The Drosophila Argos protein is the only known extracellular inhibitor of the epidermal growth factor receptor (EGFR). It is structurally related to the activating ligands, in that it is a secreted protein with a single epidermal growth factor (EGF) domain. To understand the mechanism of Argos inhibition, we have investigated which regions of the protein are essential. A series of deletions were made and tested in vivo; furthermore, by analyzing chimeric proteins between Argos and the activating ligand, Spitz (a transforming growth factor-␣-like factor), we have examined what makes one inhibitory and the other activating. Our results reveal that Argos has structural requirements that differ from all known EGFR activating ligands; domains flanking the EGF domain are essential for its function. We have also defined the important regions of the atypical Argos EGF domain. The extended B-loop is necessary, whereas the C-loop can be replaced with the equivalent Spitz region without substantially affecting Argos function. Comparison of the argos genes from Drosophila melanogaster and the housefly, Musca domestica, supports our structure-function analysis. These studies are a prerequisite for understanding how Argos inhibits the Drosophila EGFR and provide a basis for designing mammalian EGFR inhibitors.
The epidermal growth factor receptor (EGFR)1 controls many aspects of animal growth, development, and cell proliferation (1). How these different roles are coordinated and their relative importance in normal development is not yet well understood, but it is clear that overactivation of the receptor in mammals is implicated in many forms of cancer (2-5). This overactivation can be caused by amplification of the receptor gene, by activating mutations in the receptor, or frequently by overexpression of activating ligands, which causes inappropriate autocrine signaling. The EGFR is a tyrosine kinase; it has an extracellular ligand binding domain and an intracellular kinase domain. As with other receptor tyrosine kinases, it forms dimers upon activation, and these then transphosphorylate each other, causing the activation of downstream signal transduction pathways (6). There are four members of the EGFR family in mammals (ErbB1-ErbB4), and they act in a complex set of overlapping functions, both as homo-and heterodimers (7). They are activated by several classes of ligand, including EGF itself, transforming growth factor-␣ (TGF␣), neuregulins, and others. The motif responsible for receptor activation in all known ligands is an EGF domain, comprising six characteristically spaced cysteines and a few other essential amino acids (8). Antagonists of the EGFR might have therapeutic potential, and much effort has been made to understand ligand binding and activation, to help design such inhibitors (9 -14). Although several important regions have been identified, effective antagonists have remained elusive because of the apparent inability to uncouple receptor binding and activation; all mutants that still bind efficiently are acti...