title: Identification of a determinant of epidermal growth factor receptor ligand-binding specificity using a truncated, high-affinity form of the ectodomain.
abstract: Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF), and human transforming growth factor alpha (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 mEGF (and hEGF) and hTGF-alpha 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-alpha 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 equimolar ratio of ligand and receptor, leading to the formation of a 2:2 EGF:sEGFR501 dimer 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-alpha 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 Arg(45) of hEGF.
authors.affiliation: CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia.
authors.forename: T C
authors.initials: TC
authors.lastname: Elleman
authors.forename: T
authors.initials: T
authors.lastname: Domagala
authors.forename: N M
authors.initials: NM
authors.lastname: McKern
authors.forename: M
authors.initials: M
authors.lastname: Nerrie
authors.forename: B
authors.initials: B
authors.lastname: Lönnqvist
authors.forename: T E
authors.initials: TE
authors.lastname: Adams
authors.forename: J
authors.initials: J
authors.lastname: Lewis
authors.forename: G O
authors.initials: GO
authors.lastname: Lovrecz
authors.forename: P A
authors.initials: PA
authors.lastname: Hoyne
authors.forename: K M
authors.initials: KM
authors.lastname: Richards
authors.forename: G J
authors.initials: GJ
authors.lastname: Howlett
authors.forename: J
authors.initials: J
authors.lastname: Rothacker
authors.forename: R N
authors.initials: RN
authors.lastname: Jorissen
authors.forename: M
authors.initials: M
authors.lastname: Lou
authors.forename: T P
authors.initials: TP
authors.lastname: Garrett
authors.forename: A W
authors.initials: AW
authors.lastname: Burgess
authors.forename: E C
authors.initials: EC
authors.lastname: Nice
authors.forename: C W
authors.initials: CW
authors.lastname: Ward
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D015374:Biosensing Techniques; D016466:CHO Cells; D002460:Cell Line; D002645:Chickens; D006224:Cricetinae; D019281:Dimerization; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D066246:ErbB Receptors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006131:Growth Inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000096:biosynthesis / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D017384:Sequence Deletion; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000378:metabolism
chemical_list: D006131:Growth Inhibitors; D008024:Ligands; D010446:Peptide Fragments; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D066246:ErbB Receptors