Epidermal Growth Factor-related Peptides Activate Distinct Subsets of ErbB Receptors and Differ in Their Biological Activities

Numerous epidermal growth factor (EGF)-related peptide binding members of the ErbB family of receptor tyrosine kinases have been described. While several EGF agonists bind and activate ErbB-1/EGF receptor, neu differentiation factor (NDF) functions as a ligand for ErbB-3 and ErbB-4. However, it is currently unknown which specific subsets of ErbB receptors become activated in response to each of these ligands. The present study addresses this issue using the T47D breast tumor cell line, which expresses moderate levels of all the presently known ErbB receptors. We show that all the EGF agonists, but not NDF, stimulated tyrosine phosphorylation of ErbB-1. In contrast, all the EGF-related factors except amphiregulin were able to induce tyrosine phosphorylation of ErbB-2. The ability to induce tyrosine phosphorylation of ErbB-3 varied dramatically among the different EGF-related peptides. While EGF, transforming growth factor (TGF)-α, and amphiregulin only had a moderate effect, NDF dramatically increased the ErbB-3 phosphotyrosine content. Most notably, heparin binding EGF-related growth factor (HB-EGF) and betacellulin (BTC) were more effective than other EGF agonists. Consequently, only NDF, HB-EGF, and BTC significantly stimulated association of phosphatidylinositol kinase activity with ErbB-3. Among the EGF agonists, HB-EGF induced a low level of ErbB-4 tyrosine phosphorylation, while BTC was as efficient as NDF in activating ErbB-4. The BTC activation of ErbB-4 appears to be independent of ErbB-1, as shown by pretreatment of cells with an antibody that inhibits binding of EGF agonists to ErbB-1. As a result of the differential activation of ErbB receptors, most of the EGF-related growth factors had distinguishable biological activities on cultured mammary epithelial cell lines. Numerous epidermal growth factor (EGF)-related peptide binding members of the ErbB family of receptor tyrosine kinases have been described. While several EGF agonists bind and activate ErbB-1/EGF receptor, neu differentiation factor (NDF) functions as a ligand for ErbB-3 and ErbB-4. However, it is currently unknown which specific subsets of ErbB receptors become activated in response to each of these ligands. The present study addresses this issue using the T47D breast tumor cell line, which expresses moderate levels of all the presently known ErbB receptors. We show that all the EGF agonists, but not NDF, stimulated tyrosine phosphorylation of ErbB-1. In contrast, all the EGF-related factors except amphiregulin were able to induce tyrosine phosphorylation of ErbB-2. The ability to induce tyrosine phosphorylation of ErbB-3 varied dramatically among the different EGF-related peptides. While EGF, transforming growth factor (TGF)-α, and amphiregulin only had a moderate effect, NDF dramatically increased the ErbB-3 phosphotyrosine content. Most notably, heparin binding EGF-related growth factor (HB-EGF) and betacellulin (BTC) were more effective than other EGF agonists. Consequently, only NDF, HB-EGF, and BTC significantly stimulated association of phosphatidylinositol kinase activity with ErbB-3. Among the EGF agonists, HB-EGF induced a low level of ErbB-4 tyrosine phosphorylation, while BTC was as efficient as NDF in activating ErbB-4. The BTC activation of ErbB-4 appears to be independent of ErbB-1, as shown by pretreatment of cells with an antibody that inhibits binding of EGF agonists to ErbB-1. As a result of the differential activation of ErbB receptors, most of the EGF-related growth factors had distinguishable biological activities on cultured mammary epithelial cell lines.