Tc. Elleman et al., Identification of a determinant of epidermal growth factor receptor ligand-binding specificity using a truncated, high-affinity form of the ectodomain, BIOCHEM, 40(30), 2001, pp. 8930-8939
Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (
hEGF), mouse EGF (mEGF), and human transforming growth factor alpha (hTGF-a
lpha) with high affinity despite the significant differences in the amino a
cid sequences of the ligands and the receptors. In contrast, the chicken EG
FR can discriminate between mEGF (and hEGF) and hTGF-alpha and binds the EG
Fs 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-len
gth 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 po
tent 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 ge
nerate three mutants with single position substitutions at Glu(367), Gly(44
1). 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 b
y Mab528. However, mutant Gly(441)Lys showed markedly reduced binding to hE
GF, implicating Gly441, in the L2 domain, as part of the binding site that
recognizes Arg(45) of hEGF.