The ErbB signaling network consists of four transmembrane receptor tyrosine kinases and more than a dozen ligands sharing an epidermal growth factor (EGF) motif. The multiplicity of ErbB-specific ligands is incompletely understood in terms of signal specificity because all ErbB molecules signal through partially overlapping pathways. Here we addressed the action of epiregulin, a recently isolated ligand of ErbB-1. By employing a set of factor-dependent cell lines engineered to express individual ErbBs or their combinations, we found that epiregulin is the broadest specificity EGF-like ligand so far characterized: not only does it stimulate homodimers of both ErbB-1 and ErbB-4, it also activates all possible heterodimeric ErbB complexes. Consistent with its relaxed selectivity, epiregulin binds the various receptor combinations with an affinity that is approximately 100-fold lower than the affinity of ligands with more stringent selectivity, including EGF. Nevertheless, epiregulin's action upon most receptor combinations transmits a more potent mitogenic signal than does EGF. This remarkable discrepancy between binding affinity and bioactivity is permitted by a mechanism that prevents receptor down-regulation, and results in a weak, but prolonged, state of receptor activation.Various biological processes are controlled by intercellular interactions that are mediated by polypeptide growth factors. Examples include embryonic development, neuronal functions, hematopoiesis, and pathological situations, like wound healing and malignant transformation. The mechanism transmitting extracellular signals ultimately starts with binding of the growth factor to a cell surface receptor, that in many cases carries an intrinsic tyrosine kinase activity (1). These receptors fall into several subgroups sharing structural and functional characteristics. Each subgroup of receptors specifically recognizes a family of structurally homologous growth factors. Perhaps the most striking multiplicity of related growth factors is exemplified by the epidermal growth factor (EGF) 1 family of molecules (2). This six cysteine-containing motif of 45-60 amino acids is shared by all members of the family, and it functions as the receptor binding portion of the molecule. Currently there are four known receptors for EGF-like ligands, constituting the ErbB subgroup of receptor tyrosine kinases (also known as HER, or type I receptor tyrosine kinases (3)). Whereas ErbB-1 binds many ligands, including EGF, transforming growth factor ␣ (TGF␣), and amphiregulin, both ErbB-3 and ErbB-4 bind to a family of isoforms, collectively known as neuregulins (also called Neu differentiation factors, heregulins, glial growth factors, and acetylcholine receptor inducing activity) (4). A related group of molecules, termed NRG2, binds to the same two receptors (5-7), and a third molecule, NRG3, exclusively binds to ErbB-4 (8). Two other ligands, betacellulin (9), and the heparin-binding EGF-like growth factor (10, 11) bind to both ErbB-1 and ErbB-4. Interestingly, the m...

Heregulin is a ligand for the erbB3 and erbB4 receptors, with a region of high homology to epidermal growth factor (EGF). Despite this homology, these ligands bind to their corresponding receptors with great specificity. We report here the synthesis of heregulin beta 177-241 and show that a region consisting of amino acids 177-226 is sufficient both for binding and stimulation of receptor phosphorylation. Studies of chimeric EGF/heregulin peptides revealed that amino acids 177-181 of heregulin provide the specificity for binding to the heregulin receptor. The substitution of amino acids 177-181 of heregulin for the N terminus of EGF produced a unique bifunctional agonist that binds with high affinity to both the EGF receptor and the heregulin receptor.

The two isoforms of neuregulin 2 (NRG2), NRG2-␣ and NRG2-␤, were accidentally mislabeled. The names should therefore be exchanged throughout the paper, except in Fig. 9.

The recently isolated second family of neuregulins, NRG2, shares its primary receptors, ErbB-3 and ErbB-4, and induction of mammary cell differentiation with NRG1 isoforms, suggesting functional redundancy of the two growth factor families. To address this possibility, we analyzed receptor specificity of NRGs by using an engineered cellular system. The activity of isoform-specific but partly overlapping patterns of specificities that collectively activate all eight ligand-stimulatable ErbB dimers was revealed. Specifically, NRG2-␤, like NRG1-␣, emerges as a narrow-specificity ligand, whereas NRG2-␣ is a pan-ErbB ligand that binds with different affinities to all receptor combinations, including those containing ErbB-1, but excluding homodimers of ErbB-2. The latter protein, however, displayed cooperativity with the direct NRG receptors. Apparently, signaling by all NRGs is funneled through the mitogen-activated protein kinase (MAPK). However, the duration and potency of MAPK activation depend on the identity of the stimulatory ligand-receptor ternary complex. We conclude that the NRG-ErbB network represents a complex and nonredundant machinery developed for fine-tuning of signal transduction.One of the relatively simple systems of signal transduction by a polypeptide growth factor is the mechanism controlling vulva formation in the nematode Caenorhabditis elegans (reviewed in reference 33). The most ancient epidermal growth factor (EGF)-like ligand, Lin-3, which is expressed by the anchor cell, binds to the Let-23 transmembrane tyrosine kinase on the surface of the closely apposed vulva precursor cell. The latter is then directed to a vulval fate through a biochemical cascade that sequentially activates a small GTP binding protein and a series of protein kinases, culminating in the mitogen-activated protein kinase (MAPK). A remarkably expanded version of this signaling module exists in mammals (reviewed in reference 6). Four receptors, whose structures are homologous to Let-23, and a few dozen ligands, all sharing the three-loop structure of EGF, form an interactive system with a large potential for signal diversification. In addition to the multiplicity of components, the modern version of the module is characterized by diversity: one ErbB protein, ErbB-3, is devoid of tyrosine kinase activity (25), and another, ErbB-2, binds no known EGFlike factor with high affinity (28, 61). Likewise, the various ligands carry, in addition to the EGF-like motif, a variety of structural domains thought to allow interaction with extracellular components. For example, the heparin binding EGF-like factor includes a heparan sulfate binding moiety (26), and the Neu differentiation factor (NDF, also called neuregulin 1 [NRG1], or heregulin) carries an immunoglobulin (Ig) domain (27,37,63).A combination of in vitro experiments and gene targeting in mice implies that the mammalian ErbB module, like its invertebrate counterparts in worms and in flies (46), is involved with fate determination of several cell lineages. Thus, ErbB-1, and...

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