Neuregulins and Their Receptors: A Versatile Signaling Module in Organogenesis and Oncogenesis
Authors: Steve Burden, Yosef Yarden
Journal: Neuron (1997)

There has been rapid growth in our understanding of neuregulins (NRGs), a large group of structurally related polypeptide factors, and their receptors, four transmembrane 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 efforts 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 differentiation 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. Intensive 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 organized in the Banbury Center at the Cold Spring Harbor Laboratory by Mark A. Marchionni (Cambridge NeuroScience) and Greg Lemke (Salk Institute, La Jolla, CA) on "Neuregulins and Neuregulin Receptors." Neuregulins (NRGs) and their receptors are evolutionarily 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 combinatorial protein interactions. Individual organs and cell lineages, whose development is critically regulated by NRGs, are then described in light of recent gene targeting, by in vivo and in vitro experiments. 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 domains. Likewise, the single ligand is synthesized as a transmembrane precursor containing a characteristic epidermal growth factor (EGF) -like motif with six cysteine residues arranged in three loops (reviewed by24Kornfeld K. Vulval development in Caenorbaditis elegans.Trends Genetics. 1997; 13: 55-61Abstract Full Text PDF PubMed Scopus (157) Google Scholar). The Drosophila relative of ErbB (DER), however, binds four different ligands, one of which, vein, has no transmembrane precursor, but like certain NRG isoforms, carries an immunoglobulin (Ig) -like motif. Another ligand, argos, is an antagonist (reviewed by36Perrimon N. Perkins L.A. There must be 50 ways to rule the signal the case of the Drosophila EGF receptor.Cell. 1997; 89: 13-16Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). 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 invertebrates and mammals, the signaling module is necessary for development of multiple and seemingly unrelated 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. Over the last 800 million years, the ErbB-NRG module has undergone remarkable expansion through gene duplication events to reach its present state of four receptors and a few dozen ligands. It is likely that this expansion 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 domains. 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. 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, Millennium Pharmaceuticals, Boston). Like NRG isoforms, these ligands come in several variants, which probably arose via alternative splicing from a gene on chromosome 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, α and β, 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 cysteine-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 polypeptide. According to a new nomenclature, the various isoforms are grouped into three types: type I includes variants with an Ig loop and a glycosylation domain (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; 19Ho W.H. Armanini M.P. Nuijens A. Phillips H.S. Osheroff P.L. Sensory and motor neuron-derived factor. A novel heregulin variant highly expressed in sensory and motor neurons.J. Biol. Chem. 1995; 270: 14523-14532Crossref PubMed Scopus (130) Google Scholar), are now termed type III NRGs. The cytoplasmic tails of some proNRG isoforms are longer than some of the extracellular portions, and their lengths are variable. They may function in "reverse signaling" since the region common to all transmembrane NRG1s appears to associate specifically with certain cytoplasmic signaling proteins (D. Falls, Emory University, Atlanta). Further, the transmembrane isoform proNRG1-β2 has recently been isolated in a screen for apoptosis-inducing genes, but its function is apparently cell autonomous (17Grimm S. Leder P. An apoptosis-inducing isoform of Neu differentiation factor (NDF) identified using a novel screen for dominant, apoptosis-inducing genes.J. Exp. Med. 1997; 185: 1137-1142Crossref PubMed Scopus (33) Google Scholar). 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 ligand, ErbB-1 binds six mammalian EGF-like molecules: EGF, transforming growth factor α, heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, epiregulin, and betacellulin. The latter, along with HB-EGF (12Elenius K. Paul S.G.A. Sun J. Klagsbrun M. Activation of HER4 by heparin-binding EGF-like growth factor stimulates chemotaxis but not proliferation.EMBO J. 1997; 16: 1268-1278Crossref PubMed Scopus (315) Google Scholar) and possibly additional ligands, also binds to ErbB-4 (D. Stern, Yale University, New Haven, CT). The exact role of the structural heterogeneity 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 β isoform stabilizes both ErbB-3/ErbB-2 and ErbB-3/ErbB-1 heterodimers, whereas the α isoform recruits 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 Cincinnati). 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 present 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 ventricular zone of the telencephalon. In addition, stromal cells underlying many epithelial cell layers of parenchymal organs are a rich source of certain NRGs. 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 ligand 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 comparison to the other three receptors (4Baulida J. Kraus M.H. Alimandi M. Di Fiore P.P. Carpenter G. All ErbB receptors other than the epidermal growth factor receptor are endocytosis impaired.J. Biol. Chem. 1996; 271: 5251-5257Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). 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 receptor 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, including 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 heterodimers are preferred, and heterodimers that can be driven by two types of ligands (e.g., ErbB-1/ErbB-3 complexes) are better stabilized by NRG than by EGF (46Tzahar E. Waterman H. Chen X. Levkowitz G. Karunagaran D. Lavi S. Ratzkin B.J. Yarden Y. A hierarchical network of inter-receptor interactions determines signal transduction by NDF/neuregulin and EGF.Mol. Cell. Biol. 1996; 16: 5276-5287Crossref PubMed Scopus (843) Google Scholar). How ligand binding promotes different receptor combinations is currently an open question. Ligand 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 complexes 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 antibody and the corresponding ErbB-IgG, Sliwkowski supported the possibility that a conformational alteration 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 dimerization site (Stern). According to an alternative model (Y. Yarden, Weizmann 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 studies (20Jacobsen N.E. Abadi N. Sliwkowski M.X. Reilly D. Skelton N.J. Fairbrother W.J. High-resolution solution structure of the EGF-like domain of heregulin-alpha.Biochemistry. 1996; 35: 3402-3417Crossref PubMed Scopus (63) Google Scholar) and application of phage display technology to NRG (Sliwkowski) indirectly support 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 also2Barbacci E.G. Guarino B.C. Stroh J.G. Singleton D.H. Rosnack K.J. Moyer J.D. Andrews G.C. The structural basis for the specificity of epidermal growth factor and heregulin binding.J. Biol. Chem. 1995; 270: 9585-9589Crossref PubMed Scopus (97) Google Scholar, whereas several C-terminal residues dictate binding to both ErbB-3 and ErbB-4. Consistent with bivalency of another ErbB ligand, EGF, recent biophysical measurements determined several possible EGF:ErbB-1 stoichiometries in solution, of which the 2:2 complex predominates (29Lemmon M.A. Bu Z. Ladbury J.E. Zhou M. 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Presumably, each NRG isoform is characterized by a unique combination of the two sites, thereby favoring certain receptor combinations. As is the case for other receptor tyrosine kinases, ligand-induced autophosphorylation of ErbB proteins on specific tyrosine residues establishes docking sites for cytoplasmic 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 proteins 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 (31Levkowitz G. Klapper L.N. Tzahar E. Freywald A. Sela M. Yarden Y. Coupling of the c-Cbl protooncogene product to ErbB-1/EGF-receptor but not to other ErbB proteins.Oncogene. 1996; 12: 1117-1125PubMed Google Scholar) and the phosphatidylinositol-specific phospholipase (PLCγ), are recruited only to ErbB-1, and possibly ErbB-2 (9Cohen B.D. Kiener P.K. Green J.M. Foy L. Fell H.P. Zhang K. The relationship between human epidermal growth-like factor receptor expression and cellular transformation in NIH-3T3 cells.J. Biol. Chem. 1996; 271: 30897-30903Crossref PubMed Scopus (106) Google Scholar), 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 repertoire of these receptors is genetically manipulated. Nevertheless, analyses of several cellular systems expressing individual, or pairs of, ErbB proteins have already reached a consensus on the complementarity of various pairs (39Riese D.J. van Raaij T.M. Plowman G.D. Andrews G.C. Stern D.F. 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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 development was derived from expression studies that localized 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 trabeculae. Interestingly, inactivation of NRG in chick embryos, using a novel ribozyme-based antisense strategy, resulted in trabeculation defects similar to those observed 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 develops into valves. Indeed, the hearts of ErbB-3−/− 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, Genentech). 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 aspects of cardiac development, trabeculation and valvuloseptal formation, are critically regulated by distinct heterodimers of NRG receptors. Apparently, by requiring heterodimers, cardiac development gains an additional 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 hypertrophy, 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. Most myelin-forming cells of the peripheral nervous system develop from the neural crest in a multistep process requiring contact with neurons. NRGs are important neuron-derived factors, which control Schwann cell differentiation, and are likely to have a critical role in regulating Schwann cell number. In this case, a heterodimer of ErbB-2 with ErbB-3, rather than with ErbB-4, is the active receptor (30Levi A.D. Bunge R.P. Lofgren J.A. Meima L. Hefti F. Nikolics K. Sliwkowski M.X. The influence of heregulins on human Schwann cell proliferation.J. Neurosci. 1995; 15: 1329-1340Crossref PubMed Google Scholar, 18Grinspan J.B. Marchionni M.A. Reeves M. Coulaloglou M. Scherer S.S. Axonal interactions regulate Schwann cell apoptosis in developing peripheral nerve neuregulin receptors and the role of neureguins.J. Neurosci. 1996; 16: 6107-6118Crossref PubMed Google Scholar). 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 precursors, 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 (35Murphy P. Topilko P. Schneider-Maunoury S. Seitanidou T. Evercooren B.V. Charnay P. The regulation of Krox-20 expression reveals important steps in the control of peripheral glial cell development.Development. 1996; 122: 2847-2857PubMed Google Scholar). 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 precursor survival, mice lacking NRG expression displayed severely reduced numbers of presumptive Schwann cell precursors along trunk projections of nerves that normally express NRG and become ensheathed by Schwann cells. Unlike mice that are homozygous for the EGF-like domain of NRG (NRGEGF−/−), mice that are homozygous mutant in the Ig-like domain (NRGIg−/−) but express other NRG isoforms, contain Schwann cells along axonal projections 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 isoform 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. Interestingly, Schwann cells are present in trigeminal and dorsal root ganglia of ErbB-3−/− animals (E13.5), but their numbers are reduced in both the ganglia and their nerve projections (Erickson). In addition, sciatic nerves from adult ErbB-3+/− mice displayed reduced conduction 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 (45Syroid D.E. Maycox P.R. Burrola G.P. Liu N. We D. Lee K.-F. Lemke G. Kilpatrick T.J. Cell death in the Schwann cell lineage and its regulation by neuregulin.Proc. Natl. Acad. Sci. USA. 1996; 93: 9229-9234Crossref PubMed Scopus (211) Google Scholar). Although mature Schwann cells synthesize certain transmembrane isoforms of NRG and display constitutive phosphorylation of ErbB-3 (42Rosenbaum C. Karyala S. Marchionni M.A. Kim H.A. Krasnoselsky A.L. Happel B. Isaacs I. Brackenbury R. Ratner N. Schwann cells express NDF and SMDF/n-ARIA mRNAs, secrete neuregulin, and show constitutive activation of erbB3 receptors evience for a neuregulin autocrine loop.J. Exp. Neurol., in press. 1997; Google Scholar), 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 directed migration of Schwann cells cultured in vitro on peripheral nerves (32Mahanthappa N.K. Anton E.S. Matthew W.D. Glial growth factor 2, a soluble neuregulin, directly increases Schwann cell motility and indirectly promotes neurite outgrowth.J. Neurosci. 1996; 16: 4673-4683Crossref PubMed Google Scholar). NRG expression and NRG-mediated signaling change following nerve injury. Wallerian degeneration of the sciatic nerve causes Schwann cells to up-regulate expression of certain NRG isoforms, along with ErbB-3 and ErbB-2 (7Carroll S.L. Miller M.L. Fohnert P.W. Kim S.S. Corbett J.A. Expression of neuregulins and their putative receptors, ErbB2 and ErbB3, is induced during Wallerian degeneration.J. Neurosc. 1997; 17: 1642-1659PubMed Google Scholar). 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 increased NRG signaling. Schwann cells at the neuromuscular synapse provide another example for a trophic neural effect, as will be discussed below. Like Schwann cells, differentiation of oligodendrocytes, the myelin-forming cells of the central nervous system, is profoundly influenced by neurons. While migrating from the subventricular zone to tracts of the white matter, the progenitor cell progressively assumes a mature 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 oligodendrocyte 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; 8Cheng L. Mudge A.W. Cultured Schwann cells constitutively express the myelin protein P0.Neuron. 1996; 16: 309-319Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). 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 regulated by axonal NRG during early postnatal development. After axotomy, which removes the source of axonal NRG, they observed enhanced apoptosis of premyelinating Schwann cells, and the increase in apoptosis was completely prevented by the application of exogenous NRG (1