ErbB tyrosine kinases and the two neuregulin families constitute a ligand-receptor network.

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-alpha [corrected], like NRG1-beta [corrected], emerges as a narrow-specificity ligand, whereas NRG2-beta [corrected] 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.

Epiregulin is a potent pan-ErbB ligand that preferentially activates heterodimeric receptor complexes.

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.

Heregulin induces in vivo proliferation and differentiation of mammary epithelium into secretory lobuloalveoli.

Mammary gland development and differentiation is mediated through the combined activities of systemic hormones and locally synthesized growth factors. To determine the in vivo response of mammary epithelium to heregulin (HRG), we implanted Elvax pellets containing HRG alpha or HRG beta within the mammary glands of prepubescent female mice in the presence or absence of exogenous estradiol and progesterone (E/ P). Mice treated in the same way with transforming growth factor alpha (TGF-alpha) were included as a positive control. Each growth factor treatment induced epithelial ductal branching in the presence or absence of E/P. In the absence of E/P, HRG beta did not effect terminal end bud formation, mammary epitheilum branching, or ductal migration. In contrast, TGF-alpha and HRG alpha induced ductal branching and HRG alpha induced ductal migration in the absence of E/P. The overall mammary response to growth factors was potentiated by the concomitant presence of E/P. In every case, the in vivo mammary epithelial responses to HRG alpha were more robust than TGF-alpha. Limited lobuloalveolar development was also observed in growth factor-treated mammary glands when E/P was present. Histological examination of growth factor-induced lobuloalveoli revealed secretory products within the lumen of HRG alpha and HRG beta lobuloalveoli. TGF-alpha-induced lobuloalveoli lacked similar secretory products.

The structural basis for the specificity of epidermal growth factor and heregulin binding.

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.

Use of capillary electrophoresis-electrospray ionization mass spectrometry in the analysis of synthetic peptides.

We have constructed a capillary electrophoresis (CE) system with UV detection and have successfully interfaced it to an electrospray ionization mass spectrometry (ES-MS) system. A synthesized fragment of heregulin-beta (212-226) was thought to be a single component by re-injection into an HPLC system, but results from CE-UV-ES-MS indicated that a dehydration product was present in the desired peptide sample. A synthetic heregulin-alpha (177-241) was isolated by preparative HPLC, but re-injection on an analytical system indicated a tailing peak. CE-UV-ES-MS indicated a mixture whose two major components were of the same nominal molecular mass (within experimental error), suggesting the presence of an isomer or a deamidation product. The results show that CE-UV-ES-MS can be used as an orthogonal analytical technique to solve practical problems encountered in peptide synthesis laboratories.