Analogs of human epidermal growth factor which partially inhibit the growth factor‐dependent protein‐tyrosine kinase activity of the epidermal growth factor receptor

Matsunami, Risë K.; Campion, Stephen R.; Niyogi, Salil K.; Stevens, Audrey

doi:10.1016/0014-5793(90)80776-f

Cited by 34 publications

(23 citation statements)

References 17 publications

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“…Although all mutant species were able to stimulate this response, the mutant proteins with the two lowest receptor affinities appeared to stimulate this activity to a somewhat lower maximal velocity, approximately 70% that of the wild type and the other mutant species. A similar although more pronounced effect has been seen before with other mutant EGF species (28).…”

supporting

confidence: 87%

Aromaticity at position 37 in human epidermal growth factor is not obligatory for activity.

Engler¹,

Hauser²,

Cook³

et al. 1991

Mol. Cell. Biol.

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The third disulfide loop (amino acids 33 to 42) of human epidermal growth factor (hEGF) encompasses the region of highest amino acid conservation among all of the EGF-like family of molecules. The importance of some of these highly conserved residues for the maintenance of biological activity, especially the aromatic amino acid tyrosine at position 37, has until now been considered essential on the basis of previous studies with the EGF-like molecule transforming growth factor alpha. Variants at the Tyr-37 position of hEGF were constructed by site-directed mutagenesis. The substituting amino acids were phenylalanine, histidine, serine, alanine, aspartic acid, arginine, and glycine. The variants were tested for their ability to competitively displace native ['25I]hEGF from its receptor and to stimulate the protein-tyrosine kinase activity of the receptor; the order of efficacy of substituting amino acids was Phe > His > Ser > Ala > Asp > Arg > Gly in both assays. All were effective, with no or only moderate reduction in potency, in stimulating the incorporation of [3H]thymidine into acid-insoluble material of quiescent mouse A31 cells. Only Tyr-37-*Ala, Tyr-37--Arg and Tyr-37-*Gly were slightly less potent in the cell assay. Thus, neither tyrosine nor another aromatic amino acid at position 37 in hEGF is essential for full biological activity.

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supporting

confidence: 87%

Aromaticity at position 37 in human epidermal growth factor is not obligatory for activity.

Engler¹,

Hauser²,

Cook³

et al. 1991

Mol. Cell. Biol.

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“…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)(3)(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)(6)(7)(8)(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.…”

mentioning

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

Lenferink

Vugt

et al. 1995

Journal of Biological Chemistry

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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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“…Because they are highly conserved and are required for activity, it was concluded that these residues interact directly with the receptor. While replacement of Ile23 and Leu26 produced less dramatic effects on receptor affinity, more extensive studies demonstrated that these two residues can be replaced only by another nonpolar residue if activity is to be maintained, and that Ile23 is involved in direct receptor interactions (Engler et al, 1988;Campion et al, 1990;Matsunami et al, 1990;Koide et al, 1992Koide et al, , 1994.…”

Section: Discussionmentioning

confidence: 99%

“…1). Site-directed mutagenesis has been applied to probe numerous residues within this N-terminal subdomain of hEGF, and has demonstrated the importance of the nonpolar residues Ile23, Leu26, and the aromatic residues Tyr22 and Tyr29 for receptor binding (Engler et al, 1988Campion et al, 1990Campion et al, , 1993Koide et al, 1992;Matsunami et al, 1990). Replacement of other residues of hEGF, including Vail9, Met21, Gtu24, and Asp27, had little effect on EGF-receptor interactions (Engler et al, 1988), while replacement of Ala25 by Val and of Lys28 by Arg produced hEGF variants with increased receptor affinities relative to that for the wild-type protein .…”

Section: Introductionmentioning

confidence: 99%

Structure-function studies of mEGF: Probing the type Iβ-turn between residues 25 and 26

Lester

Wang

et al. 1995

J Protein Chem

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The interaction between epidermal growth factor (EGF) and its receptor molecule is not completely understood and has received much attention recently. Studies combining site-directed mutagenesis and NMR spectroscopy have identified a number of EGF residues that are required for activity and are believed to interact directly with the receptor. Instead of focusing on these residues, this study combines site-directed mutagenesis and NMR spectroscopy to probe the role of the type I beta-bend located between residues 25 and 26 of the N-terminal subdomain of the protein. Ser25 of murine EGF is replaced by Pro in an attempt to stabilize this turn conformation to produce a variant of mEGF with increased activity relative to that for the native protein. Ser25 is also replaced by Ala, which is found at position 25 in human EGF (hEGF), as a more conservative replacement. Receptor binding studies demonstrate that both mutations produce about a 30% reduction in binding affinity, which is shown to result from local changes within the loop or minor perturbations of residues neighboring the loop rather than from long-range perturbations of the beta-sheet of the N-terminal subdomain. The type I beta-turn appears to remain intact in both mutants; however, replacement with Pro seems to introduce more flexibility into this region of the protein. These results demonstrate that perturbation of this beta-turn has little effect on EGF-receptor interactions.

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Analogs of human epidermal growth factor which partially inhibit the growth factor‐dependent protein‐tyrosine kinase activity of the epidermal growth factor receptor

Cited by 34 publications

References 17 publications

Aromaticity at position 37 in human epidermal growth factor is not obligatory for activity.

Aromaticity at position 37 in human epidermal growth factor is not obligatory for activity.

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

Structure-function studies of mEGF: Probing the type Iβ-turn between residues 25 and 26

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