Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P04626 (erbB-2)
 5,251 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Eight analogues of human epidermal growth factor (hEGF) having specific amino acid substitutions in the beta-sheet structure (residues 19-31) of the amino-terminal domain were generated by site-directed mutagenesis. Affinity of the epidermal growth factor (EGF) receptor for each of these mutant hEGF analogues was measured by both radioreceptor competition binding and receptor tyrosine kinase stimulation assays. The relative binding affinities obtained by these two methods were generally in agreement for each hEGF species. The results indicate that hydrophobic residues on the exposed surface of the beta-sheet structure of the amino-terminal domain of hEGF have an important role in the formation of the active EGF-receptor complex. The substitution of hydrophobic amino acid residues, Val-19----Gly, Met-21----Thr, Ile-23----Thr, and Leu-26----Gly, resulted in decreased binding affinity, with the most severe reductions observed with the last two mutants. The mutations Ala-25----Val and Lys-28----Arg introduced amino acid residues resulting in slightly increased receptor binding affinity. Similar to previous results with acidic residues in this region [Engler, D.A., Matsunami, R.K., Campion, S.R., Stringer, C.D., Stevens, A., & Niyogi, S.K. (1988) J. Biol. Chem. 263, 12384-12390], removal of the positive charge in the Lys-28----Leu substitution had almost no effect on binding affinity, indicating the lack of any absolute requirement for ionic interactions at this site. Substitution of Tyr-22, which resulted in decreased receptor binding affinity, provides further indication of the importance of aromatic residues in this region of the molecule, as found earlier with Tyr-29 (cf. reference above).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biochemical properties of site-directed mutants of human epidermal growth factor: importance of solvent-exposed hydrophobic residues of the amino-terminal domain in receptor binding. 227 34

The protein kinase associated with the purified epidermal growth factor (EGF) receptor from membrane (Mr = 150,000) or vesicle (Mr = 170,000) preparations of A-431 cells was shown to catalyze the phosphorylation of the peptide Leu-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly at the tyrosine residue. EGF enhanced peptide phosphorylation by 3-5-fold. The steady state kinetic analysis of the purified kinase from membranes showed that the reaction mechanism was of the sequential type in either the presence of absence of EGF. Thus, the peptide and ATP must bind to the enzyme before any product is released. Both neurotensin 8-13 and kyotorphin were inhibitors but not substrates of the protein kinase. Kyotorphin was a linear noncompetitive inhibitor with ATP as the variable substrate and a linear competitive inhibitor with peptide as the variable substrate. ADP, a product of the kinase reaction, was a linear noncompetitive inhibitor with respect to ATP and a linear competitive inhibitor with respect to peptide. Based on these data, it can be suggested that the tyrosine protein kinase from A-431 cells catalyzes a Ordered Bi Bi reaction where peptide is the first substrate to bind and ADP is the last product to be released.
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PMID:The kinetics of tyrosine phosphorylation by the purified epidermal growth factor receptor kinase of A-431 cells. 610 Dec 63

Single-chain Fv fusions with C-terminal cysteinyl peptides (sFv') have been engineered using model sFv proteins based upon the 26-10 anti-digoxin IgG and 741F8 anti-c-erbB-2 IgG monoclonal antibodies. As part of the 741F8 sFv construction process, the PCR-amplified 741F8 VH gene was modified in an effort to correct possible primer-induced errors. Genetic replacement of the N-terminal beta-strand sequence of 741F8 VH with that from the FR1 of anti-c-erbB-2 520C9 VH resulted in a dramatic improvement of sFv folding yields. Folding in urea-glutathione redox buffers produced active sFv' with a protected C-terminal sulfhydryl, presumably as the mixed disulfide with glutathione. Disulfide-bonded (sFv')2 homodimers were made by disulfide interchange or oxidation after reductive elimination of the blocking group. Both 26-10 (sFv')2 and 741F8 (sFv')2 existed as stable dimers that were well behaved in solution, whereas 741F8 sFv and sFv' exhibited considerable self-association. The 741F8 sFv binds to the extracellular domain (ECD) of the c-erbB-2 oncogene protein, which is often overexpressed in breast cancer and other adenocarcinomas. The recombinant ECD was prepared to facilitate the analysis of 741F8 binding site properties; the cloned ECD gene, modified to encode a C-terminal Ser-Gly-His6 peptide, was transfected into Chinese hamster ovary cells using a vector that also expressed dihydrofolate reductase to facilitate methotrexate amplification. Optimized cell lines expressed ECD-His6 at high levels in a cell bioreactor; after isolation by immobilized metal affinity chromatography, final ECD yields were as high as 47 mg/l. An animal tumor model complemented physicochemical studies of 741F8 species and indicated increased tumor localization of the targeted 741F8 (sFv')2 over other monovalent 741F8 species.
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PMID:Engineering disulfide-linked single-chain Fv dimers [(sFv')2] with improved solution and targeting properties: anti-digoxin 26-10 (sFv')2 and anti-c-erbB-2 741F8 (sFv')2 made by protein folding and bonded through C-terminal cysteinyl peptides. 747 92

Twenty-one invasive squamous-cell carcinomas (SCC) of the bladder from Schistosoma-hematobium-infected patients were examined immunohistochemically for the expression of p53, Rb, EGFR and c-erbB-2 proteins; and screened by single-strand conformation polymorphism and sequencing for mutations in the ras (H, N, K) codon hotspots (12, 13, 61) and p53 (exons 4-9) genes. Positive staining for p53, EGFR and c-erbB-2 was reported in 38, 67 and 28% of tumors respectively. Only one of the tumors, the only one that was poorly differentiated, displayed an absence of nuclear Rb staining. Ras alterations were detected in the H-ras gene in 3 tumors, 2 of which harbored a codon-13 (Gly-->Arg) and one a codon-12 (Gly-->Ser) point mutation. p53 mutations were recorded in 12 tumors (57%), 6 of which stained positively for p53. Four tumors had exon-7 mutations (codons 235, 241 and 249; one tumor had 2 exon-7 mutations). Eight tumors were mutated in exon 8 (codons 264, 271, 273, 285, 286, 288 and 294), 5 of which harbored multiple mutations. One tumor had an insertion/deletion event in exon 9. The frequency of detection of over-expression of EGFR and c-erbB-2 in bilharzial-bladder lesions is comparable to that reported in TCC, contrasting with the infrequent loss of Rb expression found in invasive lesions associated with schistosomiasis infection. However, the detection of multiple p53 mutations in these lesions is suggestive of the involvement of a carcinogenic agent with maintenance of preferential activation of the H-ras gene.
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PMID:Molecular events underlying schistosomiasis-related bladder cancer. 762 66

We have shown previously that the epidermal growth factor (EGF) receptor is phosphorylated at Ser-1002 and that this phosphorylation is associated with desensitization of the EGF receptor. Ser-1002 is followed immediately by Pro-1003, a residue that may promote the adoption of a specific conformation at this site or severe as a recognition element for the interaction of the EGF receptor with other proteins. To examine these possibilities, we have mutated Pro-1003 of the EGF receptor to a Gly residue and have analyzed the effect of this mutation on EGF-stimulated signaling. Cells expressing the P1003G EGF receptors exhibited higher EGF-stimulated autophosphorylation and synthetic peptide phosphorylation compared to cells expressing wild-type EGF receptors. In addition, the ability of EGF to stimulate PI 3-kinase activity and mitogen-activated protein kinase activity was enhanced in cells expressing the P1003G EGF receptor. Cells expressing P1003G receptors also demonstrated an increased ability to form colonies in soft agar in response to EGF. These results indicate that mutation of Pro-1003 leads to a potentiation of the biological effects of EGF. The findings are consistent with the hypothesis that Pro-1003 plays a role in a form of regulation that normally suppresses EGF receptor function.
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PMID:Mutation of proline-1003 to glycine in the epidermal growth factor (EGF) receptor enhances responsiveness to EGF. 781 43

The hypothesis of structural alteration in transmembrane helices for signal transduction process is viewed by molecular dynamics simulation techniques. For the c-erbB-2 transmembrane domain involved in oncogenicity, the occurrence of conformational changes has been previously described as transition from the alpha to pi helix. This dynamical feature is thoroughly analyzed for the wild phenotype and oncogenic sequences from a series of 18 simulations carried out on one nanosecond time scale. We show that these structural events do not depend upon the conditions of simulations like force field or starting helix coordinates. We demonstrate that the oncogenic mutations Val659 Glu, Gln and Asp do not prevent the transition. Furthermore, we show that beta branched residues, in conjunction with Gly residues in the c-erbB-2 sequence, act as destabilizers for the alpha helix structure, pi deformations are tightly related to other local structural motifs found in soluble and membrane proteins. These structural alterations are discussed in term of structure-activity relationships for the c-erbB-2 activating mechanism mediated by transmembrane domain dimerization.
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PMID:Insight into signal transduction: structural alterations in transmembrane helices probed by multi-1 ns molecular dynamics simulations. 944 2

We reported previously that a conformation-specific antibody, Ab P2, to a 16-amino acid peptide (Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg) of the cytoplasmic domain of the beta-type platelet-derived growth factor receptor also recognizes the epidermal growth factor (EGF) receptor. Although the antibody is not directed to phosphotyrosine, it recognizes in immunoprecipitation the activated and hence phosphorylated form of both receptors. In P2 peptide, there are two tripeptide sequences, Asp-Glu-Glu and Tyr-Gln-Gln, that are also present in the EGF receptor. Our present studies using either EGF receptor C-terminal deletion mutants or point mutations (Tyr-->Phe) and our previous studies on antibody inhibition by P2-derived peptides suggest that Gln-Gln in combination with Asp-Glu-Glu forms a high-affinity complex with Ab P2 and that such complex formation is dependent on tyrosine phosphorylation. Of the five phosphate acceptor sites in the EGF receptor, clustered in the extreme C-terminal tail, phosphorylation of three tyrosine residues (992, 1068, and 1086) located between Asp-Glu-Glu and Gln-Gln is necessary for Ab P2 binding. In contrast, the acceptor sites Tyr 1173 and 1148 play no role in the conformation change. Asp-Glu-Glu and Gln-Gln are located 169 amino acids apart, and it is highly likely that the interactions among three negatively charged phosphotyrosine residues in the receptor C terminus may result in the bending of the peptide chain in such a way that these two peptides come close to each other to form an antibody-binding site. Such a possibility is also supported by our finding that receptor dephosphorylation results in complete loss of Ab P2-binding activity. In conclusion, we have identified a domain within the cytoplasmic part of the EGF receptor whose conformation is altered by receptor phosphorylation; furthermore, we have identified the tyrosine residues that positively regulate this conformation.
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PMID:Phosphorylation of tyrosine 992, 1068, and 1086 is required for conformational change of the human epidermal growth factor receptor c-terminal tail. 1006 1

The phage library derived, nonphosphorylated and thioether-cyclized peptide, termed G1TE, cyclo(CH(2)CO-Glu(1)-Leu-Tyr(3)-Glu-Asn-Val-Gly-Met-Tyr-Cys(10))-amid e, represents a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, with an IC(50) of 20 microM. The retention of binding affinity is very sensitive with respect to peptide ring-size alterations and Ala mutations. We demonstrated previously that the Glu(1) side chain and its closely related analogs partially compensate for the absence of the phosphate functionality on Tyr(3), and, based on molecular modeling, these acidic side-chains complex with the Arg67 and Arg86 side-chains of the protein in the binding cavity. In this study we judiciously altered and incorporated various natural and unnatural amino acids as Tyr replacements within the -YEN- motif, and we demonstrate the functional importance and structural requirement of Tyr(3) for effective binding of this novel non-phosphorylated ligand to the Grb2-SH2 domain. The phenyl side-chain moiety and a polar functional group with specific orientation in position Y(3) of the peptide are particularly required. Using SPR binding assays, a submicromolar inhibitor (IC(50) = 0.70 microM) was obtained when Glu(1) was replaced with alpha-aminoadipate and Tyr(3) was replaced with 4-carboxymethyl-Phe, providing peptide 14, G1TE(Adi(1), cmPhe(3)). Peptide 14 also inhibited Grb2/p185(erb)(B-2) protein association in cell homogenates of erbB-2-overexpressing MDA-MA-453 cancer cells at near one micromolar concentrations.
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PMID:Structural requirements for Tyr in the consensus sequence Y-E-N of a novel nonphosphorylated inhibitor to the Grb2-SH2 domain. 1054 28

Female FVB/N HER-2/neu transgenic mice from the age of 2 months were subcutaneously injected with saline, the peptide Epitalon(R) (Ala-Glu-Asp-Gly) or with the peptide Vilon(R) (Lys-Glu) in a single dose of 1 microg/mouse for 5 consecutive days every month. Epitalon treatment reduced the cumulative number and the maximum size of tumors (p < 0.05). Furthermore, the number of mice bearing 1 mammary tumor was increased, whereas the number of mice bearing 2 or more mammary tumors was reduced in Epitalon-treated in comparison to saline-treated animals (p < 0.05). The size but not the number of lung metastases was reduced in Epitalon-treated compared to saline-treated mice (p < 0.05). The treatment with Vilon produced significant negative effects when compared to the control group, with an increased incidence of mammary cancer development (p < 0.05), a shorter mean latent period of tumors (p < 0.05) and an increased cumulative number of tumors (p < 0.05). A 3.7-fold reduction in the expression of HER-2/neu mRNA was found in mammary tumors from HER-2/neu transgenic mice treated with Epitalon compared to control animals. The expression of mRNA for HER-2/neu was also partially reduced in Vilon-treated mice, but it remained significantly higher in Vilon- than in Epitalon-treated animals (1.9-fold increase). The data demonstrate the inhibitory effect of Epitalon in the development of spontaneous mammary tumors in HER-2/neu mice, suggesting that a downregulation of HER-2/neu gene expression in mammary adenocarcinoma may be responsible, at least in part, for the antitumor effect of the peptide.
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PMID:Inhibitory effect of the peptide epitalon on the development of spontaneous mammary tumors in HER-2/neu transgenic mice. 1220 81

Female transgenic FVB/N mice carrying the breast cancer gene HER-2/neu received epithalon (Ala-Glu-Asp-Gly) in a dose of 1 mg subcutaneously 5 times a week to from the 2nd month of life to death. Epithalon prolonged the average and maximum lifetimes of mice by 13.5 (p<0.05) and 13.9%, respectively. The peptide prolonged the average lifetime of animals without neoplasms (by 34.2%, p<0.05). Epithalon decelerated the development of age-related disturbances in reproductive activity and suppressed the formation of neoplasms. The peptide decreased the incidence of breast adenocarcinomas, lungs metastases (by 1.6 times, p<0.05), and multiple tumors (by 2 times). Epithalon 3.7-fold increased the number of mice without breast tumors (p<0.05), while the number of animals with 6 or more breast tumors decreased by 3 times (p<0.05). Epithalon prolonged the lifetime of mice with breast tumors by 1.4 times (p<0.05). These results indicate that Epithalon possesses geroprotective activity and inhibits breast carcinogenesis in transgenic mice, which is probably related to suppression of HER-2/neu expression.
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PMID:Epithalon decelerates aging and suppresses development of breast adenocarcinomas in transgenic her-2/neu mice. 1245 48


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