Argos inhibits epidermal growth factor receptor signalling by ligand sequestration.

The epidermal growth factor receptor (EGFR) has critical functions in development and in many human cancers. During development, the spatial extent of EGFR signalling is regulated by feedback loops comprising both well-understood activators and less well-characterized inhibitors. In Drosophila melanogaster the secreted protein Argos functions as the only known extracellular inhibitor of EGFR, with clearly identified roles in multiple stages of development. Argos is only expressed when the Drosophila EGFR (DER) is activated at high levels, and downregulates further DER signalling. Although there is ample genetic evidence that Argos inhibits DER activation, the biochemical mechanism has not been established. Here we show that Argos inhibits DER signalling without interacting directly with the receptor, but instead by sequestering the DER-activating ligand Spitz. Argos binds tightly to the EGF motif of Spitz and forms a 1:1 (Spitz:Argos) complex that does not bind DER in vitro or at the cell surface. Our results provide an insight into the mechanism of Argos function, and suggest new strategies for EGFR inhibitor design.

Molecular examination of the transmembrane requirements of the platelet-derived growth factor beta receptor for a productive interaction with the bovine papillomavirus E5 oncoprotein.

The small transmembrane E5 protein of bovine papillomavirus (BPV) transforms cells by forming a stable complex with and activating the platelet-derived growth factor beta receptor (PDGFbetaR). The E5/PDGFbetaR interaction is thought to involve specific physical contacts between the transmembrane domains of the two proteins. Lys(499) at the extracellular juxtamembrane position and Thr(513) within the transmembrane domain of the PDGFbetaR are required for the interaction and are predicted to contact analogously positioned residues in the E5 protein. Here, mutagenic analysis of the transmembrane region of the PDGFbetaR was performed to further characterize the nature of the E5/PDGFbetaR interaction. We show that the receptor transmembrane domain, with minimal extracellular and intracellular sequence, is sufficient for the interaction. In addition, we provide evidence that the polar nature of Thr(513) as well as its positioning along the transmembrane alpha-helix is important for the interaction. We also identify the receptor transmembrane amino acids Ile(506) and Leu(520) as additional requirements for the interaction. Because Lys(499), Thr(513), Ile(506), and Leu(520) all align along the same face of the predicted PDGFbetaR transmembrane alpha-helix, our data support the model that the PDGFbetaR contacts the E5 protein via multiple amino acids along a single alpha-helical interface.

Multiple transmembrane amino acid requirements suggest a highly specific interaction between the bovine papillomavirus E5 oncoprotein and the platelet-derived growth factor beta receptor.

The bovine papillomavirus E5 protein activates the cellular platelet-derived growth factor beta receptor (PDGFbetaR) tyrosine kinase in a ligand-independent manner. Evidence suggests that the small transmembrane E5 protein homodimerizes and physically interacts with the transmembrane domain of the PDGFbetaR, thereby inducing constitutive dimerization and activation of this receptor. Amino acids in the receptor previously found to be required for the PDGFbetaR-E5 interaction are a transmembrane Thr513 and a juxtamembrane Lys499. Here, we sought to determine if these are the only two receptor amino acids required for an interaction with the E5 protein. Substitution of large portions of the PDGFbetaR transmembrane domain indicated that additional amino acids in both the amino and carboxyl halves of the receptor transmembrane domain are required for a productive interaction with the E5 protein. Indeed, individual amino acid substitutions in the receptor transmembrane domain identified roles for the extracellular proximal transmembrane residues in the interaction. These data suggest that multiple amino acids within the transmembrane domain of the PDGFbetaR are required for a stable interaction with the E5 protein. These may be involved in direct protein-protein contacts or may support the proper transmembrane alpha-helical conformation for optimal positioning of the primary amino acid requirements.