A Single Amino Acid Exchange, Arg-45 to Ala, Generates an Epidermal Growth Factor (EGF) Mutant with High Affinity for the Chicken EGF Receptor

Abstract

The finding that human epidermal growth factor (hEGF) and human transforming growth factor (hTGF) ␣ bind with similar affinity to the human EGF receptor but differ in their affinity for the chicken EGF receptor was used as a model system to study ligand-receptor interaction of EGF receptor agonists. We previously constructed domain-exchange mutants of hEGF and hTGF␣ and found that the region COOH-terminal of the sixth cysteine residue in hTGF␣ is important for high affinity binding to the chicken EGF receptor (Kramer, R. H., Lenferink, A. E. G., Lammerts van Bueren-Koornneef, I., van der Meer, A., van Human epidermal growth factor (hEGF) 1 and human transforming growth factor (hTGF) ␣ belong to the same family of growth factors. They both bind with high affinity to the human EGF receptor, but hEGF has a 10 -50-fold lower affinity for the chicken EGF receptor than hTGF␣ (1). All members of the EGF family are characterized by the presence of six identically spaced cysteine residues, which form three intramolecular disulfide bridges. Together with some highly conserved glycine residues they are essential for the correct three-dimensional structure of the growth factor and for high affinity binding to the EGF receptor (2-4). Several other amino acids in hEGF like Leu-47 (Leu-48 in hTGF␣) and Arg-41 (Arg-42 in hTGF␣), which are not involved in maintaining structural integrity, have been shown to be crucial for high affinity binding to the EGF receptor, which suggests that they form part of the binding domain (5-9). The crystal structure of hEGF or hTGF␣ is not available, and most of the information on the structure of these growth factors has come from detailed 1 H NMR studies. Based on the observation that amino acids surrounding the second cysteine residue are in close contact with amino acids near the sixth cysteine residue, it has been postulated that Tyr-13/Leu-15/His-16 together with Arg-41/Gln-43/Leu-47 form the binding site in hEGF (10 -12). The exact region involved in binding to the receptor is still not known, however, and this has hampered the design of receptor antagonists.To gain more insight in the way hEGF and hTGF␣ bind to their receptor, we recently used the difference in binding affinity of these growth factors for the chicken EGF receptor as a model system. A total of 10 hEGF/hTGF␣ chimeras were constructed in which regions bordered by the highly conserved cysteine residues were exchanged, and their relative binding affinity for the chicken EGF receptor was assessed (13). Introduction of the region COOH-terminal of the sixth cysteine residue of hTGF␣ into hEGF appeared to be sufficient to confer high affinity binding characteristics to hEGF, and, in line with this, an exchange of the same region in hTGF␣ with the corresponding hEGF sequence caused hTGF␣ to lose its high affinity for the chicken EGF receptor. These data indicate that the COOH-terminal region in EGF receptor agonists plays an important role in receptor binding. In a recent 1 H NMR study (14), it has been shown that this region of ...

The Asn-420-linked Sugar Chain in Human Epidermal Growth Factor Receptor Suppresses Ligand-independent Spontaneous Oligomerization

“…The analyses, which used a series of chimeric chicken/human EGFRs, indicated that domain III of the chicken receptor is responsible for this lower affinity to EGF (22). Although structural determinants for the distinct binding properties of the chicken receptor have been investigated in terms of the difference in the primary structure of domain III (32)(33)(34), this could, in part, be explained by the lack of the equivalent sugar chain whose loss leads to the impairment of EGF binding in human EGFR. Nevertheless, the issue of whether the sugar chain is directly associated with the binding currently remains unclear, and it is also possible that the sugar chain may be involved in the correct folding of the domain and/or is required to maintain the conformation of domain III, so that efficient EGF binding can take place.…”

Superagonistic activation of ErbB-1 by EGF-related growth factors with enhanced association and dissociation rate constants

“…In a previous study we have shown that chimeras of hEGF and hTGFα, obtained by exchanging domains bordered by their conserved cysteine residues, all have similar binding affinity for ErbB-1 (23). In the nomenclature used, a chimera such as E4T stands for a ligand with hEGF sequences N-terminal and hTGFα sequences C-terminal of the fourth cysteine (19).…”

“…The yield of purified fusion protein was determined in an ELISA-like binding competition on IgG antibodies using biotinlabeled protein A as competitor (22), while the biological activity of the samples was determined in a binding competition assay with iodinated murine (m)EGF on HER-14 cells (23 …”

Structural Analysis of an Epidermal Growth Factor/Transforming Growth Factor-α Chimera with Unique ErbB Binding Specificity

“…T1E not only maintains high affinity for ErbB1, the natural receptor for both EGF and TGF␣, but has in addition gained the ability to bind to ErbB2⅐ErbB3 heterodimers with nearly identical affinity as NRG-1 (26). Subsequent mutation analysis showed that the TGF␣ residues His 4 and Phe 5 in the N terminus and the EGF residue Leu 28 at the tip of the B-loop are essential for the high affinity of T1E for ErbB2⅐ErbB3 heterodimers. Replacement of Leu 28 for the Glu residue present at the equivalent position in TGF␣ strongly impaired binding to ErbB2⅐ErbB3 heterodimers without affecting binding to ErbB1 (26).…”

“…Subsequent mutation analysis showed that the TGF␣ residues His 4 and Phe 5 in the N terminus and the EGF residue Leu 28 at the tip of the B-loop are essential for the high affinity of T1E for ErbB2⅐ErbB3 heterodimers. Replacement of Leu 28 for the Glu residue present at the equivalent position in TGF␣ strongly impaired binding to ErbB2⅐ErbB3 heterodimers without affecting binding to ErbB1 (26). These results indicated that the combination of residues in the N terminus of TGF␣ and in the B-loop region of EGF mediate the enhanced binding affinity for ErbB2⅐ErbB3 heterodimers.…”