Identification of the Minimal Requirements for Binding to the Human Epidermal Growth Factor (EGF) Receptor Using Chimeras of Human EGF and an EGF Repeat of Drosophila Notch

Abstract

Many proteins contain so-called epidermal growth factor (EGF)-like domains that share the characteristic spacing of cysteines and glycines with members of the EGF family. They are, however, functionally unrelated, despite the fact that the three-dimensional structure of these EGF-like domains, also, is often very similar to that of the EGF receptor agonists. In the present study, we linked an EGF-like repeat from the Drosophila Notch protein to the N-and C-terminal linear tail sequences of human EGF (hEGF), and we showed that this chimera (E1N6E) is unable to bind or activate the hEGF receptor. This recombinant protein was then used as a basic construct for identifying the minimal requirements for high affinity EGF receptor binding and activation. We selectively reintroduced a limited number of important hEGFderived residues, and by using this unique approach, we were able to make hEGF/Notch chimeras that, compared with wild type hEGF, showed nearly 100% binding affinity and mitogenic activity on HER-14 cells expressing the hEGF receptor.Polypeptide growth factors acting through their tyrosine kinase receptors play a central role in the outgrowth of tumors. Many tumor cells are able to secrete such growth factors, to which they can respond in an autocrine fashion. High expression of members of the epidermal growth factor (EGF) 1 family, in particular of transforming growth factor ␣ (TGF␣), has been found in a variety of human cancers including breast and prostate cancers; oral and head and neck cancers; tumors of the gastrointestinal tract; and colon, lung, liver, kidney, and ovarian cancers (1-4). Because most of these tumors also express the EGF receptor, there is evidence that TGF␣ cause autocrine growth stimulation in these tumors. In order to interfere with such autocrine processes, it might be of great interest to develop an EGF receptor antagonist, an EGF-like protein with high receptor binding affinity that is, however, unable to activate the receptor. Although attempts to design such an antagonist have as yet been unsuccessful, much progress has been made in elucidating the requirements for interaction of EGF with its receptor.

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

“…Mutant forms of EGF have been identified with only low ErbB-1 binding affinity that show similar dose-dependent mitogenic activity as wild-type EGF, particularly on cells overexpressing ErbB-1 (14)(15)(16)(17)(18). This shows that there is no direct correlation between the affinity of EGF-like growth factors and their mitogenic potential.…”

A Bispecific Enediyne-Energized Fusion Protein Containing Ligand-Based and Antibody-Based Oligopeptides against Epidermal Growth Factor Receptor and Human Epidermal Growth Factor Receptor 2 Shows Potent Antitumor Activity

“…The fusion protein was created by linking two oligopeptides (Ec and Hr) specific for EGFR and HER2 to lidamycin. Research showed that the COOH-terminal region of EGF is sufficient for high-affinity receptor binding (16); therefore the C-loop of EGF (22 amino acids of EGF COOH terminal) was used as EGFR targeting molecule. Because HER2 has no natural ligand and some experiments proved that the V H CDR3 loop is the central, most accessible antigen-binding segment in an intact antibody (17)(18)(19), the V H CDR3 loop of anti-erbB2 antibody C6.5 was used as a HER2 targeting molecule.…”

Ligand depletion negatively controls the mitogenic activity of epidermal growth factor

“…However, when reintroducing amino acids from the base of the B-loop of hEGF into this chimera, the affinity for ErbB1 is gradually restored, such that E3N4E/MY has a binding affinity of 1.6%, E3N4E/MA of 29% and E3N4E/MYA of 87% compared to wild-type hEGF [22]. Growth stimulation experiments on NIH3T3 cells transfected with human ErbB1 (HER-14 cells) showed that in spite of these differences in binding affinity EGF, E3N4E/MYA, E3N4E/MA and E3N4E/MY give very similar dose-response curves for mitogenic activation, while only E3N4E itself is clearly less potent [22].…”

“…In order to understand the relation between ErbB1 binding and mitogenic activity in more detail, we have analysed in this study a set of EGF-Notch chimeras, which show a gradual decrease in ErbB1 binding affinity with increasing Notch content but maintain a high mitogenic activity [22]. Here, we show that the mitogenic activity of these low affinity EGF mutants strongly depends on the ErbB1 density of the cell.…”

“…Chimeras between hEGF and EGF repeat 13 of Drosophila Notch were generated by exchanging domains bordered by the shared cysteine residues as described previously [22]. E3N4E is a chimera composed of EGF in which the B-loop (amino acids 21-30) has been exchanged for the corresponding 8 amino acids HDKINGFK sequence from Notch.…”