Chicken Epidermal Growth Factor Receptor Research Articles

Roles of transforming growth factor-alpha and epidermal growth factor in chick limb development.

We have examined the distribution of transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and the chicken EGF receptor (c-erbB), in embryonic chick limbs. Prior to limb budding, TGF-alpha is present in prospective limb-forming mesoderm and in prospective apical ectodermal ridge (AER)-forming ectoderm, but is not detected in non-limb-forming flank mesoderm or ectoderm, nor in presumptive non-AER-forming limb ectoderm, suggesting possible roles in initial limb formation and AER induction. Consistent with this possibility, TGF-alpha is present in the mesoderm of the wing buds of the amelic chick mutants limbless and wingless, which form and bud normally, but is absent from limbless and wingless ectoderm, which fails to form an AER. TGF-alpha and EGF are present in the AER of the developing limb, and TGF-alpha, EGF, and c-erbB are present in the underlying subridge mesoderm, suggesting possible roles in reciprocal AER/subridge mesoderm interactions required for limb outgrowth. We found that exogenous TGF-alpha and EGF can promote the outgrowth of limb mesoderm in the absence of the AER in vitro and can also promote the outgrowth of limbless and wingless wing bud explants. EGF is present in ventral but not dorsal limb ectoderm, suggesting a role for EGF in specification of ventral ectoderm. TGF-alpha and EGF are not detected in the differentiating cartilaginous elements or muscle primordia of the limb, suggesting that cessation of TGF-alpha and EGF expression may be required for cartilage and muscle formation. We have found that exogenous TGF-alpha and EGF inhibit chondrogenesis and myogenesis of limb mesenchyme in vitro. Together these results indicate that signaling through the EGF receptor via endogenous TGF-alpha and EGF may be important for initial limb formation, AER induction, outgrowth of limb mesoderm, and regulation of limb chondrogenic and myogenic differentiation.

The Interaction of an Epidermal Growth Factor/Transforming Growth Factor α Tail Chimera with the Human Epidermal Growth Factor Receptor Reveals Unexpected Complexities

It has been assumed that substitution of homologous regions of transforming growth factor alpha (TGF-alpha) into epidermal growth factor (EGF) can be used to probe ligand-receptor recognition without detrimental effects on ligand characteristics for the human EGF receptor (EGFR). We show that a chimera of murine (m) EGF in which the carboxyl-terminal tail is substituted for that of TGF-alpha (mEGF/TGF-alpha44-50) results in complex features that belie this initial simplistic assumption. Comparison of EGF and mEGF/TGF-alpha44-50 in equilibrium binding assays showed that although the relative binding affinity of the chimera was reduced 80-200-fold, it was more potent than EGF in mitogenesis assays using NR6/HER cells. This superagonist activity could not be attributed to differences in ligand processing or to binding to other members of the c-erbB family. It appeared to be due, in part, to choice of an EGFR-overexpressing target cell where high receptor number compensated for the low affinity of the ligand; it also appeared to be related to the ability of the chimera to activate the EGFR tyrosine kinase. Thus, when EGFR autophosphorylation was measured, mEGF/TGF-alpha44-50 was more potent than EGF, despite its low affinity. When tested using chicken embryo fibroblasts, substitution of the TGF-alpha carboxyl-terminal tail into mEGF failed to enhance its binding affinity for chicken EGFRs; however, the chimera was intermediate in potency between TGF-alpha and mEGF in mitogenesis assays. Our results suggest a contextual requirement for EGFR recognition which is ligand-specific. Further, the unpredictable responses to chimeric ligands underline the complex nature of the processes of ligand recognition, receptor activation, and the ensuing cellular response.

The Significance of Valine 33 as a Ligand-specific Epitope of Transforming Growth Factor α

Although binding of epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) to the EGF receptor (EGFR) is mutually competitive, their binding is not identical, and their biological activities are not always equivalent. To probe for ligand-specific interactions, we have synthesized analogues of TGFalpha with modifications to the residue lying between the fourth and fifth cysteines (the "hinge"). Although this residue lies in a structurally conserved region of the protein, it is not conserved within the EGFR ligand family. Our results show that in TGFalpha there is a preference for a bulky hydrophobic hinge residue; this contrasts with EGF, for which a hydrogen bond donor functionality is preferred. Sequence analysis of the human EGFR ligands revealed that the nature of the hinge residue correlated with the sequence in the B-loop beta-sheet. As this region is an important determinant in recognition of TGFalpha by the chicken EGFR, we assessed the mitogenicity of the TGFalpha hinge mutants, as well as the other EGFR ligands, using chicken embryo fibroblasts. The preference of the chicken EGFR for TGFalpha hinge mutants with hydrophobic side chains paralleled that of the human EGFR. Betacellulin and heparin-binding EGF-like growth factor also possess an hydrophobic hinge; both were at least as potent as TGFalpha for chicken embryo fibroblasts. EGF and amphiregulin, both with hydrogen bond donor functionalities at their hinge, displayed markedly decreased in potency by comparison with TGFalpha. We propose that EGFR ligands can be subclassified into TGFalpha-like and EGF-like and that this is of functional significance, identifying a potential mechanism whereby EGFR can discriminate between its ligands.

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) 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.

Contribution of the Transforming Growth Factor α B-loop β-Sheet to Binding and Activation of the Epidermal Growth Factor Receptor

We have exploited the differences in binding affinities of the chicken epidermal growth factor (EGF) receptor for EGF and transforming growth factor alpha (TGF alpha) to study the role of the B-loop beta-sheet of these ligands in receptor recognition and activation. Although EGF and TGF alpha share similar secondary and tertiary structures imposed by three highly conserved intramolecular disulfide bonds, they have only 30-40% overall sequence identity. The B-loop beta-sheet is the major structural element in EGF and TGF alpha, but sequence similarity in this region is low. To investigate its role in receptor binding, we constructed two chimeric growth factors (mEGF/hTGF alpha 21-30 and mEGF/hTGF alpha 21-32) composed of the murine EGF (mEGF) amino acid sequence with residues 21-30 of the B-loop beta-sheet replaced by the equivalent residues of human TGF alpha (hTGF alpha); in chimera mEGF/hTGF alpha 21-32, asparagine 32, which lies at the boundary of the amino and carboxyl domains of mEGF, was also replaced by its hTGF alpha counterpart (valine). In initial studies using unpurified medium, it was found that the recombinant growth factors exhibited differing mitogenic potencies (mEGF/hTGF alpha 21-32 > mEGF/hTGF alpha 21-30 > mEGF) when assayed on chicken fibroblasts, even though they were equivalent in mitogenesis assays using cells expressing the human EGF receptor. After purification, mEGF/hTGF alpha 21-32 was found to be 50 times more potent than mEGF in the chick fibroblast mitogenesis assay and exhibited a 10-fold increase in relative affinity for the chicken EGF receptor; both growth factors still exhibited equivalent mitogenic and receptor binding activity when tested on cells expressing human EGF receptors. We conclude that the B-loop beta-sheet of hTGF alpha is an important determinant of EGF receptor binding affinity and biological activity.

Induction of renal adenocarcinoma by a nonmutated erbB oncogene.

Oncogenicity tests have revealed that a nonmutated erbB oncogene induces renal adenocarcinoma in addition to erythroblastosis. The erbB oncogene is a truncated form of the chicken epidermal growth factor receptor that lacks the extracellular ligand-binding domain. Previously, the nonmutated erbB oncogene has been reported to cause only erythroblastosis. The expansion of the disease potential of erbB to additional neoplasms has been associated with mutations (truncations, deletions, and point mutations) within the erbB gene. Our results indicate that a nonmutated virally expressed erbB oncogene (REB-c) causes a 100% incidence of renal neoplasia.

Protein tyrosine kinase activities of the epidermal growth factor receptor and ErbB proteins: correlation of oncogenic activation with altered kinetics.

We have compared the protein tyrosine kinase activities of the chicken epidermal growth factor receptor (chEGFR) and three ErbB proteins to learn whether cancer-activating mutations affect the kinetics of kinase activity. In immune complex assays performed in the presence of 15 mM Mn2+, ErbB proteins and the chEGFR exhibited highly reproducible tyrosine kinase activity. Under these conditions, the ErbB and chEGFR proteins had similar apparent Km [Km(app)] values for ATP. The ErbB proteins appeared to be activated, as they had at least 3-fold-higher relative Vmax(app) for autophosphorylation and approximately 2-fold higher relative Vmax(app) for the phosphorylation of the exogenous substrate TK6 (a bacterially expressed fusion protein containing the C-terminal domain of the human EGFR). The ErbB kinases had both higher Km(app) and higher Vmax(app) for the phosphorylation of the exogenous substrate TK6 than did the chEGFR. The ratios of the Vmax(app) to the Km(app) for TK6 phosphorylation suggested that the ErbB proteins had lower catalytic efficiencies for the exogenous substrate than did the chEGFR. The three tested ErbB proteins had cytoplasmic domain mutations that conferred distinctive disease potentials. These mutations did not affect the kinetics for the phosphorylation of the exogenous substrate TK6. Two of the ErbB proteins contained all of the sites used for autophosphorylation. In these, a mutation that broadened oncogenic potential to endothelial cells caused an additional increase in Vmax(app) for autophosphorylation. Thus, mutations that change the EGFR into an ErbB oncogene cause multiple changes in the kinetics of protein tyrosine kinase activity.

Noncontiguous regions in the extracellular domain of EGF receptor define ligand-binding specificity.

Murine epidermal growth factor (EGF) binds with approximately 250-fold higher binding affinity to the human EGF receptor (EGFR) than to the chicken EGFR. This difference in binding affinity enabled the identification of a major ligand-binding domain for EGF by studying the binding properties of various chicken/human EGFR chimera expressed in transfected cells lacking endogenous EGFR. It was shown that domain III of EGFR is a major ligand-binding region. Here, we analyze the binding properties of novel chicken/human chimera to further delineate the contact sequences in domain III and to assess the role of other regions of EGFR for their contribution to the display of high-affinity EGF binding. The chimeric receptors include chicken EGFR containing domain I of the human EGFR, chicken receptor containing domain I and III of the human EGFR, and two chimeric chicken EGFR containing either the amino terminal or the carboxy terminal halves of domain III of human EGFR, respectively. In addition, the binding of various human-specific anti-EGFR monoclonal antibodies that interfere with EGF binding is also compared. It is concluded that noncontiguous regions of the EGFR contribute additively to the binding of EGF. Each of the two halves of domain III has a similar contribution to the binding energy, and the sum of both is close to that of the entire domain III. This suggests that the folding of domain III juxtaposes sequences that together constitute the ligand-binding site. Domain I also provides a contribution to the binding energy, and the added contributions of both domain I and III to the binding energy generate the high-affinity binding site typical of human EGFR.

Human Epidermal Growth Factor (EGF) Receptor Sequence Recognized by EGF Competitive Monoclonal Antibodies: Evidence for the localization of the egf-binding site

Epitopes recognized by three epidermal growth factor (EGF) competitive monoclonal antibodies, LA22, LA58, and LA90, have been localized to a 14-amino acid region in the extracellular domain of the human EGF receptor. The binding of each of these mutually competitive antibodies to A431 epidermoid carcinoma cells was inhibited up to 87% by EGF. Furthermore, binding to A431 cells was inhibited 100% by the EGF competitive monoclonal antibody 528 IgG. The EGF receptor monoclonal antibody 455 IgG, which recognizes a blood group A-related carbohydrate modification of A431 receptors and does not inhibit EGF binding, did not inhibit the binding of these three antibodies to A431 cells. Antibodies LA22, LA58, and LA90 were unusual in that they bound to recognized denatured and endoglycosidase F-treated antigenic determinants in Western blots. This suggested that the antibodies recognized continuous peptide epitopes. The epitopes for these antibodies were first localized in cyanogen bromide- and V8 protease-generated fragments of a truncated form of the EGF receptor secreted by A431 cells. In experiments with synthetic peptides, all three antibodies were found to bind to the 14 amino acids from Ala-351 to Asp-364 of the mature human EGF receptor. These amino acids are located between the two Cys-rich regions of the extracellular domain of the receptor, and they include an Arg-Gly-Asp-Ser recognition site for adhesion molecule receptors. The homologous sequence in the chicken EGF receptor, which binds mouse EGF with a 100-fold lower affinity than the human EGF receptor, contains four amino acid differences including two in the Arg-Gly-Asp-Ser tetramer. The mutually competitive binding of EGF and antibodies LA22, LA58, and LA90 implied that the amino acids between Ala-351 and Asp-364 participated in the formation of the EGF-binding site of the human EGF receptor.

Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.

The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors.

Structure and expression of the chicken epidermal growth factor receptor gene locus.

Similarity between the carboxyl-terminal portion of the human epidermal growth factor (EGF) receptor and the deduced protein sequence of the chicken-derived oncogene v-erbB, of avian erythroblastosis virus strain H, has suggested that the chicken cellular erbB locus, c-erbB, might be part of a longer EGF-receptor gene in the chicken, whose entire coding capacity remained to be defined. The c-erbB locus spans more than 20 X 10(3) base pairs (20 kbp) of DNA and contains at least 1.8 kbp homologous to the v-erbB oncogene. We show here that human EGF receptor cDNA and chicken genomic DNA share homology not only within the c-erbB locus but also within a 25.1-kbp DNA region situated 5' to this locus. The 3' region of the EGF receptor overlaps, in sequence homology, the c-erbB locus. The EGF receptor/c-erbB locus in chicken generates six related but distinctly different mRNAs of sizes 12, 9, 5, 3.6, 3.2 and 2.6 kb. The transcripts of 12, 9, and 3.6 kb contain sequences coding for both the extracellular EGF-binding domain of the receptor and the intracellular tyrosine kinase domain. The 12-kb and 9-kb transcripts, which have already been shown to contain the sequences coding for the v-erbB, were found to possess, in addition, sequences that encode the entire chicken EGF receptor. The 3.2-kb and 2.6-kb mRNAs are homologous only to the 5' portion of the EGF receptor gene. These results therefore indicate that the c-erbB locus, initially defined by homology to the viral transforming gene, corresponds to the 3' region of the EGF receptor gene in the chicken genome. The multiple, related, chicken EGF receptor RNA transcripts reported here are reminiscent of the various human EGF receptor RNA transcripts observed in normal and transformed cells.