Identification of the high affinity binding site of transforming growth factor-alpha (TGF-alpha) for the chicken epidermal growth factor (EGF) receptor using EGF/TGF-alpha chimeras.

Kramer, R.H.; Lenferink, Anne E.G.; Bueren-Koornneef, I L van; Meer, Arnold van der; Poll, M L van de; Zoelen, Everardus J. van

doi:10.1016/s0021-9258(17)37025-4

Cited by 42 publications

(11 citation statements)

References 26 publications

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“…sEGFR501 was also employed to investigate the residue responsible for the differential binding between hTGF-R and hEGF observed with chicken EGFR (9). The region on hEGF resulting in poor binding to the chicken receptor has been previously mapped to the C-terminal residues 43-53, by examining the binding properties of 10 hEGF/ hTGF-R chimeras (16). Subsequent analysis of a series of truncated ligands or multiple site/single site mutations showed that Arg 45 was critically important (17).…”

Section: Discussionmentioning

confidence: 99%

Identification of a Determinant of Epidermal Growth Factor Receptor Ligand-Binding Specificity Using a Truncated, High-Affinity Form of the Ectodomain

et al. 2001

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Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF), and human transforming growth factor alpha (hTGF-alpha) with high affinity despite the significant differences in the amino acid sequences of the ligands and the receptors. In contrast, the chicken EGFR can discriminate between mEGF (and hEGF) and hTGF-alpha and binds the EGFs with approximately 100-fold lower affinity. The regions responsible for this poor binding are known to be Arg(45) in hEGF and the L2 domain in the chicken EGFR. In this study we have produced a truncated form of the hEGFR ectodomain comprising residues 1-501 (sEGFR501), which, unlike the full-length hEGFR ectodomain (residues 1-621, sEGFR621), binds hEGF and hTGF-alpha with high affinity (K(D) = 13-21 and 35-40 nM, respectively). sEGFR501 was a competitive inhibitor of EGF-stimulated mitogenesis, being almost 10-fold more effective than the full-length EGFR ectodomain and three times more potent than the neutralizing anti-EGFR monoclonal antibody Mab528. Analytical ultracentrifugation showed that the primary EGF binding sites on sEGFR501 were saturated at an equimolar ratio of ligand and receptor, leading to the formation of a 2:2 EGF:sEGFR501 dimer complex. We have used sEGFR501 to generate three mutants with single position substitutions at Glu(367), Gly(441), or Glu(472) to Lys, the residue found in the corresponding positions in the chicken EGFR. All three mutants bound hTGF-alpha and were recognized by Mab528. However, mutant Gly(441)Lys showed markedly reduced binding to hEGF, implicating Gly(441), in the L2 domain, as part of the binding site that recognizes Arg(45) of hEGF.

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Section: Discussionmentioning

confidence: 99%

Identification of a Determinant of Epidermal Growth Factor Receptor Ligand-Binding Specificity Using a Truncated, High-Affinity Form of the Ectodomain

et al. 2001

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“…The gap was then filled in using double-stranded oligonucleotides. The genes were linked at the 5′-end to the sequence coding for the recognition sequence of the proteolytic enzyme factor X a [Ile-Glu-Gly-Arg; see Nagai and Thogersen (1987)], and the FX/growth factor constructs were cloned into the expression vector pEZZ18 (Pharmacia, Uppsala, Sweden) 3′ of the sequence coding for two synthetic protein A-derived IgG binding domains (so-called Z domains) as described previously (Van de Poll et al, 1995;Kramer et al, 1994).…”

Section: Methodsmentioning

confidence: 99%

“…Nonconservative substitutions of I23 and A30 cause a significant reduction in binding affinity [see for a recent review Groenen et al (1994)], but EGF molecules with truncated forms of the B-loop have been claimed to be biologically active (Taggart et al, 1993). Domain-exchange studies between EGF and TGFR have also not given definite conclusions in this respect (Kramer et al, 1994;Richter et al, 1995).…”

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confidence: 99%

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

et al. 1997

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Recently, it has been shown that the activation of the Drosophila EGF receptor (DER) by its natural ligand Spitz is inhibited by Argos [Schweitzer, R., et al. (1995) Nature 376, 699-702]. Argos and Spitz both have an EGF-like domain which in the case of Argos differs from that of Spitz and other EGF receptor agonists in that it has an extended B-loop of 20 amino acids instead of 10 amino acids which in addition contains an unusual cluster of charged residues. To investigate whether B-loop sequences are an important determinant for receptor activation and play a causal role in the antagonistic activity of Argos, three human (h)EGF mutants were constructed in which amino acids derived from the Argos B-loop were introduced. In one mutant (E3A4E/B10), replacement of four amino acids in the B-loop of hEGF (123, E24, D27, and K28) by the corresponding Argos residues neither altered the binding affinity of the growth factor for the hEGF receptor nor did it change its ability to induce a mitogenic response. Insertion of 2 additional Argos residues (E3A4E/B12) or extension of the B-loop by 10 amino acids (E3A4E/B20) resulted, however, in a significant loss of binding affinity. In spite of this, both E3A4E/B12 and E3A4E/B20 appeared to be strong agonists for the hEGF receptor with similar dose-response curves for mitogenic activity and MAPK activation as wild-type hEGF. These data show that several nonconservative substitutions in the hEGF B-loop are tolerated without affecting receptor binding or activation. Furthermore, they show that receptor binding and receptor signaling efficiency can be uncoupled which is a prerequisite for the development of receptor antagonists.

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“…TGF-a is closely related to epidermal growth factor (EGF) and binds to the same receptor, EGFR (Kramer et al, 1994). This binding induces activation of the classical mitogen-activated protein kinase (MAPK, ERK) pathway, a module of three protein kinases that is organized in a hierarchical fashion and is known to regulate basic cellular functions such as proliferation, differentiation, and migration (Marshall, 1994).…”

mentioning

confidence: 99%

Activation of Epidermal Growth Factor Receptor/ERK Signaling Correlates with Suppressed Differentiation in Malignant Acanthosis Nigricans

Haase

Hunzelmann

2002

Journal of Investigative Dermatology

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Identification of the high affinity binding site of transforming growth factor-alpha (TGF-alpha) for the chicken epidermal growth factor (EGF) receptor using EGF/TGF-alpha chimeras.

Cited by 42 publications

References 26 publications

Identification of a Determinant of Epidermal Growth Factor Receptor Ligand-Binding Specificity Using a Truncated, High-Affinity Form of the Ectodomain

Identification of a Determinant of Epidermal Growth Factor Receptor Ligand-Binding Specificity Using a Truncated, High-Affinity Form of the Ectodomain

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

Activation of Epidermal Growth Factor Receptor/ERK Signaling Correlates with Suppressed Differentiation in Malignant Acanthosis Nigricans

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