Neuron, Vol.
18, 847–855, June, 1997, Copyright 1997 by Cell Press
Neuregulins and Their Receptors:
A Versatile Signaling Module
in Organogenesis and Oncogenesis
Steve Burden* and Yosef Yarden†
* Molecular Neurobiology Program
Skirball Institute
New York University Medical Center
New York, New York 10016
† Department of Molecular Cell Biology
The Weizmann Institute of Science
Rehovot 76100
Israel
There has been rapid growth in our understanding of
neuregulins (NRGs), a large group of structurally related
polypeptide factors, and their receptors, four transmem-
brane tyrosine kinases of the ErbB family. This new
knowledge reveals a complex signaling system, used in
different tissues to regulate cell survival, proliferation,
and differentiation. Only 5 years ago, independent ef-
forts to identify activities responsible for stimulating
Neu/ErbB-2 phosphorylation, Schwann cell proliferation,
and acetylcholine receptor synthesis resulted in the
striking discovery that the peptide factors, Neu differen-
tiation factor (NDF/heregulin), glial growth factor (GGF),
and acetylcholine receptor inducing activity (ARIA) were
the products of the same gene. The name neuregulin,
originally introduced by Mark Marchionni, represents
a composite of the initial names and recognizes the
importance of these factors in the nervous system. In-
tensive efforts have now clarified not only an enormous
functional diversity of the NRG-ErbB signaling network,
but also its role in midgestation organogenesis as a key
determinant in differentiation of several cell lineages
(Figure 1). Instructive illustrations of this diversity were
provided by presentations at a recent workshop orga-
nized in the Banbury Center at the Cold Spring Harbor
Laboratory by Mark A. Marchionni (Cambridge Neuro-
Science) and Greg Lemke (Salk Institute, La Jolla, CA)
on “Neuregulins and Neuregulin Receptors.”
Neuregulins (NRGs) and their receptors are evolution-
arily conserved in both structure and function and were
present more than a billion years ago. Therefore, we
start this review with lessons learned from invertebrates.
We then discuss the structural basis of signaling by
these ligands and their receptors, including a newly
discovered second NRG gene, and review current
thoughts on how diversity is generated through combi-
natorial protein interactions. Individual organs and cell
lineages, whose development is critically regulated by
NRGs, are then described in light of recent gene tar-
geting, by in vivo and in vitro experiments.
Invertebrate ErbB Signaling Modules
The simplest version of the NRG-ErbB network is found
in C. elegans; it is comprised of a single receptor, the
product of the let-23 gene, and a single ligand encoded
by lin-3. Nevertheless, the major structural determinants
of mammalian NRGs and ErbBs are already present: the
receptor’s ectodomain is a duplicated structure carrying
two cysteine-rich blocks, and its cytoplasmic portion
is a tyrosine kinase flanked by noncatalytic regulatory
Meeting Report
domains. Likewise, the single ligand is synthesized as
a transmembrane precursor containing a characteristic
epidermal growth factor (EGF) -like motif with six cyste-
ine residues arranged in three loops (reviewed by Korn-
feld, 1997). The Drosophila relative of ErbB (DER), how-
ever, binds four different ligands, one of which, vein,
has no transmembrane precursor, but like certain NRG
isoforms, carries an immunoglobulin (Ig) -like motif. An-
other ligand, argos, is an antagonist (reviewed by Perri-
mon and Perkins, 1997). A mammalian antagonist has
yet to be identified. Like their mammalian counterparts,
the invertebrate ErbB receptors also signal primarily
through the Ras-Raf-MAP-kinase pathway. In both in-
vertebrates and mammals, the signaling module is nec-
essary for development of multiple and seemingly unre-
lated cell lineages and organs, and its requirement
coincides with more than one developmental phase.
Another lesson that is mirrored in mammals is the tightly
controlled availability of the ligands, whose range of
action is relatively short; the synthesizing cell and the
responsive tissue are juxtaposed or only a few cell layers
apart.
The ErbB Signaling Network of Mammals
Over the last 800 million years, the ErbB-NRG module
has undergone remarkable expansion through gene du-
plication events to reach its present state of four recep-
tors and a few dozen ligands. It is likely that this expan-
sion reflects an increasing physiological demand for
control of additional cell lineages. The diversification
potential of the module is enhanced by combinatorial
protein–protein interactions. The diversity of responses
is generated by three levels of interacting molecules:
first, there are multiple ligands whose receptor-binding
motifs, an EGF-like domain, are flanked by variable do-
mains. Second, all ligands bind to a set of receptors
that can generate all possible homo- and heterodimers.
Third, each different receptor dimer can couple to a
distinct set of cytoplasmic signaling proteins.
Ligands
A newly isolated family of NRG-like ligands, termed
NRG2, was described by three groups at the workshop
(H. Chang, Stanford University, Palo Alto, CA; C. Lai,
Scripps Institute, La Jolla, CA; and A. Goodearl, Millen-
nium Pharmaceuticals, Boston). Like NRG isoforms,
these ligands come in several variants, which probably
arose via alternative splicing from a gene on chromo-
some 5, which is distinct from the human NRG gene
(located on chromosome 8 and now termed NRG1). The
structure of these new ligands includes an EGF-like
motif, which has two variants, a and b, as well as a
collection of other recognizable extracellular motifs that
are variably present: a signal peptide, two variants of a
cysteine-containing N-terminal domain, either a cyste-
ine-rich domain (CRD) or a domain distantly related to
a kringle motif, an Ig-like loop, a glycosylation domain,
and a transmembrane domain. The presence or absence
of the latter domain determines whether the precursor
is made as a transmembrane protein or as a secreted
Neuron
848

Figure 1. Physiological Functions of the NRG-ErbB Signaling Module

The center panel schematically shows the four ErbB proteins crossing the plasma membrane (horizontal bar). ErbB-3 and ErbB-4 are the
direct NRG receptors, whereas ErbB-1 and ErbB-2 function as coreceptors. The tyrosine kinase domain of ErbB-3 is inactive (crossed). The
color code of the receptors is maintained in the other panels, each presenting one of the known roles of NRGs (functions symbolized by thick
arrows). Note juxtaposition of the sources of NRG (blue) and the responsive cells (red). AChR, acetylcholine receptor; SC, Schwann cell; and
NRG, neuregulin.

polypeptide. According to a new nomenclature, the vari-
ous isoforms are grouped into three types: type I in-
cludes variants with an Ig loop and a glycosylation do-
main (e.g., isoforms originally named NDF and ARIA);
NRGs with an Ig loop but no glycosylation domain are
termed type II (e.g., the NRG isoform originally called
GGF-II); and the CRD domain-containing factors, which
were originally named sensory and motor neuron–
derived factor (SMDF; Ho et al., 1995), are now termed
type III NRGs. The cytoplasmic tails of some proNRG
isoforms are longer than some of the extracellular por-
tions, and their lengths are variable. They may function
in “reverse signaling” since the region common to all
transmembrane NRG1s appears to associate specifi-
cally with certain cytoplasmic signaling proteins (D.
Falls, Emory University, Atlanta). Further, the transmem-
brane isoform proNRG1-b2 has recently been isolated in
a screen for apoptosis-inducing genes, but its function is
apparently cell autonomous (Grimm and Leder, 1997).
Thus, the transmembrane precursor forms of NRGs may
also function independently of ErbB proteins.
Both NRG1 and NRG2 isoforms bind directly to ErbB-3
and to ErbB-4, but they recruit ErbB-1 and ErbB-2 as
coreceptors. Whereas ErbB-2 has no known direct li-
gand, ErbB-1 binds six mammalian EGF-like molecules:
EGF, transforming growth factor a, heparin-binding
EGF-like growth factor (HB-EGF), amphiregulin, epi-
regulin, and betacellulin. The latter, along with HB-EGF
(Elenius et al., 1997) and possibly additional ligands,
also binds to ErbB-4 (D. Stern, Yale University, New
Haven, CT). The exact role of the structural heterogene-
ity of NRGs is still unknown. Best understood is the
variation in the EGF-like receptor-binding domain. The
C-terminal loop of NRG1 determines the strength of
receptor binding and has a role in determining which
receptor heterodimers will form: the higher affinity b
isoform stabilizes both ErbB-3/ErbB-2 and ErbB-3/
ErbB-1 heterodimers, whereas the a isoform recruits
Meeting Review
849
only the former complex (Pinkas-Kramarski et al., 1996).
In addition, each NRG isoform has a distinct spatial
and temporal pattern of expression (C. Birchmeier, Max
Delbrück Center, Berlin; N. Ratner, University of Cincin-
nati). For example, type I isoforms, which are the only
NRG1 isoforms detectable at early embryonic ages, are
the predominant species expressed in the endocardium,
while type II NRGs are the major isoforms expressed in
embryonic skeletal muscle, and type III NRGs are pres-
ent in sympathetic, motor, and sensory neurons (X. Yang
and L. Role, Columbia University, New York). On the
other hand, all NRG1 types are expressed in the ventric-
ular zone of the telencephalon. In addition, stromal cells
underlying many epithelial cell layers of parenchymal
organs are a rich source of certain NRGs.
Receptors
The two direct NRG receptors, ErbB-3 and ErbB-4, and
their two coreceptors, ErbB-1 and ErbB-2, qualify as
distinct components of a signaling network in that they
differ in most functional aspects, yet are capable of
extensive intermolecular interactions. In addition to li-
gand specificity and affinity, expression patterns, and
intracellular trafficking following ligand binding, the four
NRG receptors differ in kinase activity and substrate
selectivity. Most remarkable is ErbB-3, whose catalytic
function and endocytosis are both impaired by compari-
son to the other three receptors (Baulida et al., 1996).
The deficiency in catalytic function, however, can be
overcome by the process of ligand-induced receptor
dimerization, an essential step that precedes receptor
phosphorylation and intracellular signaling. Although
ErbB-3 homodimers are functionally defective, this re-
ceptor is recruited into heterodimers by the three other
ErbB proteins. Thus, ErbB-3 signaling depends not only
on NRG binding, but also requires association with other
ErbB proteins. Ligand-induced formation of homo- and
heterodimers is shared by the other ErbB proteins, and
all 10 possible receptor combinations can exist, includ-
ing a homodimer of the ligand-less ErbB-2, which is
formed as a consequence of either overexpression or an
oncogenic mutation within the transmembrane domain.
Selection of a coreceptor by a ligand-occupied direct
receptor is clearly a nonrandom process that follows
hierarchical rules. For example, ErbB-2-containing het-
erodimers are preferred, and heterodimers that can be
driven by two types of ligands (e.g., ErbB-1/ErbB-3 com-
plexes) are better stabilized by NRG than by EGF (Tzahar
et al., 1996). How ligand binding promotes different re-
ceptor combinations is currently an open question. Li-
gand affinities of the various dimer combinations differ
significantly, even when dimers are artificially stabilized
with immunoglobulin Fc tails (ErbB-IgG; M. Sliwkowski,
Genentech). This is exemplified by heterodimeric com-
plexes of ErbB-3 or ErbB-4 with ErbB-2, whose affinities
for NRGs exceed those of other combinations, including
an ErbB-3/ErbB-4 heterodimer. Studying the ErbB-2/
ErbB-3 heterodimer with an anti-ErbB-3 monoclonal an-
tibody and the corresponding ErbB-IgG, Sliwkowski
supported the possibility that a conformational alter-
ation takes place after NRG binding, exposing a cryptic
dimerization site that recruits ErbB-2. Replacements of
specific residues of ErbB-2 with a cysteine indicate that
the juxtamembrane portion of the extracellular domain
is a dimer interface, and therefore may act as a dimeriza-
tion site (Stern).
According to an alternative model (Y. Yarden, Weiz-
mann Institute, Rehovot, Israel), NRGs are bivalent
ligands; they first bind a direct receptor (either ErbB-3
or ErbB-4) through an N-terminally located high affinity
site, and then a second site of NRG, whose affinity is
lower and specificity broader, selects a coreceptor with
some preference for ErbB-2. High resolution NMR stud-
ies (Jacobsen et al., 1996) and application of phage
display technology to NRG (Sliwkowski) indirectly sup-
port the existence of two receptor contact sites. His2
(178) and Leu3, two invariant residues located at the
N-terminus of the EGF-like domain, are essential for
NRG binding to ErbB-3 (see also Barbacci et al., 1995),
whereas several C-terminal residues dictate binding to
both ErbB-3 and ErbB-4. Consistent with bivalency of
another ErbB ligand, EGF, recent biophysical measure-
ments determined several possible EGF:ErbB-1 stoi-
chiometries in solution, of which the 2:2 complex pre-
dominates (Lemmon et al., 1997). In addition, affinity
labeling with EGF indicates that the C-terminus of this
ligand contacts the receptor’s N-terminus, whereas the
C-tail of EGF is juxtaposed to the sequence intervening
the two cysteine-rich domains of ErbB-1 (Summerfield
et al., 1996). Thus, NRGs may drive receptor dimerization
through a mechanism that combines conformational al-
terations in receptors with bivalent ligand interactions
generating a series of molecular complexes, including
a stable quaternary complex of two ligands bridging two
receptors. Presumably, each NRG isoform is character-
ized by a unique combination of the two sites, thereby
favoring certain receptor combinations.
Effector Signaling Pathways
As is the case for other receptor tyrosine kinases, ligand-
induced autophosphorylation of ErbB proteins on spe-
cific tyrosine residues establishes docking sites for cyto-
plasmic signaling proteins sharing a phosphotyrosine
binding motif (SH2 or PTB domains). Due to differences
in the amino acid sequences surrounding phosphorylation
sites of each receptor, different sets of signaling pro-
teins become associated with each of the 10 possible
ErbB complexes. Several substrates, like Grb-2 and Shc,
are shared by most ErbB species. However, others like
the negative regulator c-Cbl (Levkowitz et al., 1996) and
the phosphatidylinositol-specific phospholipase (PLCg),
are recruited only to ErbB-1, and possibly ErbB-2 (Co-
hen et al., 1996), resulting in calcium mobilization only
in response to EGF.
ErbB specificity of other substrates, such as Src and
PI-3 kinase, must still be examined in cells whose reper-
toire of these receptors is genetically manipulated.
Nevertheless, analyses of several cellular systems ex-
pressing individual, or pairs of, ErbB proteins have already
reached a consensus on the complementarity of various
pairs (Riese et al., 1995; Cohen et al., 1996; Pinkas-
Kramarski et al., 1996; Tzahar et al., 1996; Zhang et al.,
1996). Homodimers are in general less active than hetero-
dimers in stimulating cell proliferation, and ErbB-2-con-
taining heterodimers are superior to other heterodimeric
complexes. The least effective and most unstable dimer
appears to be the NRG-driven ErbB-3/ErbB-4 complex,
and dimers formed between ErbB-1 and either NRG
Neuron
850
receptor display an intermediate signaling potency. The
remarkable mitogenic superiority of the ligand-less pro-
tein, ErbB-2, attracted significant attention because this
protein was implicated, more than other members of the
family, in cancer development. Functional inactivation of
ErbB-2 demonstrated that ErbB-2 amplifies and pro-
longs signaling by NRG and EGF through deceleration
of the rate of ligand dissociation (Graus-Porta et al.,
1995; Karunagaran et al., 1996). Presumably, complexes
containing a ligand bound to a direct receptor and ErbB-2
as a coreceptor are long-lived due to retarded ligand
dissociation and a relatively slow rate of ErbB-2 endocy-
tosis. On the other hand, because ErbB-2 is a strong
activator of the MAP-kinase pathway, its heterodimers
are most potent in signaling.
NRGs in Cardiac Development
Quite unexpectedly, but in agreement with the notion
that NRGs act through a signaling network rather than
via a direct receptor, targeting of the genes encoding
NRG (C. Birchmeier), ErbB-2 (K.-F. Lee, Salk Institute),
and ErbB-4 (M. Gassman, Salk Institute) in mice resulted
in an almost identical cardiac malformation (Gassmann
et al., 1995; Lee et al., 1995; Meyer and Birchmeier,
1995; Kramer et al., 1996). Homozygous embryos die in
utero at around embryonic day 10, presumably due to
defects resulting from the absence of heart trabeculae,
the finger-like extensions of the heart’s wall. The only
previous clue for involvement of NRGs in cardiac devel-
opment was derived from expression studies that local-
ized NRG to the endothelial cells of the endocardium and
both ErbB-2 and ErbB-4 to the adjacent heart muscle of
the ventricular wall, the myocardium. In the absence of
any of the three components, the inductive effect of the
endocardium on the myocardium is defective: the wall
does not protrude into the chamber to form the trabecu-
lae. Interestingly, inactivation of NRG in chick embryos,
using a novel ribozyme-based antisense strategy,
resulted in trabeculation defects similar to those ob-
served in mice (Lemke). The short-range inductive action
of NRG in mammalian heart development absolutely
depends on the presence of both the receptor, ErbB-4,
and the coreceptor, ErbB-2. Thus, contrary to in vitro
studies implying that ErbB-4 can transmit signals in the
absence of ErbB-2, homodimers of ErbB-4 are inactive
in ventricle differentiation.
The other direct NRG receptor, ErbB-3, is not involved
in trabeculation as its expression is restricted to the
mesenchyme of the endocardial cushion, which devel-
ops into valves. Indeed, the hearts of ErbB-32/2 mice
display delayed but otherwise normal trabeculation, but
their atrioventricular valves are rudimentary and thinned,
leading to death in utero at E13.5 (S. Erickson, Genen-
tech). As the ErbB-3-defective mice survive longer than
the other mutants, many additional sites of NRG-ErbB-3
functions were observed in these embryos (see below).
The endocardium-derived NRG mediates formation of
cushion mesenchyme, from which valve primordia evolve.
The identity of the ErbB-3 coreceptor in valvuloseptal
differentiation is unknown, as neither ErbB-1 nor ErbB-2
are expressed in the cushion. Thus, two different as-
pects of cardiac development, trabeculation and valvu-
loseptal formation, are critically regulated by distinct
heterodimers of NRG receptors. Apparently, by requir-
ing heterodimers, cardiac development gains an addi-
tional control level: the availability of a coreceptor. As
for other growth factors, a clinical connection is in sight.
Since expression levels of NRG, ErbB-2, and ErbB-3
increase following treatments that induce cardiac hyper-
trophy, an adaptive or pathological thickening of the
heart’s wall, the possibility that this hypertrophy may
be stimulated by increased NRG/ErbB signaling was
raised by K.-F. Lee.
NRGs in Schwann Cell and
Oligodendrocyte Differentiation
Most myelin-forming cells of the peripheral nervous sys-
tem develop from the neural crest in a multistep process
requiring contact with neurons. NRGs are important
neuron-derived factors, which control Schwann cell dif-
ferentiation, and are likely to have a critical role in regu-
lating Schwann cell number. In this case, a heterodimer
of ErbB-2 with ErbB-3, rather than with ErbB-4, is the
active receptor (Levi et al., 1995; Grinspan et al., 1996).
In fact, ErbB-2 involvement in proliferation of Schwann
cells was inferred long ago on the basis of Schwannoma
induction by an oncogenic mutant of the rodent ErbB-2.
In cell culture, NRGs are the only factors able to mimic
neurites in supporting survival of Schwann cell precur-
sors, and a soluble ErbB-4 can block neuronal support
(K. Jessen, University College, London). The precursors
first appear around embryonic day 12.5 in the mouse,
and by embryonic day 15, they complete differentiation
to Schwann cells expressing the calcium binding protein
S100 and the transcription factor Krox-20 (Murphy et al.,
1996). The latter, along with the cyclic-AMP responsive
element binding protein (CREB), is activated by NRG in
Schwann cells. Consistent with the role of NRG in precur-
sor survival, mice lacking NRG expression displayed se-
verely reduced numbers of presumptive Schwann cell pre-
cursors along trunk projections of nerves that normally
express NRG and become ensheathed by Schwann cells.
Unlike mice that are homozygous for the EGF-like do-
main of NRG (NRGEGF2/2), mice that are homozygous
mutant in the Ig-like domain (NRGIg2/ 2) but express other
NRG isoforms, contain Schwann cells along axonal pro-
jections of cranial nerves (C. Birchmeier). These results
indicate that Schwann cells do not depend on the Ig-
like isoform of NRG. Since the most abundant NRG iso-
form in motor and sensory neurons, CRD-NRG, lacks
the Ig domain, but instead has a CRD (Yang and Role),
it seems likely that isoforms containing the CRD are
sufficient to initiate Schwann cell differentiation. Inter-
estingly, Schwann cells are present in trigeminal and
dorsal root ganglia of ErbB-32/2 animals (E13.5), but
their numbers are reduced in both the ganglia and their
nerve projections (Erickson). In addition, sciatic nerves
from adult ErbB-31/2 mice displayed reduced conduc-
tion velocity, in agreement with a Schwann cell defect.
On maturation, Schwann cells respond to NRGs by
enhanced proliferation, probably reflecting the ability of
axons to maintain an appropriate ratio to myelin-forming
Schwann cells (Syroid et al., 1996). Although mature
Schwann cells synthesize certain transmembrane iso-
forms of NRG and display constitutive phosphorylation
Meeting Review
851
of ErbB-3 (Rosenbaum et al., 1997), factor concentration
may not be sufficiently high to maintain proliferation via
an autocrine mechanism (Ratner), indicating that other
factors may have a role in regulating postnatal cells.
Nevertheless, NRG at low concentrations increases di-
rected migration of Schwann cells cultured in vitro on
peripheral nerves (Mahanthappa et al., 1996).
NRG expression and NRG-mediated signaling change
following nerve injury. Wallerian degeneration of the sci-
atic nerve causes Schwann cells to up-regulate expres-
sion of certain NRG isoforms, along with ErbB-3 and
ErbB-2 (Carroll et al., 1997). Because these Schwann
cells are more intensely stained with antibodies specific
for a major tyrosine autophosphorylation site in ErbB-2
(C.D. Stiles, Dana Farber Cancer Institute, Boston),
nerve degeneration is apparently accompanied by in-
creased NRG signaling. Schwann cells at the neuromus-
cular synapse provide another example for a trophic
neural effect, as will be discussed below.
Like Schwann cells, differentiation of oligodendro-
cytes, the myelin-forming cells of the central nervous
system, is profoundly influenced by neurons. While mi-
grating from the subventricular zone to tracts of the white
matter, the progenitor cell progressively assumes a ma-
ture oligodendrocyte phenotype. J. Salzer (New York
University) reported that NRG is mitogenic to both cell
types in vitro. Moreover, NRG reverses differentiation of
myelin-forming oligodendrocytes, and under certain in
vitro conditions, it can inhibit commitment to both an oligo-
dendrocyte lineage (at the pro-oligodendrocyte stage) and
to type 2 astrocytes. Strikingly, NRG similarly inhibits
expression of myelin-specific proteins by Schwann cells
(A. Mudge, University College, London; Cheng and
Mudge, 1996). While these activities should be verified
in vivo, they suggest that NRG has a common role in
the myelinating cells of both the central and peripheral
nervous systems. It enables neurons to enlarge the pool
of premyelinating glial cells, and only after NRG-ErbB
signaling has been attenuated can myelination proceed.
Indeed, studies presented by J. Grinspan and S. Scherer
(University of Pennsylvania, Philadelphia) indicate that
the pool of premyelinated Schwann cells is the one regu-
lated by axonal NRG during early postnatal develop-
ment. After axotomy, which removes the source of axonal
NRG, they observed enhanced apoptosis of premy-
elinating Schwann cells, and the increase in apoptosis
was completely prevented by the application of exoge-
nous NRG (Grinspan et al., 1996). Similar rescue of
Schwann cells was observed at neuromuscular junc-
tions, Golgi tendon organs, and Pacinian corpuscle (W.
Thompson, University of Texas, Austin).
The interaction of NRG with myelinating cells has im-
plications for demyelinating disorders of the nervous
system. Evidence was presented indicating that NRG
may have a clinically beneficial role as a therapeutic
agent for multiple sclerosis. C. Raine (Albert Einstein
College of Medicine, New York) used an adoptive trans-
fer model of experimental allergic encephalomyelitis
(EAE) to determine whether NRG might reduce the rate
or extent of demyelination. In this model, clinical re-
lapses are episodic, and they are associated with inflam-
mation, demyelination, and remyelination. Raine re-
ported that repeated injections of NRG at different times
during the disease reduced the frequency of clinical
relapses. The mechanism of action is unclear; indeed,
it is possible that NRG acts indirectly on immune cells
rather than directly on oligodendrocytes. Nevertheless,
NRG clearly enhanced the extent of myelination in these
mice, suggesting the need for further study of a potential
therapeutic role for NRG in multiple sclerosis.
An interesting interplay between NRG and sonic
hedgehog (Shh) is emerging from studies of oligoden-
drocyte development in the chick spinal cord (R.H.
Miller, Case Western Reserve University, Cleveland).
Since oligodendrocyte precursors are located in the
ventral neural tube, oligodendrocytes do not develop in
explants of the dorsal portion of the tube. Although nei-
ther NRG nor low concentrations of Shh could induce
oligodendrocytes in explants of dorsal neural tube, the
two ligands together collaborated to induce oligoden-
drocyte differentiation in these explants.
NRGs in the Neuromuscular Synapse
NRGs play an important role at neuromuscular syn-
apses, where they are thought to act as a signal that
stimulates transcription of certain genes, including
those encoding acetylcholine receptor (AChR) subunits,
in nuclei near the synaptic site. The first in vivo evidence
supporting this notion was presented by G. Fischbach
(Harvard Medical School), who reported that mice that
are heterozygous for the Ig allele of NRG (NRGIg1/2) are
mildly myasthenic. These heterozygous mice have fewer
(50%) AChRs at their neuromuscular synapses, support-
ing the idea that NRG has a role in stimulating AChR
synthesis in synaptic nuclei, and that the concentration
of NRG at the synapse is limiting. The mutant mice
apparently compensate for the deficit in AChR number
by releasing more acetylcholine from their nerve termi-
nals, measured as an increase in quantal content, and
this result may explain why the heterozygous mice are
behaviorally normal. Nevertheless, tetanic stimulation
of their motor nerves in vitro showed that synaptic trans-
mission fails more readily in NRGIg1/ 2 mice, further indi-
cating that NRG has a role in neuromuscular function.
The Ig isoform of NRG is not the major NRG isoform
in motor neurons, and indeed the spinal cord of NRGIg 1/2
mice have nearly normal levels of NRG. Since the Ig-
like domain is crucial for binding to heparan sulfate pro-
teoglycans, it may promote accumulation of this isoform
in the extracellular matrix of the synaptic cleft, even
during stages of development in which the abundance
of this mRNA is relatively low (Fischbach). An alternative
explanation for the apparent paradox of reduced neuro-
muscular function in mice containing nearly normal lev-
els of NRG in their spinal cord was raised by S. Burden
(Skirball Institute, New York). NRG is synthesized not
only by motor neurons but also by skeletal muscle cells
(Moscoso et al., 1995; Rimer et al., 1996, Soc. Neurosci.
abstract), and the persistence of the membrane-bound
form of NRG at denervated synapses demonstrates that
muscle cells contribute NRG to the synaptic site (Rimer
et al., 1996, Soc. Neurosci. abstract). Because the Ig iso-
form is the major NRG isoform in skeletal muscle cells
(X. Yang and Burden), the phenotype of the NRGIg1/ 2
mice may be explained by a reduction in muscle-derived
Neuron
852
rather than in motor neuron-derived NRG. Moreover,
since muscle cells synthesize NRG and ErbBs, and local-
ize both ligands and receptors to the synaptic site, NRG-
ErbB signaling at the synapse may function, at least
in part, by an autocrine mechanism (Burden), involving
activation of a Ras/ERK2-dependent pathway (Altiok et
al., 1997).
Agrin is an important signal for neuromuscular syn-
apse formation, since mice lacking agrin or MuSK, a
component of its receptor complex, lack neuromuscular
synapses. Because mice lacking agrin (Gautam et al.,
1996) or MuSK (DeChiara et al., 1996) fail to cluster
ErbBs, NRG-ErbB signaling is likely to depend on agrin/
MuSK signaling. Agrin is sufficient to cluster NRG and
ErbBs in skeletal muscle, since forced ectopic expres-
sion of neural agrin at nonsynaptic sites in skeletal mus-
cle cells caused not only AChRs but also ErbBs and
pro-NRG to cluster at the ectopic site (Rimer et al., 1996,
Soc. Neurosci. abstract). Thus, agrin/MuSK signaling
regulates the distribution of muscle-derived NRG and
ErbBs.
Identification of an ErbB-binding protein, which, like
the ErbBs, is concentrated at neuromuscular synapses,
was described by K. Carraway (Harvard). This heregulin
receptor binding (HRB) protein, identified in a yeast two-
hybrid screen using the cytoplasmic domain of ErbB-3
as bait, is expressed in heart, skeletal muscle, and brain.
The protein is myristolated, has an imperfect ring finger
domain, and can bind both ErbB-3 and ErbB-4 in vitro,
although the binding does not appear to depend on
ErbB phosphorylation. Like rapsyn-induced clustering
of AChRs in nonmuscle cells, HRB appears to regulate
the clustering of ErbB-3 in transfected COS cells.
The role of NRG in regulating the nonmyelinating ter-
minal Schwann cells at the neuromuscular synapse was
addressed by W. Thompson. In mammalian muscle, syn-
aptic sites are usually organized in an endplate band
adjacent to the main intramuscular nerve. Schwann cells
within skeletal muscle are restricted to these intramus-
cular nerves and to synaptic sites. Injection of NRG into
skeletal muscle of neonatal mice, however, resulted in
the proliferation and redistribution of Schwann cells
throughout the muscle. Motor axons in NRG-injected
muscle are likewise found throughout the muscle. Since
prior studies by Thompson indicated that Schwann cells
are likely to have an important role in guiding motor
axons to denervated synaptic sites (Son and Thompson,
1995), these new results raise the possibility that NRG,
possibly provided by skeletal muscle, indirectly regu-
lates the position of motor axons by regulating the num-
ber and position of Schwann cells in muscle.
Although NRG2, like NRG1, binds to ErbB-3 and to
ErbB-4 and concomitantly stimulates their association
with other ErbB family members, resulting in ligand-
stimulated tyrosine phosphorylation, NRG1, but not
NRG2, activates expression of AChR genes in muscle
cell lines (Lai, Yang, and Burden; A. Goodearl, Millenium
Pharmaceuticals). Similarly, NRG1 but not NRG2 stimu-
lates Schwann cell proliferation. These unexpected re-
sults raise the possibility that the extent or duration of
signaling by NRG1 and NRG2 may differ and account
for the different biological responses observed in mus-
cle and in Schwann cells.
NRG Actions on Neurons and Cranial Nerves
Because NRGs and their direct receptors are widely
expressed by neurons of the central and peripheral ner-
vous systems, paracrine and autocrine regulation of
nerves by NRG is possible. Disruption of all NRG iso-
forms, or only Ig-containing isoforms, resulted, however,
in neural defects that were limited to cranial nerves at
the time of death of mutant animals. Specifically, the
neural crest–derived component of cranial ganglia V (tri-
geminal), and to a lesser extent that of ganglia VII–IX,
were severely reduced in size. In addition, these ganglia
were disconnected, either completely or partially, from
the hindbrain. Apparently, NRG controls survival, rather
than migration, of cells originating in the NRG-express-
ing hindbrain rhombomeres 2, 4, and 6 (Meyer and Birch-
meier, 1995). These neural crest–derived cells are al-
ready committed to a neural fate, and their survival
specifically depends on an Ig-containing NRG (C. Birch-
meier). Survival is mediated by ErbB-2 and ErbB-3, but
not an active ErbB-4, as ErbB-22/2 and ErbB-32/2mice
shared a similar cranial nerve phenotype with NRG2/2
mice (Lee; Erickson), but both ganglia size and hindbrain
connections were not affected in ErbB-42/ 2 mice (M.
Gassman, Salk Institute). Instead, in the latter animals,
ganglia V, VII, and VIII are almost fused, and their con-
nections are mistargeted: the trigeminal and facial gan-
glia are each connected to both rhombomeres 2 and 4,
instead of their normal single links to rhombomeres 2
and 4, respectively. The reason for mistargeting is cur-
rently unknown, but it probably relates to the localized
expression of ErbB-4 in the intervening odd numbered
rhombomeres 3 and 5 (Lemke, 1996). Alternatively, mis-
targeting is not seen in NRG2/2 embryos as a result of the
extensive cell loss in their cranial ganglia (Gassmann).
Another possible explanation for the discordant NRG
and ErbB-4 phenotypes is the existence of additional
ErbB-4 ligands, such as betacellulin (Riese et al., 1996),
HB-EGF, or NRG2. Alternatively, reciprocal mechanisms
involving other neurotrophic factors may be involved.
For example, sympathetic neuroblast-derived NRG can
induce synthesis of nerotrophin-3 (NT-3) by nonneuronal
cells, thereby affecting in an indirect way the survival
of neurons (Verdi et al., 1996). Potentially, this or a similar
mechanism may underlie the observation that NRG pro-
motes survival and neurite outgrowth of cultured embry-
onic retinal cells (O. Bermingham-McDonogh, University
of Washington, Seattle). Neurons isolated from neonates
(P4) lost responsiveness to NRG, in parallel to a de-
creased expression of ErbB-2 (Bermingham-McDonogh
et al., 1996). Thus, neurite outgrowth in retinal cells, as
well as in the more extensively studied PC-12 cell line
(Gamett et al., 1995), correlates with ErbB-2 expression,
reemphasizing a receptor-coded specificity biological
effect of NRGs.
Reciprocal interactions involving NRG also play a role
in neural migration. E. Anton (Yale) and G. Corfas (Har-
vard; Rio et al., 1997) studied migration in the cerebral
cortex and in the cerebellum, respectively, and con-
cluded that neuron-derived NRG instructs the underly-
ing glia, either radial glia or the cerebellar Bergmann
glia, to promote directed neuron movement. In the cortex,
glia-expressed ErbB-2 is involved, perhaps in collabora-
tion with ErbB-3 and ErbB-4; in Bergmann glia, NRG
Meeting Review
853
induced CREB phosphorylation. Migration could be
blocked either by an anti-NRG antibody added to ex-
plants of the cerebral cortex or by a dominant-negative
mutant of ErbB-4 ectopically expressed in glia cells. In
both the developing cerebral cortex and cerebellum,
NRG promoted the neuron-primed elongation of glial
cells, but not their adhesion to neuronal cells, indicating
that NRG directly induces radial morphology of the glia,
and this cell reciprocally promotes neuron migration
through an unknown mechanism.
Acoustic trauma (exposure to excessive noise for a
long time, or to certain antibiotics and diuretics) may
lead to loss of mechanosensory hair cells from the co-
chlea, resulting in irreversible deficits in hearing. In cer-
tain species, these damaged epithelial cells can be re-
placed by division of supporting cells, which share some
features with Schwann cells. Jeff Corwin (University of
Virginia, Charlottesville) reported that postnatal rat sup-
porting cells undergo mitosis in response to NRG, and
to a lesser extent to one of the ErbB-1 ligands (TGFa),
suggesting yet another clinical application of these
growth factors.
NRGs in Epithelial Morphogenesis
The wealth of information on NRGs as primary mediators
of nerve communication with neighboring nonneuronal
cells may be paralleled by initial reports of similar inter-
actions in other epithelial organs, whose cellular hetero-
geneity and complex anatomical connections are reminis-
cent of the nervous system. Morphogenesis of epithelial
organs is controlled, in part, by multiple polypeptide
factors secreted by the underlying stromal mesen-
chyme. With the exception of ErbB-4, whose expression
is limited to certain epithelia, the three other receptors
and coreceptors of NRGs are present in most epithelial
cell layers. Moreover, mesenchymal cells are the richest
source, outside of the nervous system, of the corre-
sponding ligands, both NRGs and ErbB-1-specific li-
gands. An example of NRG action in mesenchyme–
epithelial interactions is provided by the mammary gland
(C., and W. Birchmeier, Max Delbrück Center, Berlin).
The gland is one of a few organs in which major morpho-
genesis takes place after birth. During puberty, it in-
volves branching and elongation that result in a ductal
tree. A second budding phase, during pregnancy, gener-
ates the milk-producing lobuloalveolar structures. Anal-
yses of organ cultures of the mouse mammary gland
suggest that NRG, whose expression peaks at preg-
nancy, regulates the second morphogenic phase by
stimulating lobuloalveolar budding and milk production.
Of note, synthesis of milk components (e.g., casein and
neutral lipids) in response to NRG is retained in vitro by
certain mammary cell lines.
Interestingly, when pellets releasing NRG were im-
planted within the mammary glands of prepubescent
female mice, NRG-a, but not NRG-b, could induce ductal
branching and migration in the absence of exogenous
steroid hormones, but both isoforms were active in in-
duction of secretory lobuloalveoli (Jones et al., 1996).
Mice engineered to constitutively overexpress NRG-b2
in the mammary gland displayed persistence of the
mammary end bud structures, which normally disappear
during puberty, and also promoted mammary tumors
(Krane and Leder, 1996). Thus, NRGs appear to induce
growth and inhibit differentiation of the primitive epithe-
lial duct, but later in development these factors enhance
mammary differentiation.
The reason that epithelial defects are not prominently
detectable in NRG-, ErbB-2-, and ErbB-4-targeted mice
is probably due to the early embryonic death of the
mutant mice, and the relatively late onset of epithelial
organogenesis. Indeed, major defects in epithelia of
skin, lung, hair, placenta, and other organs were de-
tected in ErbB-1-defective mice (which live longer than
NRG2/2 mice). Likewise, ErbB-3-defective mice (which
live 3 days longer than NRG2/2 mice) already display
some abnormalities in stomach and in pancreas (Erick-
son). Another reflection of the role of ErbB-3 in epithelial
differentiation was derived from an in vivo model of
wound healing, where NRG was found to mediate both
epidermal migration and up-regulation of keratinocyte
differentiation markers (Danilenko et al., 1995). As ex-
pected from other inductive actions of NRG, the ligand
is secreted by skin fibroblasts and affects nearby kera-
tinocytes, whose expression of ErbB proteins changes
during reepithelialization.
The dynamic pattern of ErbB expression during cell
fate determination and morphogenic processes appar-
ently dictates the nature of cellular response. This ap-
pears to be true in all in vitro models of NRG actions,
including Schwann cells. As oligodendrocytes differenti-
ate, ErbB-4 is down-regulated, and ErbB-2 expression
is increased (Canoll et al., 1996), whereas during skeletal
muscle differentiation, ErbB-3 expression is strongly in-
duced while ErbB-2 expression remains unchanged (Jo
et al., 1995). Dramatic examples of how the repertoire
of ErbB proteins might affect cell function are provided
by several types of epithelial cancers (carcinomas):
some tumors overexpress ErbB-2 at levels 100-fold
greater than normal, and in some types of cancers, the
extent of overexpression predicts disease outcome and
response to hormone- and chemotherapy. Apparently,
ErbB-2 overexpression in epithelial cells biases forma-
tion of its more potent heterodimers, leading to en-
hanced proliferation at the expense of a differentiated
phenotype. On the other hand, blocking ErbB-2 action
by using anti-ErbB-2 monoclonal antibodies results in
retardation of cell proliferation, an effect that has been
recently used in breast cancer patients to inhibit mam-
mary tumor growth (Baselga et al., 1996).
Conclusions
Results obtained in the few years since the NRG family
was discovered already make it clear that this signaling
module is involved in a myriad of physiological and de-
velopmental processes, as well as in pathological
states. This multiplicity of biological functions likely re-
flects the enormous biochemical potential of the NRG-
ErbB network to diversify signals. The available NRG2
isoforms, together with the possible existence of addi-
tional ErbB receptors and a direct ErbB-2 ligand, may
extend the ensuing complexity even further. Results
from mice with targeted mutations support the possibil-
ity that this signaling complexity, which has been estab-
lished by expression studies and in vitro experiments, is
Neuron
854
utilized in vivo. Thus, in the near future, we will probably
witness parallel biochemical and physiological decod-
ing of the many distinct signaling pathways employed
by ErbB proteins. Conceivably, lessons learned in this
subfamily of growth factor receptors will be applicable
to other subtypes of receptor tyrosine kinases.
Acknowledgments
We thank the organizers of the workshop and many participants for
helpful comments, and Iris Alroy for help with the figure.
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