Growth factor receptors of the epidermal growth factor (EGF) receptor family play pivotal roles in the regulation of cell proliferation and differentiation and are involved in the development of human cancers. It has been well documented that these receptors undergo growth factor-stimulated homo-and heterodimerization as a first step in the initiation of signaling cascades. Here we provide evidence for a new mechanism for growth factor-stimulated receptor dimer formation, designated secondary dimerization. The growth factor-induced dimerization and ensuing receptor trans-autophosphorylation results in the dissociation of the original (primary) receptor dimer. Each phosphorylated receptor monomer then interacts with a new (nonphosphorylated) receptor to form a secondary dimer. Treatment of cells with EGF yields Neu-ErbB3 secondary dimers, and heregulin treatment induces the formation of Neu-EGF receptor (secondary) dimers. The ability of EGF and heregulin to stimulate a cascade of dimerization events points to a novel mechanism by which multiple signaling activities and diverse biological responses are initiated by members of the EGF receptor family.

Eleven recently isolated erbB-transducing viruses as well as avian erythroblastosis virus (AEV)-R (ES4) and AEV-H have been characterized for the type of disease they cause, their ability to transform fibroblasts in culture, their ability to cause disease in pedigrees of chicken that differ in susceptibility to erbB-induced erythroblastosis, and the structure of their erbB genes. Differences in each of the biological parameters correlated with differences in erbB sequences encoding the C-terminal domain of the epidermal growth factor receptor (EGFR). Seven viruses were strain restricted in their ability to induce erythroblastosis and did not transform fibroblasts. These seven viruses contained v-erbB genes encoding the complete C terminus of the EGFR. AEV-R and AEV-H were not pedigree restricted in their ability to induce erythroblastosis and could transform fibroblasts. These viruses contain v-erbB genes that lack codons for the immediate C terminus of the EGFR. Three viruses caused angiosarcoma and one caused fibrosarcoma. The angiosarcoma and fibrosarcoma-inducing viruses were not strain restricted and did not cause erythroblastosis. The v-erbB genes of each of these viruses contained extensive internal deletions or 3' truncations in sequences encoding the C-terminal domain of the EGFR.

We have reported that overexpression of Neu leads to heregulin-stimulated neurite outgrowth and the tyrosine-phosphorylation of Neu and other cellular proteins in PC12 cells. Considering that Neu/ErbB2 alone is not able to functionally couple to heregulin, we looked for the possible involvement of ErbB3 in these neurite outgrowth and tyrosine phosphorylation responses. We found that heregulin stimulates the tyrosine phosphorylation of endogenous ErbB3 protein in PC12 cells and that this phosphorylation, like that of Neu, is greatly enhanced in cells that overexpress Neu. Furthermore, overexpression of ErbB3 in PC12 cells led to heregulin-stimulated neurite extension. In addition to becoming tyrosine-phosphorylated, Neu/ErbB2 and ErbB3 associate with each other, and each associates with the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase in a heregulin-dependent manner. Thus, Neu/ErbB2 and ErbB3 appear to cooperate to mediate the heregulin signal in PC12 cells. Like heregulin, epidermal growth factor (EGF) also stimulate the tyrosine phosphorylation of both Neu and ErbB3. However, there are clear differences between the EGF- and heregulin-stimulated phosphorylations of ErbB3. In the heregulin response, two tyrosine-phosphorylated forms of ErbB3 are detected. Of these, only the more quickly migrating form (on SDS-polyacrylamide gel electrophoresis) is found to be associated with Neu, whereas the other, more slowly migrating form is uniquely capable of forming stable complexes with p85. In the EGF response, at least two tyrosine-phosphorylated forms of ErbB3 are detected, but these phosphoproteins have distinctly lower apparent molecular weights compared with the heregulin-stimulated ErbB3 phosphoproteins and do not complex with p85. Thus the formation of a stable ErbB3-p85 complex in PC12 cells is a unique outcome of heregulin signaling that correlates with the differences in cell morphology induced by the activated EGF receptor and the Neu tyrosine kinase.

Retroviral vectors pDOL/NeuN and pDOL/NeuT were used to express normal and transforming rat neu cDNAs in PCI2 cells. DOL/ NeuT-infected cells exhibited a high frequency of spontaneous neurite outgrowth while DOL/NeuN-infected cells showed neurite outgrowth in the presence of heregulin, a putative ligand for the neu receptor tyrosine kinase. In both cases, neurite outgrowth was preceded by phosphorylation of p185 ncu and several other cellular proteins. Thus the neu tyrosine kinase can elicit morphological and biochemical changes resembling, but distinct from, those stimulated by NGF, and heregulin stimulates neu to elicit these effects in PCI2 cells.