Identifiation of a Determinant of Epidermal Growth Factor Receptor Ligand-Binding Specificity Using a Truncated, High-Affinity Form of the Ectodomain

摘要

Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), m6use EGF (rnEGF), and human transforming growth factor 0. (hTGF-#alpha#) with high affinity despite the significant differences in the amino acid sequences of the ligands and the receptors. In contrast, the chicken EGFR can discriminate between rnEGF (and hEGF) and hTGF-o. and binds the EGFs with approximately 100-fold lower affinity. The regions responsible for this poor binding are known to be Arg~(45) in hEGF and the L2 domain in the chicken EGFR. In this study we have produced a truncated form of the hEGFR ectodomain comprising residues 1-501 (sEGFR501), which, unlike the full-length hEGFR ectodomain (residues 1-621, sEGFR621), binds hEGF and hTGF-o. with high affinity (K_D = 13-21 and 35-40 nM, respectively). sEGFR501 was a competitive inhibitor of EGF-stimulated mitogenesis, being almost 10- fold more effective than the full-length EGFR ectodomain and three times more potent than the neutralizing anti-EGFR monoclonal antibody Mab528. Analytical ultracentrifugation showed that the primary EGF binding sites on sEGFR501 were saturated at an equifiolar ratio of ligand and receptor, leading to the formation of a 2:2 EGF:sEGFR501 dimmer complex. We have used sEGFR501 to generate three mutants with single position substitutions at Glu~(367), Gly~(441), or Glu~(472) to Lys, the residue found in the corresponding positions in the chicken EGFR. All three mutants bound hTGF-o. and were recognized by Mab528. However, mutant Gly~(441) Lys showed markedly reduced binding to hEGF, implicating Gly~(441), in the L2 domain, as part of the binding site that recognizes Arg45 of hEGF.