The interaction of a chimeric EGF containing the C-tail of TGFa with the EGF receptor reveals hidden ligand complexities

It has been assumed that substitution of homologous regions of TGFû into EGF can be used to probe ligand:receptor recognition without detrimental effects on ligand characteristics for the human EGFR. We show that a chimera of EGF in which the C-tail is substituted for that of TGFa results in complex features which belie this initial simplistic assumption. Comparison of EGF and the chimera in equilibrium binding assays showed that the relative binding affinity of the chimera was reduced 60 fold. Surprisingly, the chimera was more potent than EGF in mitogenesis assays using NR6/HER cells. This superagonist activity appeared to be due, in part, to choice of an EGFR overexpressing target cell where high receptor number compensated for the low affinity of the ligand. However, the enhanced mitogenic activity relative to EGF suggested a further unusual property of the chimera. Examination of EGFR tyrosine kinase activity showed that the chimera was at least as potent as EGF, despite its low affinity. Our results suggest a contextual requirement for EGFR recognition which is ligand specific. Further, the unpredictable responses to chimeric Hgands underlines the complex nature of the processes of ligand recognition, receptor activation and the ensuing cellular response.