Epigen, the last ligand of ErbB receptors, reveals intricate relationships between affinity and mitogenicity

Four ErbB receptors and multiple growth factors sharing an epidermal growth factor (EGF) motif underlie transmembrane signaling by the ErbB family in development and cancer. Unlike other ErbB proteins, ErbB-2 binds no known EGF-like ligand. To address the existence of a direct ligand for ErbB-2, we applied algorithms based on genomic and cDNA structures to search sequence data bases. These searches reidentified all known EGF-like growth factors including Epigen (EPG), the least characterized ligand, but failed to identify novel factors. The precursor of EPG is a widely expressed transmembrane glycoprotein that undergoes cleavage at two sites to release a soluble EGF-like domain. A recombinant EPG cannot stimulate cells singly expressing ErbB-2, but it acts as a mitogen for cells expressing ErbB-1 and co-expressing ErbB-2 in combination with the other ErbBs. Interestingly, soluble EPG is more mitogenic than EGF, although its binding affinity is 100-fold lower. Our results attribute the anomalous mitogenic power of EPG to evasion of receptor-mediated depletion of ligand molecules, as well as to inefficient receptor ubiquitylation and down-regulation. In conclusion, EPG might represent the last EGF-like growth factor and define a category of low affinity ligands, whose bioactivity differs from the more extensively studied high affinity ligands.