Substitution of lysine for arginine at position 42 of human transforming growth factor-alpha eliminates biological activity without changing internal disulfide bonds.

Defeo-Jones, Deborah; Tai, J Y; Vuocolo, G A; Wegrzyn, Ronald; Schofield, T L; Riemen, Mark W.; Oliff, Allen

doi:10.1128/mcb.9.9.4083

Cited by 40 publications

(12 citation statements)

References 30 publications

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“…hEPR is a much weaker antagonist of the ErbB-1 and ErbB-4 receptors with IC 50 values of 2800 nM and Ͼ5 M, respectively (34), indicating that 9E5 IgG binds to hEPR more strongly than ErbB-1 and ErbB-4. According to previous studies, mutational analysis and chemical regeneration suggest that the guanidinium group of Arg E40 in hEPR is essential for binding of the ErbB receptor (40,41). These results support that 9E5(Fab) acts as not only the simple capturer of EPR but also the competitive neutralization antibody against EGFR with inhibition of the functional residue Arg E40 .…”

Section: Discussionsupporting

confidence: 70%

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5

Kado

Mizohata

Nagatoishi

et al. 2016

Journal of Biological Chemistry

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Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementaritydetermining regions (CDRs), CDR1-3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cistrans isomerization of Pro 103 . A molecular dynamics simulation and mutational analyses revealed that Arg 40 in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nM. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)⅐EPR on the known complex structure of EGF⅐EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)⅐EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer.Recently, antibody therapy has been attracting considerable attention as a possible cure for several types of diseases. For instance, trastuzumab is a humanized IgG1 monoclonal antibody that is targeted for the human epidermal growth factor (EGF) receptor (EGFR) 6 2 (HER2, ErbB-2), which is used in the treatment of metastatic breast cancer (1).Initially, the EPR precursor protein is expressed as a type I transmembrane protein. A disintegrin and metalloproteinase 17 (ADAM17) catalyzes ectodomain shedding of the EPR precursor protein, which produces mature EPR (2). EPR induces dimerization of EGFR and promotes autophosphorylation in the intracellular kinase domain of EGFR (3). EGFR phosphorylation activates several types of intracellular signaling pathways, such as the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/Akt, and STAT5 pathways (4 -6). As a result, proliferation, cell survival, and angiogenesis are induced in the cell.Although the expression of EPR is suppressed in most adult normal tissues, EPR is overexpressed in human colon, breast, and ovarian cancers (7-10). Therefore, normalization of EGF signaling is expected to cure these cancers. Recently, humanized anti-EPR antibodies with high affinity targeted cytotoxicity have been prepared and characterized (11), and these antibodies have the potential to act as anticancer drugs.The structure of EPR was first determined by NMR (12

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Section: Discussionsupporting

confidence: 70%

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5

Kado

Mizohata

Nagatoishi

et al. 2016

Journal of Biological Chemistry

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“…Arg-42, which appears to be less important for binding based on the NOE criterion, may play a structural role in preserving the local conformation of Phe-15, which is a critical residue based upon the NOE data. Structural studies of the inactive R42K mutant exclude any gross conformational changes; however, do not rule out subtle effects that alter the microenvironment of the phenylalanine (17,36).…”

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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“…(ii) The binding data indicate the requirement for a polar, isosteric sidechain at position 16 of hEGF that functions as both hydrogen-bond donor and acceptor. The reduced binding affinity of the H18K hTGF␣ mutant [Defeo-Jones et al, 1989] suggests that H18 in hTGF␣ may play a similar role. (iii) As the H16 mutants are similar to wild-type hEGF in their native conformation (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Partially purified Pseudomonas exotoxin A-hEGF chimeras in which H16 was replaced with Q display reduced receptor binding affinity (19% of wildtype) and cytotoxic activity [Shiah et al, 1992]. In another report, a highly purified H18K hTGF␣ mutant was shown to retain 22% relative binding affinity and 7% relative mitogenic potency [Defeo-Jones et al, 1989]. This suggests a stringent requirement for H since semiconservative changes to either Q or K result in loss of biological activity.…”

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confidence: 99%

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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Substitution of lysine for arginine at position 42 of human transforming growth factor-alpha eliminates biological activity without changing internal disulfide bonds.

Cited by 40 publications

References 30 publications

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5

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

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

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