Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:O76050 (neu)
 3,969 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Enhanced levels of disulfide-linked dimers of the neu oncogene product have been suggested to be associated with the transformed state [Weiner DB, Liu J, Cohen JA, Williams WV, Greene MI: Nature 338:230-231, (1989)]. We, therefore, investigated the properties of the dimeric forms of p185HER-2/neu from the human breast carcinoma cell line, SK-BR-3. We found disulfide-linked dimers as well as noncovalently associated dimers that were detected by cross-linking with bis(sulfosuccinimidyl) suberate (BS3). However, the disulfide-linked dimers did not exist in intact cells, since they were eliminated when the cells were lysed in the presence of the alkylating agent, sodium iodoacetate. Moreover, the disulfide-linked dimeric molecules were not the activated form of p185HER-2 since they incorporated about the same level of phosphate in an in vitro kinase reaction as the monomeric molecules. In contrast, the noncovalent dimers appeared to be present on the surface of intact cells and were phosphorylated at levels at least tenfold higher than monomers in an in vitro kinase reaction.
 ...
PMID:Disulfide-linked and noncovalent dimers of p185HER-2 in human breast carcinoma cells. 137 5

The neu protooncogene (also known as c-erbB2, NGL, and HER2) encodes a 185-kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity that resembles the receptor for epidermal growth factor. The p185 gene and protein were originally identified in the brain and are thought to play a critical role in neurogenesis. Aberrant c-erbB2 protein overexpression also occurs in several human adenocarcinomas. A ligand for p185, neu-activating factor (NAF), specifically binds to neu receptor and increases the p185c-neu tyrosine phosphorylation in vitro and in vivo in a dose-dependent manner. We now show that NAF specifically binds to purified p185 expressed in baculovirus. Direct binding analysis showed that NAF binds with high affinity (Kd = 1.3 nM). We have investigated changes in the structure and association state of baculovirus-produced neu holoreceptor that are induced by ligand binding. In this study, we used sucrose gradients to show that purified p185c-neu exists mainly in the monomeric form at low concentrations, whereas at higher concentrations p185c-neu exists as dimers or multimers. At low concentrations, but in the presence of ligand, p185c-neu sediments as a dimeric or multimeric form. Monomer-oligomer interconversion is absolutely ligand dependent at low receptor concentrations. The high molecular weight form of the receptor is enzymatically more active, as a consequence of ligand-driven activation of the receptor kinase. Oncogenic p185neu receptors sediment predominantly as high molecular weight forms and have constitutively active kinases.
 ...
PMID:Ligand and p185c-neu density govern receptor interactions and tyrosine kinase activation. 790 21

Single-chain Fv (scFv) molecules exhibit highly specific tumour-targeting properties in tumour-bearing mice. However, because of their smaller size and monovalent binding, the quantities of radiolabelled scFv retained in tumours limit their therapeutic applications. Diabodies are dimeric antibody-based molecules composed of two non-covalently associated scFv that bind to antigen in a divalent manner. In vitro, diabodies produced from the anti-HER2/neu (c-erbB-2) scFv C6.5 displayed approximately 40-fold greater affinity for HER2/neu by surface plasmon resonance biosensor measurements and significantly prolonged association with antigen on the surface of SK-OV-3 cells (t1/2 cell surface retention of > 5 h vs 5 min) compared with C6.5 scFv. In SK-OV-3 tumour-bearing scid mice, radioiodinated C6.5 diabody displayed a highly favourable balance of quantitative tumour retention and specificity. By as early as 4 h after i.v. administration, significantly more diabody was retained in tumour (10 %ID g(-1)) than in blood (6.7 %ID ml(-1)) or normal tissue (liver, 2.8 %ID g(-1); lung, 7.1 %ID g(-1); kidney, 5.2 %ID g(-1)). Over the next 20 h, the quantity present in blood and most tissues dropped approximately tenfold, while the tumour retained 6.5 %ID g(-1) or about two-thirds of its 4-h value. In contrast, the 24-h tumour retention of radioiodinated C6.5 scFv monomer was only 1 %ID g(-1). When diabody retentions were examined over the course of a 72-h study and cumulative area under the curve (AUC) values were determined, the resulting tumor-organ AUC ratios were found to be superior to those previously reported for other monovalent or divalent scFv molecules. In conclusion, the diabody format provides the C6.5 molecule with a distinct in vitro and in vivo targeting advantage and has promise as a delivery vehicle for therapeutic agents.
 ...
PMID:Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu. 965 55

Dimerization of the neu/ErbB-2 receptor tyrosine kinase is a necessary but not a sufficient step for signaling. Despite the efforts expended to identify the molecular interactions responsible for receptor-receptor contacts and particularly those involving the transmembrane domain, structural details are still unknown. In this work, molecular dynamics simulations of the helical transmembrane domain (TM) of neu and ErbB-2 receptors are used to predict their dimer structure both in the wild and oncogenic forms. A global conformational search method, applied to define the best orientations of parallel helices, showed an energetically favorable configuration with the specific mutation site within the interface, common for both the nontransforming and the transforming neu/ErbB-2 TM dimers. Starting from this configuration, a total of 10 simulations, about 1.4 ns each, performed in vacuum, without any constraints, show that the two helices preferentially wrap in left-handed interactions with a packing angle at about 20 degrees. The resulting structures are nonsymmetric and the hydrogen bond network analysis shows that helices experience pi local distortions that facilitate inter-helix hydrogen bond interactions and may result in a change in the helix packing, leading to a symmetric interface. For the mutated sequences, we show that the Glu side chain interacts directly with its cognate or with carbonyl groups of the facing backbone. We show that the connectivity between interfacial residues conforms to the knobs-into-holes packing mode of transmembrane helices. The dimeric interface described in our models is discussed with respect to mutagenesis studies.
 ...
PMID:Structure prediction of the dimeric neu/ErbB-2 transmembrane domain from multi-nanosecond molecular dynamics simulations. 1066 32

In immunodeficient mice antitumor single-chain Fv (scFv) molecules penetrate tumors rapidly and have rapid serum clearance, leading to excellent tumor:normal organ ratios. However, the absolute quantity of scFv retained in the tumor is low due to rapid serum clearance and monovalent scFv binding. We previously demonstrated that the presence of an additional binding site prolongs in vitro and in vivo association of scFv-based molecules with tumor cells expressing relevant antigen. The contribution of the intrinsic affinity of each component scFv to the association between a dimeric scFv and its target antigen is largely unknown. Here, we have constructed bivalent diabody molecules from three affinity mutants of the human anti-ErbB2 (HER2/neu) scFv molecule C6.5 by shortening the peptide linker between the heavy (VH) and light (VL) chains variable domains from 15 to 5 amino acids. The shorter linker prevents intramolecular pairing of VH and VL, resulting in intermolecular pairing and creation of a dimeric Mr 50,000 molecule with two antigen-binding sites. The scFv used to create the diabodies span a 133-fold range of affinity for the same epitope of ErbB2 [133 nM (C6G98A), 25 nM (C6.5), and 1 nM (C6ML3-9)] and differ by only one to three amino acids. Diabody binding kinetics were determined by surface plasmon resonance on the immobilized ErbB2 extracellular domain. The association rate constants obtained for each diabody molecule were similar to that of the parental (component) scFv. However, the dissociation rate constants obtained for the bivalent diabodies were up to 15-fold slower. The magnitude of the decrease in the bivalent dissociation rate constant was inversely proportional to the monovalent interaction, ranging from only 3-fold for that of the C6ML3-9 diabody to 15-fold for the C6G98A diabody. This resulted in only a 22-fold difference in bivalent affinity, compared with a 133-fold difference in affinity for the respective scFv. Equilibrium-binding constants obtained by surface plasmon resonance correlated well with the equilibrium-binding constants determined in vitro on ErbB2 overexpressing cells. Biodistribution studies were performed in scid mice bearing established SKOV3 tumors. At 24 h, 3-37-fold more diabody was retained in tumor compared with the parental scFv monomers. This likely results from a higher apparent affinity, because of bivalent binding, and a slower serum clearance. Surprisingly, the differences in affinity between diabodies did not result in differences in quantitative tumor retention or tumor to blood ratios. In fact, the diabody constructed from the lowest affinity scFv exhibited the best tumor-targeting properties. We conclude that, above a threshold affinity, other factors regulate quantitative tumor retention. In addition, straightforward dimerization of a low-affinity scFv leads to significantly greater tumor localization than does exhaustive scFv affinity maturation.
 ...
PMID:Targeting of bivalent anti-ErbB2 diabody antibody fragments to tumor cells is independent of the intrinsic antibody affinity. 1110 10

Confocal laser scanning microscopy (CLSM) is an extensive but reliable tool for assessing the hybridisation signals in fluorescence in situ hybridisation (FISH). Most CLSMs are equipped with an argon-laser and a helium/neon-laser illumination system with excitation wavelengths of 488, 543 and 633 nm. A protocol for an optimal nuclear counterstaining in combination with dual-colour FISH for these laser illumination systems has not been established so far. Here, we determined the suitability of eleven dimeric and monomeric cyanine nucleic acid stains on paraffin sections of breast carcinoma specimens in combination with dual-colour FISH (Her-2/neu and centromere 17) for CLSM application. Strong staining of cell nuclei was observed for TO-PRO-3 and YO-PRO-3, YOYO-1 and propidium iodide (PI), but only TO-PRO-3 showed specific staining of nuclei without any staining of the cytoplasm. A specific emission in exclusively one distinct fluorescence channel was shown for TO-PRO-3 (633 nm excitation) as well as YOYO-1, BO-PRO-1 and Sytox Green (488 nm excitation), evaluated by a CLSM and confirmed by 3-D fluorescence spectra. High stability of fluorescence intensity was shown for the far-red dyes TO-PRO-3, YO-PRO-3, YOYO-3 and Syto-59 as well as YOYO-1 and PI. Only TO-PRO-3 was due to its high specificity and stability suitable for detection of an amplification of the Her-2/neu gene by dual-colour FISH and CLSM evaluation.
 ...
PMID:TO-PRO-3 is an optimal fluorescent dye for nuclear counterstaining in dual-colour FISH on paraffin sections. 1140 57

The specific point mutation Val-->Glu664 within the transmembrane domain of the neu/erbB-2 receptor is associated with increased receptor dimerization and increased receptor tyrosine kinase activity resulting in malignant transformation of cells. It is well established that Glu and residues in proximity are necessary for receptor dimerization but many studies suggest that other intramembrane constraints, not yet elucidated, are determinant for transformation. In this work, we investigated dimer models both to understand the structural role of the Glu mutation in the transmembrane domain association and to determine helix-helix contacts required for oncogenic transformation. Different types of helix-helix association based on data resulting from Cys mutational studies of the full wild receptor and spectroscopic data of transmembrane neu peptides have been explored by molecular dynamics simulations. The study leads to propose a model for the dimeric association of the transmembrane domains of the oncogenic neu receptor showing left-handed interactions of the two helices stabilized by symmetrical hydrogen bonding interactions involving the Glu side chain on one helix and the facing carbonyl of Ala661 on the second helix. Contacting residues observed in the symmetric interface explain the transforming activity or the non transforming activity of many neu mutants. Moreover the left-handed coiled coil structure is fully consistent with recent results proving the role of rotational linkage of the transmembrane domain with the kinase domain. Comparison between the predicted dimer model and those presumed from experiments strongly suggests helix flexibility in the extracellular juxtamembrane region.
 ...
PMID:Dimer interface of transmembrane domains for neu/erbB-2 receptor dimerization and transforming activation: a model revealed by molecular dynamics simulations. 1156 46

Mixtures of dicaproyl- (DC), dimyristoyl- (DM) and 1-tetradecanoyl-2-biphenylbutanoyl-(TBB) phosphatidylcholine (PC) in water produce bicelle membranes that are oriented by magnetic fields. DMPC/DCPC systems orient such that their membrane plane is parallel to the magnetic field, whereas for TBBPC/DCPC, the plane is perpendicular to the field. Partial temperature-composition-hydration diagrams are established using solid-state 31P-NMR. DMPC/DCPC bicelles exist on a large range of composition but on a narrow temperature domain (25-45 degrees C). At converse, TBBPC/DCPC form bicelles on a narrow compositional range but over a large temperature span (10-70 degrees C). The TBBPC/DCPC bicelles are shown to be a very powerful potential tool to study the orientation of hydrophobic helices in membranes using wide line 15N-NMR. The DMPC/DCPC system that undergoes a micelle-to-bicelle transition on going from 10 degrees C to 40 degrees C may be used with circular dichroism to study the state of association of hydrophobic helices within the membrane. Results suggest that the transmembrane fragment of the neu/erbB-2 receptor is monomeric in micellar medium and dimeric/multimeric in bicelle membranes.
 ...
PMID:Bicelle membranes and their use for hydrophobic peptide studies by circular dichroism and solid state NMR. 1596 Dec 33

mAbs capable of disabling heterodimeric kinase complexes of the epidermal growth factor receptor (EGFR) and human EGFR type 2/neu have therapeutic relevance to various human cancers. In this study, we demonstrate that in addition to the dimer, EGFR and human EGFR type 2 can associate as homo- and heterotetramers. EGF-induced phosphorylation of the tetramers was significantly lower than that of the dimers, indicating that the tetrameric receptor complexes have impaired signaling activity. Targeting v-erb-b2 erythroblastic leukemia viral oncogene homolog (erbB) receptors with mAbs promoted erbB tetrameric assembly, suggesting that a component of the antitumor activity may be mediated by the ability of Abs to shift the equilibrium from active dimeric to impaired tetrameric receptor complex states. This study suggests a novel therapeutic approach to disable signaling of erbB and potentially other receptors in tumors by biologic agents capable of inducing receptor tetramerization.
 ...
PMID:Targeted antireceptor therapy with monoclonal antibodies leads to the formation of inactivated tetrameric forms of ErbB receptors. 1720 65

Vectors based on Adenovirus type 5 (Ad5) are among the most common vectors in cancer gene therapy trials to date. However, for increased efficiency and safety, Ad5 should be de-targeted from its native receptors and re-targeted to a tumor antigen. We have described earlier an Ad5 vector genetically re-targeted to the tumor antigen HER2/neu by a dimeric version of the Affibody molecule ZH inserted in the HI-loop of the fiber knob of a coxsackie and adenovirus receptor-binding ablated fiber. This virus showed almost wild-type growth characteristics and infected cells through HER2/neu. Here we generate vectors with double specificity by incorporating two different Affibody molecules, ZH (HER2/neu-binding) and ZT (Taq polymerase-binding), at different positions relative to one another in the HI-loop. Receptor-binding studies together with viral production and gene transfer assays showed that the recombinant fiber with ZT in the first position and ZH in the second position (ZTZH) bound to both its targets, whereas surprisingly, the fiber with ZHZT was devoid of binding to HER2/neu. Hence, it is possible to construct a recombinant adenovirus with dual specificity after evaluating the best position for each ligand in the fiber knob.
 ...
PMID:Re-targeted adenovirus vectors with dual specificity; binding specificities conferred by two different Affibody molecules in the fiber. 1894 96


1