The Interaction of an Epidermal Growth Factor/Transforming Growth Factor α Tail Chimera with the Human Epidermal Growth Factor Receptor Reveals Unexpected Complexities

• Published in: The Journal of biological chemistry 1996-11, Vol.271 (48), p.30392-30397
• Main Authors: Puddicombe, Sarah M., Wood, Lynn, Chamberlin, Stephen G., Davies, Donna E.
• Format: Article
• Language: English
• Subjects: Animals; Binding, Competitive; Cell Division; Chick Embryo; CHO Cells; Cricetinae; Enzyme Activation; Epidermal Growth Factor - chemistry; ErbB Receptors - chemistry; Humans; Ligands; Methionine - chemistry; Receptor Protein-Tyrosine Kinases - chemistry; Recombinant Fusion Proteins; Signal Transduction; Structure-Activity Relationship; Transforming Growth Factor alpha - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.48.30392

It has been assumed that substitution of homologous regions of transforming growth factor α (TGF-α) into epidermal growth factor (EGF) can be used to probe ligand-receptor recognition without detrimental effects on ligand characteristics for the human EGF receptor (EGFR). We show that a chimera of murine (m) EGF in which the carboxyl-terminal tail is substituted for that of TGF-α (mEGF/TGF-α44-50) results in complex features that belie this initial simplistic assumption. Comparison of EGF and mEGF/TGF-α44-50 in equilibrium binding assays showed that although the relative binding affinity of the chimera was reduced 80-200-fold, it was more potent than EGF in mitogenesis assays using NR6/HER cells. This superagonist activity could not be attributed to differences in ligand processing or to binding to other members of the c-erbB family. It 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; it also appeared to be related to the ability of the chimera to activate the EGFR tyrosine kinase. Thus, when EGFR autophosphorylation was measured, mEGF/TGF-α44-50 was more potent than EGF, despite its low affinity. When tested using chicken embryo fibroblasts, substitution of the TGF-α carboxyl-terminal tail into mEGF failed to enhance its binding affinity for chicken EGFRs; however, the chimera was intermediate in potency between TGF-α and mEGF in mitogenesis assays. Our results suggest a contextual requirement for EGFR recognition which is ligand-specific. Further, the unpredictable responses to chimeric ligands underline the complex nature of the processes of ligand recognition, receptor activation, and the ensuing cellular response.