In Vivo Analysis of Argos Structure-Function

Abstract

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...

Argos Mutants Define an Affinity Threshold for Spitz Inhibition in Vivo

“…Consistent with its role as an inhibitor of dEGFR signaling, Aos knock-outs exhibit phenotypes typical of dEGFR gain-of-function mutants, and Aos misexpression inhibits dEGFR signaling (26,29). Aos contributes importantly to the spatio-temporal regulation of dEGFR signaling through its participation in a negative feedback loop as a result of Spi-dependent dEGFR signaling.…”

“…However, recent studies have shown that Aos inhibits dEGFR signaling by binding to the activating ligand Spi rather than associating with the receptor itself (25), bringing the significance of the atypical EGF-like domain in Aos into question. As pointed out by Howes et al (26), sequences outside the putative EGF motif of Aos are strongly conserved in the house fly Musca domestica (representing an evolutionary distance of ϳ100 million years) and are required for Aos function in vivo, further suggesting that more than just the putative EGF-like domain is required for Aos function. We have expanded upon these observations by identifying Aos orthologs outside the dipteran lineage (Fig.…”

“…The NCR and CCR are defined primarily by sets of 4 and 12 conserved cysteines, respectively. Previous studies have indicated that the CCR represents the primary ligand-binding domain and can provide a measure of activity in vivo (25,26). In contrast, no direct functionality has been ascribed to the NCR.…”

“…Misexpression of wild type aos in developing photoreceptors (using sevenless-GAL4) in an aos mutant background rescues the knock-out phenotype and restores the eye to nearly wild type morphology (Fig. 5B) (26,29). Consistent with the observation that aos P372S retains considerable activity in the suppression assays, we observed significant (but not complete) rescue of the aos null phenotype with this allele (Fig.…”

“…1B) and contains a putative EGF-like domain (residues 363-424) (24). Misexpression of the entire CCR from Aos (residues 225-444) displays partial activity in vivo and is sufficient for binding Spi in vitro (albeit with a ϳ20-fold decreased affinity) (25,26). In contrast, misexpression of the putative EGF-like domain alone does not rescue Aos mutant phenotypes (26), arguing that (if it does adopt an EGFlike fold) it is not sufficient for Aos function.…”