An exact general analysis of ligand binding displacement and saturation curves

et al. 1998

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.

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Section: C1-----------------------------------------------c6 Ndmentioning

confidence: 88%

Section: Methodsmentioning

confidence: 99%

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

Poll

et al. 1998

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“…Expression and Purification of Recombinant Growth Factors-The expression of ZZ/FX/growth factor fusion protein in the periplasm was analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting, and the total amount of IgG binding activity in the periplasm was measured by a competitive enzyme-linked immunosorbent assay (19). The levels of expression of wild type and mutant growth factors appeared to be similar on Western blot as shown in Fig.…”

Section: Mutant Growth Factors-in Previous Workmentioning

confidence: 94%

“…Bacteria were grown in 2YTE (16 g of bactotrypton, 10 g of yeast, 8 g of NaCl/liter) at 28°C until an A 600 of 1.5 was reached and the periplasmic proteins were isolated as described (17). After purification on IgG-Sepharose (Pharmacia), the amount of fusion protein recovered was measured by a competitive enzyme-linked immunosorbent assay using biotin-labeled protein A (19). The fusion proteins were digested by Factor X a coupled to CNBr-activated Sepharose, and the growth factors were purified by an additional run on IgGSepharose.…”

Section: Methodsmentioning

confidence: 99%

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

Poll

et al. 1995

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 ...

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“…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 …”

Section: Ligand Productionmentioning

confidence: 99%

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

Zoelen

et al. 2000