title: The interaction of an epidermal growth factor/transforming growth factor alpha tail chimera with the human epidermal growth factor receptor reveals unexpected complexities.
abstract: It has been assumed that substitution of homologous regions of transforming growth factor alpha (TGF-alpha) 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-alpha (mEGF/TGF-alpha44-50) results in complex features that belie this initial simplistic assumption. Comparison of EGF and mEGF/TGF-alpha44-50 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-alpha44-50 was more potent than EGF, despite its low affinity. When tested using chicken embryo fibroblasts, substitution of the TGF-alpha carboxyl-terminal tail into mEGF failed to enhance its binding affinity for chicken EGFRs; however, the chimera was intermediate in potency between TGF-alpha 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.
authors.affiliation: Cancer Research Campaign Medical Oncology Unit, Southampton General Hospital, Southampton SO16 6YD, United Kingdom.
authors.forename: S M
authors.initials: SM
authors.lastname: Puddicombe
authors.forename: L
authors.initials: L
authors.lastname: Wood
authors.forename: S G
authors.initials: SG
authors.lastname: Chamberlin
authors.forename: D E
authors.initials: DE
authors.lastname: Davies
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002455:Cell Division; D002642:Chick Embryo; D006224:Cricetinae; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000737:chemistry*; D066246:ErbB Receptors / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D008715:Methionine / Q000737:chemistry; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry*; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D016211:Transforming Growth Factor alpha / Q000737:chemistry*
chemical_list: D008024:Ligands; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D008715:Methionine; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases