Identification of Regions of the Epidermal Growth Factor Receptor Involved in Ligand Binding Specificity Using a High Affinity Form of the Ectodomain

Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF) and human transforming growth factor-α (h-TGF-α) with high affinity despite significant differences in the amino acid sequences of the ligands and the receptors [1]. In contrast, the chicken EGFR can discriminate between mEGF or hEGF and hTGF-α, the EGFs having approximately 100-fold lower affinity [2]. The regions responsible for this poor binding are known to be Arg45 in hEGF [3] and the L2 domain in the chicken EGFR [4]. In this study we have produced a truncated form of the hEGFR ectodomain comprising domains L1/CR1/L2 plus the first module of the second Cys-rich region, CR2 (residues 1–501, sEGFR501). This truncated receptor was used to characterise its ligand-binding properties, including its ability to form ligand-induced dimers and to act as an inhibitor of EGF-stimulated mitogenic responses in BaF cells transfected with the EGFR [5]. In addition, based on a model of the EGFR extracellular domain [6] we have generated three mutants of sEGFR501 with single position substitutions at Glu367, Gly441 or Glu472 to Lys, the residue found in the corresponding positions in the chicken EGFR, to investigate the residues responsible for ligand discrimination.