathways used by a number of dif-
ferent cell types in Drosophila (Figure 2b), indicating the
evolutionary importance of this signaling cascade.
 Neuregulin and ErbB receptor signaling pathways Buonanno and Fischbach
Signaling in glial cells
In the central and peripheral nervous system, NRGs sig-
naling is of central importance for glial cell survival,
proliferation and differentiation (reviewed in [45,46]).
Schwann cells and oligodendrocytes express ErbB recep-
tors, as well as NRGs. In vitro and in vivo studies have
shown the importance of NRG-1 in directing neural crest
differentiation towards a glial fate. By E10.5, mice carrying
targeted mutations in NRG-1 and ErbB-2 show a severe
reduction in the number of cells destined to become
Schwann cells [45,46].
Recent studies using mutant mice that circumvent the car-
diac-related lethality and survive up to birth demonstrate
the importance of distinct NRG-1 isoforms and ErbB
receptors for Schwann cell precursor proliferation, migra-
tion and survival [11,12••,13,14••,15••,16•]. These mice
manifest an absence or severe reduction in Schwann cell
precursors, which normally accompany the peripheral sen-
sory and motorneuron axons. The axonal defasciculation
and loss of motorneurons observed in the mutant mice may
be attributed to the lack of Schwann cell-derived trophic
factors [17••]. NRG signaling is also required later in
development for promoting or maintaining the adequate
levels of Schwann cell myelination, as shown by a recent
target mutation using ErbB-2Krox-Cre mice [16•].
Although cells destined to become Schwann cells require
nerve-derived NRG for proliferation and differentiation in
the embryo, during regeneration of adult peripheral nerves,
NRGs may serve as autocrine mitogens to expand cell num-
ber in absence of axons [47]. Analogous to its effects in
Schwann cells, NRG-1 treatment in vitro modulates
oligodendroglial fate [48••], induces mitogenesis of
oligodendrocyte precursors, and promotes survival [49••].
Because these studies have mostly analyzed the effects of
NRG-1 using cultured cells, it will be important to deter-
mine in vivo how NRG-1, as well as the more neural-specific
NRG-2 and -3, affect oligodendrocyte development.
The intracellular signaling pathways activated by NRGs in
glial cells have begun to be resolved. NRG signaling in
developing and regenerating Schwann cell is mediated by
ErbB-2 and -3 receptors, and the loss of Schwann cells in
mice lacking these receptors indicate that their function is
essential and is not compensated in vivo by either ErbB-1
or -4 receptors. ErbB receptor dimerization and cross-phos-
phorylation results within seconds in the recruitment of
the adaptor protein Grb2 and the focal adhesion kinase
(FAK) [50,51]. The presence of FAK in the leading edge of
Schwann cell processes, and the observation that NRG
promotes migration of these cells [52], suggest that
FAK–ErbB receptor signaling may promote cell motility in
Schwann cells. Type II NRG-1 (GGF) and axonal contact,
which promote Schwann cell proliferation and survival
[53,54], activate both the phosphatidylinositol-3-OH
kinase (PI3K) and MAPK pathways. Whereas both of
these pathways contribute to GGF-mediated Schwann cell
291
survival, only the PI3K pathway is necessary for the axon-
mediated Schwann cell proliferation, survival and
myelination in culture [55•].
Studies of the intracellular pathways downstream of ErbB
receptor activation in oligodendrocytes have been more
limited. There have been apparent differences reported
for the combination of ErbB-2–4 receptors expressed in
oligodendrocytes O2A (+) progenitors [50,56,57], which
might result from culture differences or from the dynamic
regulation of receptors in these cells. As with Schwann
cells, PI3K pathway signaling through the serine/threonine
kinase Akt is implicated in the NRG-dependent survival
of oligodendrocytes [58]. The effectors downstream from
Akt activation or the structural genes regulated by NRGs
in glial cells remain unknown. It will be interesting to
determine whether the activity of the transcription factors
SCIP and Krox-20 (EGR-2) are modified by NRGs, given
the central role that these factors have in many aspects of
Schwann cell development.
Mechanisms enhancing NRG diversity
and specificity
NRG and ErbB diversity
Duplications of an ancestral gene have resulted in at least
four NRG genes in mammals, and alternate use of transcrip-
tional promoters and splice sites markedly increases the
number of NRG isoforms derived from these loci. Although
the EGF-like domain alone is sufficient to elicit ErbB phos-
phorylation and mimic many of the biological effects of the
full-length factors, the importance of the other NRG struc-
tural domains are beginning to be identified. For example,
the ectodomains of NRG-1 can determine whether the iso-
forms are expressed as transmembrane proteins or released
surface [59,60••], thus influencing their range of action (see
Figure 1). Moreover, mice carrying targeted mutations in the
amino-terminal regions of NRG-1, in the IgG-like domain
[13] or CRD [12••] manifest different phenotypes.
Surprisingly, NRG-1 has the highest homology with NRG-2
in the transmembrane (TM) domain (92% identical), and
with NRG-3 in the juxtamembrane domain (80% identical);
there is little sequence similarity in the cytoplasmic
domains. Although specific functions have not been
assigned to the TM domains, sequences in the cytoplasmic
tail of NRG-1 (Type I) regulate its proteolytic cleavage and
release [61,62]. As shown in Figure 2b, processing of the
Drosophila EGF-like ligand Spitz — the homologue of
transforming growth factor-α — suggest how interactions
between TM, juxtamembrane and cytoplasmic domains
may regulate pro-NRG processing in mammals [63].
Interactions of both the TM and cytoplasmic domains of
Spitz with Rhomboid (a putative seven TM domain protein)
and Star (a single-pass TM protein) are necessary for its pro-
teolytic release and subsequent activation of the EGF
receptor (DER). Rhomboid homologues have been cloned
in mammals, but their potential roles in processing EGF-
like ligands have not been determined.
292 Signalling mechanisms
The cellular distribution, tyrosine kinase activities and
NRG-binding affinities differ among the ErbB receptors.
Alternatively spliced or truncated transcripts have been
observed for all ErbB receptors, but in few cases have these
transcripts been shown to generate protein products or to
affect receptor function. An alternatively spliced ErbB-4
isoform, which lacks a consensus PI3K-binding site, fails to
promote cell survival and chemotaxis [64•]. ErbB-4 splice
variants (which vary in sequence at the extracellular jux-
tamembrane domain) are found in neurons, and have
different susceptibilities to proteolytic cleavage by the
tumor necrosis factor-α-converting enzyme in response to
phorbol ester and pervanadate stimulation [65•].
Evidence indicates that NRG-binding proteins or co-recep-
tors may increase the availability or specificity of
ligand-receptor interactions [66,67]. Heparin sulfate proteo-
glycans bind NRG-1 isoforms with the IgG-like domain,
which may serve to sequester these ligands to specific
subcellular regions. In Schwann cells, a transmembrane
glycoprotein (CD44), which is constitutively associated with
ErbB-2 and -3 receptors functions as a co-factor that
enhances NRG signaling [67]. Thus, the diversity of ligands
and receptors, and their interaction with co-receptors, are
mechanisms that enhance the signaling complexity and
specificity of this ligand–receptor network.
Different affinities of NRGs for distinct ErbB receptor
combinations
Various mechanisms have been adopted to increase the sig-
naling specificity of the NRG–ErbB network. Although
the EGF-like domains of NRGs are similar, the binding
specificities and affinities of distinct isoforms are different
for the various combinations of ErbB receptors. NRG-1
and -2 bind directly to ErbB-3 and -4 [6], whereas NRG-3
and -4 bind preferentially to the ErbB-4 receptor [9,10•].
Studies analyzing the binding of NRG-1–3 to ErbB homo-
and heterodimerized receptors expressed in transfected
myeloid cells [68], or to dimers of the soluble extracellular
regions of the receptors [69•], have demonstrated a wide
range of NRG specificities and affinities for the different
ErbB receptor dimers. In addition, specific NRG isoforms
favor the formation or the stabilization of different ErbB-1–4
heterodimeric receptors on the cell surface [70,71].
NRGs binding to the same ErbB receptor dimers elicit
different signals
The same combination of ErbB receptors can discriminate
among different NRG gene products and their splice
variants to activate distinct intracellular pathways
[6,68,72,73,74••]. For example, in response to NRG-1, -2
and -3 binding, cells engineered to express single ErbB
receptor isoforms are phosphorylated in different tyrosine
residues, resulting in the recruitment of distinct adaptor
proteins. Moreover, the distinct NRG-1 and -2 isoforms (α
and β) selectively activate or modulate the kinetic proper-
ties of different kinase signaling pathways [68,73,74••].
The interpretation of many of these studies many be lim-
ited, however, because only the EGF-like domains of
NRGs were used. This issue may be important because, as
discussed above, other sequences in the NRG ectodomain
contribute to functional specificity in vivo and in vitro.
Nonetheless, these results have implications that are espe-
cially important for central neurons, because they receive
numerous inputs that can express different NRG proteins.
Association of ErbB receptors with signaling microdomains
The subcellular distribution of ErbB receptors indicates
that they are present at microdomains associated with
caveolae and PSDs — regions that facilitate cross-talk
between distinct signaling pathways [75–77]. In cardiac
myocytes, the ErbB-4 receptor is associated with
caveolin-3, but translocates out of caveolae minutes after
NRG-1 (GGF) treatment [78]. Receptor translocation from
microdomains and/or proteolytic cleavage [79] provide
mechanisms for ligand-induced desensitization. ErbB
receptors have also been found at PSDs where they inter-
act with MAGUKs [28••,29••].
Studies in C. elegans suggest how ErbB receptors may be tar-
geted to synapses. The trafficking, targeting and/or
localization of the C. elegans ErbB receptor homologue
(LET-23) to basolateral membranes — a region important
for signaling — requires the products of three genes,
LIN-2, -7 and -10 [80]. The mammalian homologues of
these genes were cloned recently, and the proteins found to
be expressed in neurons. The proteins LIN-2/CASK [81],
LIN-7/Velis/MALS [82] and LIN-10/Mint/aX11 [83] all
contain multiple PDZ domains. The PDZ domain of LIN-7
interacts with the carboxy-terminal tail of the LET-23
receptor carrying the consensus interaction domain (T-C-L).
MALS-1 and -2 interact with PSD-95 and NMDA receptors
[82], and CASK and Mint are found in intracellular dendritic
compartments where ErbB-4 immunoreactivity is observed
(see [28••]), which suggests that this family of proteins may
target ErbB-4 receptors to PSDs (see Update).
Conclusions
The NRG family exerts a multitude of biological effects in
the nervous system, often with seemingly opposing actions
(proliferation versus differentiation), by signaling through a
limited number of ErbB receptors. Our knowledge of the
complexity of NRG ligands and their receptors, and the dis-
tinct mechanisms that they use to generate signaling
specificity, has increased greatly in the past few years as a
result of experiments performed in numerous cell types and
organisms. Studies of the prototypical EGF signaling path-
way in Drosophila and C. elegans predict that additional
proteins (see [84]), controlling processes from ligand avail-
ability and receptor binding to feedback loops that terminate
ErbB signaling, remain to be identified in mammals.
The use of proteomics and cDNA arrays promises to enhance
our understanding of how distinct NRGs promote specific
protein interactions and regulate target genes in different cell
 Neuregulin and ErbB receptor signaling pathways Buonanno and Fischbach
types. The fact that the nervous system expresses the widest
variety of NRGs, and that ErbB receptors colocalize with
neurotransmitter receptors and channels at interneuronal
synapses, suggest that cross-talk between these different sig-
naling systems may serve to couple synaptic activity to
changes associated with synaptic plasticity.
Update
Recent experiments demonstrate the importance of the
serine/threonine cylin-dependent kinase 5 (Cdk5), and its
required co-activator p35, in the NRG-dependent regula-
tion of AChRs [86••]. Cdk5 and p35 are highly expressed
in embryonic muscles, and while their levels decrease with
development, both proteins concentrate at the NMJs.
Cdk5/p35 associate with and phosphorylate serine residues
in ErbB receptors on muscle membranes. This kinase
activity is important, because Cdk5 inhibition attenuates
NRG-induced AChR mRNA expression in cultured
myotubes and overexpression of p35 increases AChR sub-
unit transcription independently from NRG treatment.
These results implicate the Cdk5/p35 kinase pathway for
NRG signaling at the neuromuscular synapse.
Similar to the association of PSD95 with ErbB-4 [28••],
ERBIN (ErbB-2 interacting protein) interacts specifically
with the C-terminus of ErbB-2 receptors via its single PDZ
domain and functions to retain and/or target ErbB-2 to baso-
lateral membranes of epithelial cells [87,88•]. The precise
eight amino acid C-terminal sequence in ErbB-2 required for
ERBIN binding was recently determined and is also found
at the C-termini of NMDA receptor NR2C and NR2B sub-
units, and the potassium channel Kv1.4 [88•]. In epithelial
cells, ErbB-2 also interacts with a different PDZ-domain pro-
tein known as PICK-1 (protein kinase C interacting protein),
and ERBIN oligomerizes with PICK-1 [88•]. In neurons,
PICK-1 interacts with glutamate (AMPA subtype) and
ephrin receptors at PSDs, and ErbB-2 receptors accumulate
at synaptic puncta ([27,28••]; A Buonanno, unpublished
data). It is interesting to speculate, based on the oligomeriza-
tion of ERBIN and PICK-1 in epithelial cells [88•], and
analogues to the association of NMDA and ErbB receptors
with PSD-95 [28••,29••], that the interactions of ErbB-2,
PICK-1 and ERBIN may regulate the AMPA receptor traf-
ficking associated with synaptic plasticity.
Acknowledgements
We thank Dorothy Turetsky and Steven Carroll for their helpful comments
on the manuscript, and K Vasudevan and S Kinsley for assistance
with figures.
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