Deregulated signaling by the four members of the epidermal growth factor receptor tyrosine kinase family (erbB family) is implicated in the genesis or progression of human cancers. However, efforts to analyze signaling by these receptors have been hampered by the diversity of ligands and extensive interreceptor cross talk. We have expressed the four human erbB family receptors, singly and in pairwise combinations, in a pro-B-lymphocyte cell line (Ba/F3) and investigated the range of interactions activated by the epidermal growth factor homology domain of the agonist neuregulin ␤. The results provide the first comprehensive analysis of the response of this receptor family to a single peptide agonist. This peptide induced complex patterns of receptor tyrosine phosphorylation and regulation of Ba/F3 cell survival and proliferation. These data demonstrate the existence of several previously undocumented receptor interactions driven by neuregulin.Deregulated signaling by the four receptor tyrosine kinases encoded by the erbB gene family (erbB-1/epidermal growth factor receptor [EGFR], neu/erbB-2/HER2, erbB-3/HER3, and erbB-4/HER4) is implicated in human mammary cancer, ovarian cancer, gastric cancer, and glioblastoma (reviewed in reference 19). Understanding the normal and pathological functions of these receptors requires that their regulation by hormones be elucidated. One complication is that there are at least 15 different agonists for erbB family receptors, including EGF, transforming growth factor ␣, amphiregulin, betacellulin, heparin-binding EGF-like growth factor, and the several differentially spliced variants of the neuregulins (NRGs), also known as gp30 (27), heregulins (18), neu differentiation factors (35, 54), glial growth factors (28), and acetylcholine receptorinducing activity (5, 12). Some of these factors bind to and activate signaling by more than one receptor. Moreover, these ligands stimulate nonadditive receptor interactions in cells expressing multiple erbB receptor family members. For example, EGF activates neu when coexpressed with the EGFR, but EGF does not bind or activate neu expressed on its own (22,50). This transmodulation activation of neu by EGFR apparently works through the formation of EGF-driven receptor heterodimers (15, 53).NRGs were initially identified as candidate neu ligands by their ability to induce neu tyrosine phosphorylation. The longest forms of NRG contain several different modular domains, including a kringle fold, a C-2 immunoglobulin-like domain, a putative heparan sulfate proteoglycan attachment site, sites for N-and O-linked glycosylation, an EGF homology domain, a hydrophobic membrane-spanning domain, and an intracellular domain of variable length (6,18,28,35,54). Tissue-specific alternative splicing of NRG transcripts from a single gene results in many NRG isoforms containing different sets of these motifs. Moreover, alternative splicing also produces two types of EGF domain, designated ␣ and ␤ (55). ␣ and ␤ isoforms have different biological activities, which...