A.E.G. Lenferink

Differential endocytic routing of homo- and hetero-dimeric ErbB tyrosine kinases confers signaling superiority to receptor heterodimers

Both homo‐ and hetero‐dimers of ErbB receptor tyrosine kinases mediate signaling by a large group of epidermal growth factor (EGF)‐like ligands. However, some ligands are more potent than others, although they bind to the same direct receptor. In addition, signaling by receptor heterodimers is superior to homodimers. We addressed the mechanism underlying these two features of signal tuning by using three ligands: EGF; transforming growth factor α (TGFα); and their chimera, denoted E4T, which act on cells singly expressing ErbB‐1 as a weak, a strong, and a very strong agonist, respectively. Co‐expression of ErbB‐2, a developmentally important co‐receptor whose expression is frequently elevated in human cancers, specifically potentiated EGF signaling to the level achieved by TGFα, an effect that was partially mimicked by ErbB‐3. Analysis of the mechanism underlying this trans‐potentiation implied that EGF‐driven homodimers of ErbB‐1 are destined for intracellular degradation, whereas the corresponding heterodimers with ErbB‐2 or with ErbB‐3, dissociate in the early endosome. As a consequence, in the presence of either co‐receptor, ErbB‐1 is recycled to the cell surface and its signaling is enhanced. This latter route is followed by TGFα‐driven homodimers of ErbB‐1, and also by E4T‐bound receptors, whose signaling is further enhanced by repeated cycles of binding and dissociation from the receptors. We conclude that alternative endocytic routes of homo‐ and hetero‐dimeric receptor complexes may contribute to tuning and diversification of signal transduction. In addition, the ability of ErbB‐2 to shunt ligand‐activated receptors to recycling may explain, in part, its oncogenic potential.

The oncogenic ErbB-2/ErbB-3 heterodimer is a surrogate receptor of the epidermal growth factor and betacellulin

The ErbB-1 receptor tyrosine kinase binds to six different growth factors, whose prototype is the epidermal growth factor (EGF). Two homologous epithelial receptors, ErbB-3 and ErbB-4, bind all isoforms of another family of growth factors, the Neu differentiation factors (NDFs/neuregulins). The fourth member of the ErbB family, ErbB-2, acts as the preferred heterodimeric partner of ligand-occupied complexes of the three other ErbB proteins. Here we report that at high concentrations, EGF can induce cell growth and differentiation in the absence of ErbB-1. This function is shared by betacellulin, but not by three other ligands, including the transforming growth factor α (TGFα). The functional receptor was identified as a heterodimer between ErbB-3 and ErbB-2, a previously identified oncogenic complex. When singly expressed, neither ErbB-3 nor ErbB-2 can mediate signaling by EGF. In addition, when co-expressed, blocking either receptor by using site-specific antibodies inhibited EGF and betacellulin activities, indicating strict cooperativity between ErbB-3 and ErbB-2. Through analysis of chimeras between EGF and TGFα, we identified the middle portion of EGF (loopu2009B) as the site that enables activation of ErbB-2/ErbB-3. In conclusion, cooperative and promiscuous binding of stroma-derived growth factors by the epithelium-expressed ErbB-2/ErbB-3 heterodimer may be significant to cancer development. The mechanistic implications of our results for a model that attributes receptor dimerization to ligand bivalency, as well as to a recently proposed mechanism of secondary dimerization, are discussed.

The EGF domain: requirements for binding to receptors of the ErbB family.

Epidermal growth factor (EGF) has been the prototype growth-stimulating peptide for many years. It has a characteristic structure with three disulfide bridges, which is essential for its activity. However, many other proteins, including both growth factors and proteins with unrelated functions, have similar EGF-like domains. This indicates that besides a characteristic conformation provided by the EGF-like domain, specific amino acids are required to provide specificity in protein functioning. Currently, more than 10 different growth factors with an EGF-like domain have been characterized which all exert their action by binding to the four members of the erbB family of receptors. In this review, studies are described on the structure-function relationship of these EGF-like growth factor molecules in an attempt to analyze the individual amino acids that determine their binding specificity to the individual members of the erbB family.

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

The finding that human epidermal growth factor (hEGF) and human transforming growth factor (hTGF) α 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 hTGFα and found that the region COOH-terminal of the sixth cysteine residue in hTGFα 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 hTGFα (designated E6ET) showed intermediate binding affinity, whereas replacement of the former region in hEGF by hTGFα was sufficient to generate a mutant (designated E6TE) with a similar high affinity for the chicken EGF receptor as wild type hTGFα. 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 hTGFα, 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.

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

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 DrosophilaNotch 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 hEGF-derived 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.

Rational Design for the Development of Epidermal Growth Factor Receptor Antagonists

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) bind with similar high affinity to the human EGF receptor. Using a domain-exchange strategy we have shown that the C-terminal linear region of these molecules is involved in high affinity receptor binding. By further single amino acid substitution in this linear C-terminal region, a putative interaction site of these ligands with their receptor has been identified. This identification of a receptor binding domain in EGF/TGF alpha provides an important initial step in the development of EGF receptor antagonists with significant clinical potential.