Structure-function analysis of human transforming growth factor-<i>α</i> by site-directed mutagenesis

Feild, John; Reid, Robert H.; Rieman, David J.; Kline, Tammy Page; Sathe, Ganesh M.; Greig, Russell; Anzano, Mario A.

doi:10.1042/bj2830091

Cited by 12 publications

(4 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus far, point mutation studies of the carboxyl-terminal region of hEGF and hTGF␣ have focused mainly on the highly conserved Asp-46 and Leu-47 (Asp-47 and Leu-48 in hTGF␣). Leu-47 and (less stringently) Asp-46 have been shown to be crucial for receptor binding and activation (5,7,9,12). By using a domain exchange strategy, however, a systematic survey of the importance of non-conserved amino acids can be made.…”

Section: Discussionmentioning

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

View full text Add to dashboard Cite

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 ...

show abstract

Section: Discussionmentioning

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

View full text Add to dashboard Cite

show abstract

“…The essential amino acid residues for the mitogenic activity were examined in detail for EGF and TGF-␣ using recombinant DNA technology. In EGF, the importance of (21,22,37,38). In these residues, which are crucial for biological activities, Arg (41 in EGF, 42 in TGF-␣) and Leu (47 in EGF, 48 in TGF-␣) are highly conserved in the EGF family.…”

Section: Resultsmentioning

confidence: 99%

Novel Betacellulin Derivatives

Itoh

Kondo

Tanaka

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Betacellulin (BTC) is a member of the epidermal growth factor family. It has two biological activities: mitogenic activity in fibroblasts and vascular smooth muscle cells, and differentiation activity for the differentiation of pancreatic acinar AR42J cells into insulinsecreting cells. The previous finding that recombinant BTC promotes the neogenesis of ␤-cells in a mouse model supports the possibility that BTC is a therapeutic protein. However, the mitogenic activity of BTC may not be needed for differentiation into ␤-cells and may cause a side effect in clinical use. We prepared several derivatives of BTC to segregate the two activities, to decrease the mitogenic activity, and to maintain the differentiation activity. We succeeded in obtaining BTC derivatives segregated by the two biological activities by preparing truncated-type derivatives. A derivative of BTC, BTC24 -76, with a truncated N-terminal 23 amino acids and C-terminal 4 amino acids, was 2.5-fold more active in differentiation and had one-tenth of the mitogenic activity. The derivatives described in the present study should be helpful in future applications as therapeutic proteins and in basic research for discovery of a BTCspecific receptor.

show abstract

“…[11][12][13] Simultaneously, those responsible for binding to ErbB3 and ErbB4 in the EGF domain of heregulin-β were studied by alanine scanning mutagenesis. 14 However, no relationship between receptor binding specificity and biological activity has been reported.…”

Section: Introductionmentioning

confidence: 99%

A Betacellulin Mutant Promotes Differentiation of Pancreatic Acinar AR42J Cells into Insulin-Producing Cells with Low Affinity of Binding to ErbB1

Nagaoka¹,

Fukuda²,

Hashizume³

et al. 2008

Journal of Molecular Biology

View full text Add to dashboard Cite

Betacellulin (BTC) is one of the members of the epidermal growth factor (EGF) ligand family of ErbB receptor tyrosine kinases. It is a differentiation factor as well as a potent mitogen. BTC promotes the differentiation of pancreatic acinar-derived AR42J cells into insulin-producing cells. It independently and preferentially binds to two type I tyrosine kinase receptors, the EGF receptor (ErbB1) and ErbB4. However, the physiochemical characteristics of BTC that are responsible for its preferential binding to these two receptors have not been fully defined. In this study, to investigate the essential amino acid residues of BTC for binding to the two receptors, we introduced point mutations into the EGF domain of BTC employing error-prone PCR. The receptor binding abilities of 190 mutants expressed in Escherichia coli were assessed by enzyme immunoassay. Replacement of the glutamic acid residue at position 88 with a lysine residue in BTC was found to produce a significant loss of affinity for binding to ErbB1, while the affinity of binding to ErbB4 was unchanged. In addition, the mutant of BTC-E/88/K showed less growth-promoting activity on BALB/c 3T3 cells compared with that of the wild-type BTC protein. Interestingly, the BTC mutant protein promoted differentiation of pancreatic acinar AR42J cells at a high frequency into insulin-producing cells compared with AR42J cells that were treated with wild-type BTC protein. These results indicate the possibility of designing BTC mutants, which have an activity of inducing differentiation only, without facilitating growth promotion.

show abstract

Structure-function analysis of human transforming growth factor-α by site-directed mutagenesis

Cited by 12 publications

References 43 publications

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

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

Novel Betacellulin Derivatives

A Betacellulin Mutant Promotes Differentiation of Pancreatic Acinar AR42J Cells into Insulin-Producing Cells with Low Affinity of Binding to ErbB1

Contact Info

Product

Resources

About