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

Monique L.M Van De Poll,Anne E.G Lenferink,Marianne J.H Van Vugt,Jacqueline J.L Jacobs,Jannie W.H Janssen,Manon Joldersma,Everardus J.J Van Zoelen

doi:10.1074/jbc.270.38.22337

Monique L.M Van De Poll, Anne E.G Lenferink + Show 5 more

Open Access

https://doi.org/10.1074/jbc.270.38.22337

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Abstract

The finding that human epidermal growth factor (hEGF) and human transforming growth factor (hTGF) alpha 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 alpha and found that the region COOH-terminal of the sixth cysteine residue in hTGF alpha 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 de Poll, M. L. M., and van Zoelen, E. J. J. (1994) J. Biol. Chem. 269, 8708-8711). To analyze this domain in more detail, we now constructed four additional chimeras in which either the region between the sixth cysteine residue and the highly conserved Leu-47 was exchanged or the region COOH-terminal of Leu-47. A mutant in which the latter region in hEGF was replaced by hTGF alpha (designated E6ET) showed intermediate binding affinity, whereas replacement of the former region in hEGF by hTGF alpha was sufficient to generate a mutant (designated E6TE) with a similar high affinity for the chicken EGF receptor as wild type hTGF alpha. Furthermore, a deletion mutant of hEGF lacking three COOH-terminal amino acids, EGF50, showed intermediate binding affinity for the chicken EGF receptor similar to E6ET, but upon additional deletions (EGF49 and EGF48), this initial gain in affinity was lost. A systematic analysis of the region between the sixth cysteine residue and Leu-47 showed that the low affinity of hEGF for the chicken EGF receptor is mainly due to the presence of Arg-45. Replacement of the positively charged Arg-45 by Ala, the corresponding amino acid in hTGF alpha, was sufficient to generate a mutant growth factor with high affinity for the chicken EGF receptor. This indicates that in hEGF Arg-45 may play an important role in receptor binding. A model is proposed in which positively charged amino acids close to or within the receptor recognition site of hEGF prohibit high affinity binding to the chicken EGF receptor due to electrostatic repulsion of positively charged amino acids in the putative ligand binding domain of the chicken EGF receptor.

Highlights

Human epidermal growth factor1 and human transforming growth factor ␣ belong to the same family of growth factors
Several other amino acids in Human epidermal growth factor (hEGF) like Leu-47 (Leu-48 in human transforming growth factor (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 [5,6,7,8,9]
Mutant Growth Factors—In previous work [13], the COOHterminal region in hTGF␣ was identified as an important domain for high affinity binding of hTGF␣ to the chicken EGF receptor

Summary

An EGF Mutant with High Affinity for the Avian EGF Receptor

Amino acids in the COOH-terminal domain are responsible for high affinity binding to the chicken EGF receptor. The binding characteristics of an additional 7 hEGF/hTGF␣ exchange mutants and 3 COOH-terminal truncated forms of hEGF were investigated. A single amino acid exchange, Arg-45 to Ala, was found to be sufficient to generate an hEGF mutant with high affinity for the chicken EGF receptor

EXPERIMENTAL PROCEDURES

RESULTS

Binding affinity

DISCUSSION