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

Engler, D A; Hauser, M R; Cook, J S; Niyogi, Salil K.

doi:10.1128/mcb.11.5.2425

Cited by 19 publications

(12 citation statements)

References 23 publications

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“…When the mitogenic potencies of the H16 mutants are compared to that of wild-type hEGF, it appears that survival may be governed by kinetically controlled events not easily correlated with receptor binding affinities determined at equilibrium. Similar conclusions have been reached in studies where the metabolic activity/viability of cell populations in response to genetically engineered EGF or TGF␣ has been assessed by 3 H-TdR [Engler et al, 1991] and 125 I-UdR [Puddicombe et al, 1996] uptake into DNA.…”

Section: Discussionsupporting

confidence: 74%

Receptor recognition by histidine 16 of human epidermal growth factor via hydrogen-bond donor/acceptor interactions

1999

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Human epidermal growth factor (hEGF) and human transforming growth factor alpha (hTGFalpha) are prototypical of structurally related polypeptide mitogens which interact with the epidermal growth factor receptor (EGFR). Several determinants of receptor recognition that specify function have been proposed on the basis of structural criteria. This study evaluates the role of one such candidate, H16 of hEGF, by site-specific mutagenesis. When assayed for receptor tyrosine kinase stimulation using (Glu4,Tyr1)n as the exogenous substrate in vitro, the relative agonist activities of position 16 mutants range from 14-263% of wild-type hEGF. The rank order of potency was found to correlate with the relative receptor binding affinities of the mutants, which range from 7-272% of wild-type, as determined by radioreceptor competition assays. The mitogenic activity of the H16 mutants is similar to that of wild-type hEGF as determined by clonogenic assays using rat tracheal epithelial cells. While the colony forming efficiencies do not reflect significant differences in growth rate or survival characteristics in the presence of the hEGF variants, it is reduced to 1.6% in control cultures which lack EGF in the medium. The results show that H16 of hEGF, although not essential for mitogenic activity, optimizes receptor recognition by hydrogen-bond donor/acceptor interactions and may share this feature with H18 of hTGFalpha.

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Section: Discussionsupporting

confidence: 74%

Receptor recognition by histidine 16 of human epidermal growth factor via hydrogen-bond donor/acceptor interactions

1999

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“…Site-directed mutagenesis of Tyr-38 in TGF-␣ and Tyr-37 in EGF apparently gives conflicting results for the significance of this residue in the receptor interaction since it was shown to be nonessential in EGF (38) and essential in TGF-␣ (16). This conflict may be the consequence of a different mechanism or site of binding with the two ligands; however, our NOE data indicate that if mutation of this residue precludes binding of TGF-␣, then it does so by altering the ligand conformation and thus probably does not contribute directly to the interaction.…”

Section: Discussionmentioning

confidence: 99%

NMR Study of the Transforming Growth Factor-α (TGF-α)-Epidermal Growth Factor Receptor Complex

McInnes

Hoyt

Harkins³

et al. 1996

Journal of Biological Chemistry

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The study of human transforming growth factor-␣ (TGF-␣) in complex with the epidermal growth factor (EGF) receptor extracellular domain has been undertaken in order to generate information on the interactions of these molecules. Analysis of 1 H NMR transferred nuclear Overhauser enhancement data for titration of the ligand with the receptor has yielded specific data on the residues of the growth factor involved in contact with the larger protein. Significant increases and decreases in nuclear Overhauser enhancement cross-peak intensity occur upon complexation, and interpretation of these changes indicates that residues of the A-and C-loops of TGF-␣ form the major binding interface, while the B-loop provides a structural scaffold for this site. Human TGF-␣ 1 is a 50-amino acid polypeptide with 40% sequence homology to epidermal growth factor (1, 2). In addition, the structural similarity of the two molecules results in their ability to compete for binding to the EGF receptor (3-5). Complexation of TGF-␣ with this receptor is believed to mediate a variety of biological effects, including embryonic development of certain tissues and wound healing (6, 7); however, the major sphere of interest of this protein lies in its role in the transformation and maintenance of various malignant tumors (8, 9).The structural features of the homologous growth factors that contain three disulfides and hence three loops (A, B, and C) have been determined by NMR (10 -13) and include a triplestranded anti-parallel ␤-sheet comprising the N-terminal region, a smaller anti-parallel double hairpin in the C terminus of the molecule, and a helical segment in the A-loop in some structures. Previous studies undertaken to elucidate the structurally important residues of TGF-␣ (and EGF) required for complexation have implicated residues including Phe-15, Tyr-38, . The consensus of a variety of structural studies including the use of synthetic peptide fragments (19 -21), recombinant chimeric proteins (22, 23), and anti-TGF-␣ and anti-EGF antibodies (24, 25) is that receptor binding occurs with multiple domains of TGF-␣, although conflicting results have been obtained concerning the involvement of the A-, B-, and C-loops. The multidomain binding model is consistent with the observation that, at present, it is not possible to reduce the size of the growth factor without significantly compromising its affinity for the EGF receptor. This was illustrated by deletion studies where the N-terminal residues outside the A-loop were truncated. This mutant had 3% of the binding affinity of the intact protein (20). Further data from receptor-bound TGF-␣ may lead to the structure-based design of reductant molecules through the precise identification of ligand binding determinants. Hoyt et al. (26) have recently demonstrated, through a study of 1 H NMR transverse and longitudinal relaxation rates for the methyl resonances of TGF-␣ in the free state and in association with the EGFR-ED, that the C-terminal residues undergo a dramatic decrease in flexibility upo...

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“…3C), is the most highly conserved region in the EGF repeat, with invariant residues Gly-36, Tyr-37, Gly-39, and Arg-41. The predicted spi protein differs from h u m a n EGF in loop C with a conservative Tyr ~ Phe change at position 37; recent work has shown that this substitution in h u m a n EGF has little or no effect on biological activity of the protein (Engler et al 1991 ). In addition, spi protein shows conservation of residues Tyr/Phe-13, Leu-15, and Arg-41, postulated to lie at the interface of the receptor and growth factor (for review, see Campbell et al 1990) and has a conservative Leu ~ Ile change at position 47.…”

Section: The Spi Gene Encodes a Putative Egf-like Growth Factormentioning

confidence: 98%

The Drosophila spitz gene encodes a putative EGF-like growth factor involved in dorsal-ventral axis formation and neurogenesis.

Rutledge¹,

Zhang²,

Bier³

et al. 1992

Genes Dev.

268

155

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We describe the molecular characterization of the Drosophila gene spitz (spi), which encodes a putative 26-kD, EGF-like transmembrane protein that is structurally similar to TGF-tx. Temporal and spatial expression patterns of spi transcripts indicate that spi is expressed throughout the embryo. Examination of mutant embryos reveals that spi is involved in a number of unrelated developmental choices, for example, dorsal-ventral axis formation, glial migration, sensory organ determination, and muscle development. We propose that spi may act as a ligand for cell-specific receptors, possibly rhomboid and/or the Drosophila EGF receptor homolog.

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

Cited by 19 publications

References 23 publications

Receptor recognition by histidine 16 of human epidermal growth factor via hydrogen-bond donor/acceptor interactions

Receptor recognition by histidine 16 of human epidermal growth factor via hydrogen-bond donor/acceptor interactions

NMR Study of the Transforming Growth Factor-α (TGF-α)-Epidermal Growth Factor Receptor Complex

The Drosophila spitz gene encodes a putative EGF-like growth factor involved in dorsal-ventral axis formation and neurogenesis.

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