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)

The Mr 185,000 glycoprotein encoded by human c-erbB-2/neu/HER2 gene, termed c-erbB-2 gene product, shows a close structural similarity with epidermal growth factor receptor and is now regarded to be a growth factor receptor for an as yet unidentified ligand. Abundant c-erbB-2 mRNA was demonstrated by Northern blot studies in the human breast cancer cell line SK-BR-3. Cellular radiolabeling experiments followed by immunoprecipitation with three different anti-c-erbB-2 gene product antibodies, recognizing extracellular domain, kinase domain, and carboxyl-terminal portion, respectively, demonstrated the production of a large amount of c-erbB-2 gene product which had the capacity to be phosphorylated. Immunization of mice with concentrated culture medium conditioned by SK-BR-3 cells always generated antibodies against c-erbB-2 gene product, demonstrating that this culture medium contained substance(s) immunologically indistinguishable from c-erbB-2 gene product. This observation was supported by the successful development of a monoclonal antibody against c-erbB-2 gene product, GFD-OA-p185-1, by immunizing mice with this culture medium. The biochemical nature of the substance(s) present in the culture medium was further characterized. When the culture medium conditioned by [35S]cysteine-labeled SK-BR-3 cells was immunoprecipitated by three different anti-c-erbB-2 gene product antibodies, only the antibody recognizing extracellular domain precipitated the [35S]-labeled protein with a molecular weight of 110,000, namely p110. The newly developed monoclonal antibody also immunoprecipitated this protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The presence of c-erbB-2 gene product-related protein in culture medium conditioned by breast cancer cell line SK-BR-3. 198 Nov 43

The activation of the epidermal growth factor (EGF) receptor tyrosine kinase activity is thought to represent a key initial step in EGF-mediated mitogenesis. The mechanisms underlying the regulation of the EGF receptor tyrosine kinase activity were examined through comparisons of the holoreceptor, purified from human placenta, and a soluble 42 kDa tyrosine kinase domain (TKD), generated by the limited trypsin proteolysis of the holoreceptor. The results of these studies highlight the importance of divalent metal ions (Me2+), i.e., Mn2+ and Mg2+, as activators of the tyrosine kinase activity. Manganese is an extremely effective activator of the holoreceptor tyrosine kinase, and under some conditions (low ionic strength) it completely alleviates the need for EGF to stimulate activity. In contrast, Mg2+ only weakly stimulates the holoreceptor tyrosine kinase activity in the absence of EGF, but promotes essentially full activity in the presence of the growth factor. Like the holoreceptor, the soluble TKD is highly active in the presence of Mn2+. However, the isolated TKD is completely inactive in the presence of Mg2+, and, in fact, Mg2+ inhibits the Mn2(+)-stimulated tyrosine kinase activity. The differences in the effects of Mn2+ and Mg2+ on the isolated TKD were further demonstrated by monitoring the effects of Me2+ on the modification of a reactive cysteine residue(s) on the TKD. While Mn2+ potentiates the inhibition by cysteine-directed reagents of the tyrosine kinase activity, Mg2+ has no effect on either the rate or the extent of the inhibition. Both the regulation by Mn2+ of the kinase activity of the TKD and the potentiation by Mn2+ of the cysteine reactivity of the TKD occur over a millimolar concentration range, which implicates a direct binding interaction by the metal ion. Overall, these results demonstrate that there are two key activator sites on the EGF receptor, i.e., the EGF binding site on the extracellular domain and a Me2+ binding site on the cytoplasmic TKD. Me2+ interactions with the cytoplasmic kinase domain apparently result in conformational changes which regulate the levels of tyrosine kinase activity, influence the degree to which this activity is responsive to EGF, and probably account for the effects of Me2+ on the aggregation state of the receptor (Carraway, K.L., III, Koland, J.G. and Cerione, R.A. (1989) J. Biol. Chem. 264, 8699-8707). In general, Mg2(+)-induced conformation changes prime the receptor for activation by EGF, while Mn2+ can fully activate the receptor tyrosine kinase and thereby short-circuit growth factor control.
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PMID:Activation of the EGF receptor tyrosine kinase by divalent metal ions: comparison of holoreceptor and isolated kinase domain properties. 235 10

Single-chain Fv proteins containing a COOH-terminal cysteine (sFv') were constructed by using an antidigoxin 26.10 sFv and an anti-c-erbB-2 741F8 sFv. The fully active sFv' proteins were prepared by expression in Escherichia coli as insoluble inclusion bodies, followed by in vitro refolding using glutathione redox buffers and purification. The COOH-terminal cysteines of the refolded sFv' proteins were protected by a blocking group presumed to be the glutathionyl peptide, which was easily and selectively removed by gentle reduction. Air oxidation of the reduced sFv' monomers resulted in the efficient formation of disulfide-linked sFv' homodimers, designated (sFv')2, which were stable under oxidizing conditions and relatively slow to be disrupted under reducing conditions. The (26-10-1 sFv')-(741F8-1 sFv') heterodimer was prepared and possessed dual-antigen specificity; the active bispecific (sFv')2 dimerized under native conditions, apparently as a manifestation of self-association by the 741F8 sFv' subunit. Biodistribution and imaging studies that were performed on mice bearing human SK-OV-3 tumor xenografts that express the c-erbB-2 as a cell surface antigen were reviewed. Radioiodinated 741F8-2 (sFv')2 homodimer localized to the tumors with high specificity, as evidenced by excellent tumor:normal tissue ratios. Sagittal section autoradiography of whole animals 24 h after administration of antibody species revealed that 741F8 (sFv')2 produced a stronger tumor image than comparable doses of the 741F8 Fab, monomeric sFv', and the 26-10 (sFv')2 control without the high nonspecific background distribution of the 741F8 IgG.
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PMID:Targeting c-erbB-2 expressing tumors using single-chain Fv monomers and dimers. 749 81

Protein tyrosine phosphatases all contain a conserved cysteine that forms an intermediate thiophosphate ester bond during tyrosine phosphate hydrolysis. A bacterial glutathione S-transferase fusion protein containing rat brain phosphatase PTP1b was constructed in which this conserved cysteine was mutated to serine. The resulting catalytically inactive enzyme was labeled in vivo to high specific activity with 35S, and the binding of this labeled fusion protein to the immunoprecipitated epidermal growth factor (EGF) receptor was evaluated. The binding was ligand-dependent, and saturation analysis revealed a nonlinear Scatchard plot, with a Kd for high affinity binding of approximately 100 nM. A number of glutathione S-transferase fusion proteins containing src homology 2 (SH2) domains attenuated phosphatase binding in a concentration-dependent manner. Phospholipase C (PLC) gamma and the GTPase-activating protein of ras were the most potent inhibitors. Tyrosine-phosphorylated EGF receptor peptide fragments were evaluated for specific inhibition of PTP1b and PLC gamma SH2 binding to the activated receptor. One such peptide, modeled on EGF receptor tyrosine 992, blocked the binding of both fusion proteins. Another phosphopeptide, modeled on tyrosine 1148, inhibited the binding of PTP1b but not the PLC gamma fusion protein. This site specificity was confirmed by analysis of equilibrium binding of the fusion proteins to EGF receptors mutated in each of these phosphorylation sites. The results revealed clear sequence specificity in the binding of proteins involved in the regulation of intracellular signaling by receptor tyrosine kinases.
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PMID:Sequence specificity in recognition of the epidermal growth factor receptor by protein tyrosine phosphatase 1B. 769 94

This investigation has utilized novel forms of the single-chain Fv (sFv), wherein a cysteine-containing peptide has been fused to the sFv carboxyl terminus to facilitate disulfide bonding or specific cross-linking of this sFv' to make divalent (sFv')2. The 741F8 anti-c-erbB-2 monoclonal antibody was used as the basis for construction of 741F8 sFv, from which the sFv' and (sFv')2 derivatives were prepared. Recombinant c-erbB-2 extracellular domain (ECD) was prepared in CHO cells and the bivalency of 741F8 (sFv')2 demonstrated by its complex formation with ECD. The tumor binding properties of 125I-labeled anti-c-erbB-2 741F8 sFv, sFv', and (sFv)2 were compared with radiolabeled antidigoxin 26-10 sFv' and (sFv')2 controls. Following intravenous administration of radiolabeled species to severe combined immune-deficient (SCID) mice bearing SK-OV-3 tumors (which over-express c-erbB-2), blood and organ samples were obtained as a function of time over 24 h. Comparative analysis of biodistribution and tumor-to-organ ratios demonstrated the 741F8 sFv, sFv', and (sFv')2 had excellent specificity for tumors, which improved with time after injection. This contrasted with nonspecific interstitial pooling in tumors observed with the 26-10 sFv, sFv', and (sFv')2, which decreased with time after administration. Tumor localization was significantly better for disulfide or peptide crosslinked 741F8 (sFv')2 having Gly4Cys tails than for monovalent 741F8 sFv' or Fab. The superior properties of the 741F8 (sFv')2 in targeting SK-OV-3 tumors in SCID mice suggests the importance of further investigations of divalent sFv analogs for immunotargeting.
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PMID:Tumor targeting in a murine tumor xenograft model with the (sFv')2 divalent form of anti-c-erbB-2 single-chain Fv. 773 32

The production of several single-chain Fv (sFv) antibody proteins was examined by three modes of mammalian cell expression. Our primary model was the 741F8 anti-c-erbB-2 sFv, assembled as either the VH-VL or VL-VH, and expressed alone, with C-terminal cysteine for dimerization, or as fusion proteins with carboxyl-terminal effector domains, including interleukin-2, the B domain of staphylococcal protein A, the S-peptide of ribonuclease S, or hexa-histidine metal chelate peptide. Constructs were expressed and secreted transiently in 293 cells and stably in CHO or Sp2/0 cell lines, the latter yielding up to 10 mg per liter. Single-chain constructs of MOPC 315 myeloma and 26-10 monoclonal antibodies were also expressed, as were hybrids comprising unrelated VH and VL regions. Our results suggest that mammalian expression is a practical and valuable complement to the bacterial expression of single-chain antibodies.
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PMID:Mammalian cell expression of single-chain Fv (sFv) antibody proteins and their C-terminal fusions with interleukin-2 and other effector domains. 776 52

Eph, Elk, and Eck are prototypes of a large family of transmembrane protein-tyrosine kinases, which are characterized by a highly conserved cysteine-rich domain and two fibronectin type III repeats in their extracellular regions. Despite the extent of the Eph family, no extracellular ligands for any family member have been identified, and hence, little is known about the biological and biochemical properties of these receptor-like tyrosine kinases. In the absence of a physiological ligand for the Elk receptor, we constructed chimeric receptor molecules, in which the extracellular region of the Elk receptor is replaced by the extracellular, ligand-binding domain of the epidermal growth factor (EGF) receptor. These chimeric receptors were expressed in NIH 3T3 cells that lack endogenous EGF receptors to analyze their signaling properties. The chimeric EGF-Elk receptors became glycosylated, were correctly localized to the plasma membrane, and bound EGF with high affinity. The chimeric receptors underwent autophosphorylation and induced the tyrosine phosphorylation of a specific set of cellular proteins in response to EGF. EGF stimulation also induced DNA synthesis in fibroblasts stably expressing the EGF-Elk receptors. In contrast, EGF stimulation of these cells did not lead to visible changes in cellular morphology, nor did it induce loss of contact inhibition in confluent monolayers or growth in semisolid media. The Elk cytoplasmic domain is therefore able to induce tyrosine phosphorylation and DNA synthesis in response to an extracellular ligand, suggesting that Elk and related polypeptides function as ligand-dependent receptor tyrosine kinases.
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PMID:Biological and biochemical activities of a chimeric epidermal growth factor-Elk receptor tyrosine kinase. 841 96

We report the use of structure-based drug design to create a selective erbB-1 (a.k.a. epidermal growth factor receptor) and erbB-2 (a.k.a. neu/her2 growth factor receptor) tyrosine kinase inhibitor. Using the X-ray crystal structure of the ternary complex of the cAMP-dependent Ser/Thr kinase together with a sequence alignment of the catalytic domains of a representative set of Ser/Thr and Tyr protein kinases, we have examined the nucleotide binding site for potential positions to attach an irreversible inhibitor. This information, combined with homology modeling of the erbB-1 and erbB-2 tyrosine kinase catalytic domains, has led to the identification of Cys797 of erbB1 and Cys805 of erbB2, which are structurally equivalent to Glu127 in the cAMP dependant Ser/Thr kinase as potential target residues. The X-ray structure of the cAMP Ser/Thr kinase shows Glu127 to be involved in a hydrogen-bonding interaction with the 2'-OH of the ribose portion of ATP. Using molecular modeling, it was predicted that the Cys side chains in erbB-1 and erbB-2 performed an analogous role, and it was postulated that the replacement of the 2'-OH of adenosine with a thiol might allow for a covalent bond to form. Since only erbB-1 and erbB-2 have a Cys at this position, the inhibitor should be selective. This model was subsequently tested experimentally by chemical synthesis of 2'-thioadenosine and assayed against the full length erbB-1 receptor and the catalytic domains of erbB-2, insulin receptor, beta-PDGF receptor, and the FGF receptor. Our results show that thioadenosine covalently inactivates erbB-1 with a second-order rate constant of k(max)/K(S) = 2000 +/- 500 M(-1) s(-1). Inactivation is fully reversed by 1 mM dithiothreitol, suggesting that inactivation involves the modification of a cysteine residue at the active site, presumably Cys797. The rate of inactivation saturates with increasing thioadenosine concentrations, suggesting that inactivation occurs through initial formation of a noncovalent complex with K(D) = 1.0 +/- 0.3 microM, followed by the slow formation of a disulfide bond with a rate constant of k(max) = (2.3 +/- 0.2) x 10(-3) s(-1). This approach may have application in the design of selective irreversible inhibitors against other members of the kinase family.
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PMID:Structure-based design of a potent, selective, and irreversible inhibitor of the catalytic domain of the erbB receptor subfamily of protein tyrosine kinases. 908 34

Small peptides, 8-10 amino acids long, derived from degradation of cytoplasmic proteins by a proteasome-proteinase complex, are usually presented and recognized by CD8+ cytolytic T lymphocytes (CTLs) associated with major histocompatibility complex (MHC) class I molecules. Recently synthetic peptides were used for the in vitro induction of tumor-specific CTLs, offering another strategy in the study of the immune-response repertoire and providing a new tool in cancer vaccination and immunotherapy. Peptides derived from otherwise normal proteins, overexpressed in many tumors as products of the protooncogene, may represent a target for an immune response. This is the case of HER-2/neu gene (also known as ErbB-2), encoding a cysteine-rich glycoprotein transmembrane receptor with tyrosine kinase activity (gp185neu). Recent data, demonstrating that HLA-A2.1-related peptides are able to stimulate in vitro CD8+ lymphocytes, Prompted us to study the binding to HLA-A2.1 molecules of several gp185 synthetic peptides containing a cystein residue and to define the relevance of this amino acid residue in the reduced or oxidated form of the sulfhydryl group. We found that monomers and their homodimers, linked by a disulfide bridge, bind to HLA-A2.1 molecules with overlapping affinity. These results suggest that additional amino acids of the nonapeptide do not prevent the binding and the HLA refolding through chemical or sterical interactions. This might be of particular relevance for the in vivo processing of cysteine-rich proteins. Because ErbB-2 molecules, as tumor-differentiation antigens in melanoma, are cysteine-rich molecules, it may be relevant to evaluate the possible role of the cystine residues interacting with the T-cell receptor. The recognition of these heterodimers by CD8+ lymphocytes will require functional in vivo studies.
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PMID:MHC-peptide binding: dimers of cysteine-containing nonapeptides bind with high affinity to HLA-A2.1 class I molecules. 940 48

Recent evidence has suggested that amplification and overexpression of erbB-2/neu is an important determinant in the initiation and progression of human breast cancer. Consistent with this assertion is the observation that transgenic mice that overexpress the neu proto-oncogene heritably develop mammary adenocarcinomas. More recently, we have demonstrated that activation of neu in many of these tumours occurs as a result of somatic mutations located within the transgene itself. Indeed, careful examination of the altered neu transcripts revealed the presence of in-frame deletions that encode aberrant Neu receptors lacking 5-12 amino acids within the extracellular domain, located adjacent to the transmembrane domain. Interestingly, the majority of the deletions analysed affect one of several conserved cysteine residues present within this region. Moreover, introduction of these activating mutations into the wild-type neu cDNA results in its oncogenic conversion. These observations suggest that this cysteine-rich region plays an important role in regulating the catalytic activity of Neu.
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PMID:Oncogenic activation of Neu/ErbB-2 in a transgenic mouse model for breast cancer. 951 19


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