Insertion of Argos Sequences into the B-Loop of Epidermal Growth Factor Results in a Low-Affinity Ligand with Strong Agonistic Activity

Poll, Monique L.M. van de; Vugt, Marianne J.H. van; Lenferink, Anne E.G.; Zoelen, E.J.J. van

doi:10.1021/bi970227f

Cited by 23 publications

(19 citation statements)

References 29 publications

(38 reference statements)

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“…7A and B), like Aos, suggesting that the C-terminal region of Aos containing the EGF-like domain is important for Aos's functions. Many studies have been performed on the structurefunction relationship of EGF-like molecules, aimed at the development of EGFR antagonists (24,62). Vein is a moderate activator of DER and can be converted into an inhibitor by exchanging its EGF domain for that of Aos (52).…”

Section: Discussionmentioning

confidence: 99%

The Interaction between the Drosophila Secreted Protein Argos and the Epidermal Growth Factor Receptor Inhibits Dimerization of the Receptor and Binding of Secreted Spitz to the Receptor

Jin

Sawamoto

Ito

et al. 2000

Molecular and Cellular Biology

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Drosophila Argos (Aos), a secreted protein with an epidermal growth factor (EGF)-like domain, has been shown to inhibit the activation of the Drosophila EGF receptor (DER). However, it has not been determined whether Aos binds directly to DER or whether regulation of the DER activation occurs through some other mechanism. Using DER-expressing cells (DER/S2) and a recombinant DER extracellular domain-Fc fusion protein (DER-Fc), we have shown that Aos binds directly to the extracellular domain of DER with its carboxyl-terminal region, including the EGF-like domain. Furthermore, Aos can block the binding of secreted Spitz (sSpi), a transforming growth factor ␣-like ligand of DER, to the extracellular domain of DER. We observed that sSpi stimulates the dimerization of both the soluble DER extracellular domain (sDER) and the intact DER in the DER/S2 cells and that Aos can block the sSpi-induced dimerization of both sDER and intact DER. Moreover, we have shown that, by directly interacting with DER, Aos and SpiAos (a chimeric protein that is composed of the N-terminal region of Spi and the C-terminal region of Aos) inhibit the dimerization and phosphorylation of DER that are induced by DER's overexpression in the absence of sSpi. These results indicate that Aos exerts its inhibitory function through dual molecular mechanisms: by blocking both the receptor dimerization and the binding of activating ligand to the receptor. This is the first description of this novel inhibitory mechanism for receptor tyrosine kinases.The epidermal growth factor (EGF) receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases (RTKs), which are composed of an extracellular domain, a transmembrane region, and a cytoplasmic domain, which includes a tyrosine kinase domain (5, 20) (see Fig. 1A). The binding of EGF to its receptor induces conformational changes in the extracellular domain (18), resulting in rapid dimerization of the receptor (3,8,25). In its dimerized state, the activated tyrosine kinase phosphorylates tyrosine in the carboxyl-terminal region of the adjacent receptor through an intermolecular mechanism (23,29,57).Like its vertebrate homologues, the Drosophila EGFR (DER) mediates various inductive signaling events in several tissues to regulate normal development (1,42,50,55). DER signaling functions principally through the Ras/mitogen-activated protein kinase (MAPK) signal transduction pathway, which is highly conserved between Drosophila and mammals (14, 40). The loss-of-function mutant phenotypes of DER indicate that DER regulates a variety of developmental processes, including the survival of embryonic ectodermal tissues, the proliferation of imaginal discs, the morphogenesis of several adult ectodermal structures, and neural differentiation (7, 55). Since DER signaling is involved in many different aspects of development, like other members of the ErbB family, its activation must be controlled precisely. Evidence from genetic and biochemical analyses indicates that both activating and inhibitory ligands reg...

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Section: Discussionmentioning

confidence: 99%

The Interaction between the Drosophila Secreted Protein Argos and the Epidermal Growth Factor Receptor Inhibits Dimerization of the Receptor and Binding of Secreted Spitz to the Receptor

Jin

Sawamoto

Ito

et al. 2000

Molecular and Cellular Biology

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“…Fractions containing low affinity B-loop mutants were identified by dot blot analysis using a polyclonal antibody (Ab-3) raised against recombinant wild type hEGF (Oncogene Science Inc., Cambridge, MA). Previous studies have shown that this antibody also recognizes hEGF mutants with altered B-loop sequences (11). Probed proteins were detected by a goat anti-rabbit antibody linked to horseradish peroxidase and visualized by enhanced chemiluminescence (Boehringer Mannhein).…”

Section: Methodsmentioning

confidence: 99%

“…2 This again emphasizes the importance of this region in receptor recognition. The B-loop, at the opposite site of the molecule, is, however, much less conserved among EGF receptor agonists, and we have recently shown, using chimeras of hEGF and the Drosophila EGF receptor antagonist Argos, that several nonconservative substitutions can be made in the B-loop of hEGF without a significant effect on binding affinity (11).…”

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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

Vugt

Lenferink

et al. 1998

Journal of Biological Chemistry

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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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“…Previous work from our group has shown a direct correlation between MAPK activation and mitogenic activity of EGF-like growth factors (15). In order to obtain more insight in the kinetics of receptor activation, we compared the doseand time dependence of MAPK phosphorylation by the superagonistic chimeras to those of the other EGF-like ligands.…”

Section: Map-kinase Activation By Egf/tgfα Chimerasmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Lenferink

Zoelen

Vugt

et al. 2000

Journal of Biological Chemistry

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Insertion of Argos Sequences into the B-Loop of Epidermal Growth Factor Results in a Low-Affinity Ligand with Strong Agonistic Activity

Cited by 23 publications

References 29 publications

The Interaction between the Drosophila Secreted Protein Argos and the Epidermal Growth Factor Receptor Inhibits Dimerization of the Receptor and Binding of Secreted Spitz to the Receptor

The Interaction between the Drosophila Secreted Protein Argos and the Epidermal Growth Factor Receptor Inhibits Dimerization of the Receptor and Binding of Secreted Spitz to the Receptor

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

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

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