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Biomedical subjects

György Vereb

Publications and source records attributed to György Vereb.

At least 19 recordsLinked to original sources
PubMed2006

Nucleolar localization of phosphatidylinositol 4-kinase PI4K230 in various mammalian cells.

BACKGROUND: Previous immunohistochemical investigations could not detect PI4K230, an isoform of mammalian phosphatidylinositol 4-kinases (also called type III alpha), in the nucleus and nucleolus of cells in spite of its predicted nuclear localization signals. METHODS: Immunofluorescent detection of PI4K230 and other PI4K isoforms was performed on formaldehyde (PFA) or ethanol fixed cells and rat brain cryosections. Costaining with nucleolin and the effect of siRNA, Triton X-100, DNase, and RNase treatments were also tested to determine the localization of PI4K230. RESULTS: PI4K230 gives a prominent signal in the nucleolus of ethanol fixed rat brain cryosections and of several cell types in addition to its presence in the nucleus and cytoplasm. The PI4K230 immunoreactivity of the nucleolus is masked in PFA fixed cells, but it can be restored by treatment of PFA fixed cells with hot wet citrate buffer or by washing the cryosections with PBS prior to PFA fixation. Nucleolar PI4K230 occurs in a Triton X-100 resistant complex. Treatment of COS-7 cells with siRNA targeting PI4K230 and permeabilized B50 cells with DNase or RNase results in the loss of PI4K230 signal from the nucleolus. CONCLUSION: These experiments suggest the participation of PI4K230 in a DNase and RNase sensitive complex with a unique localization and function in the nucleolus.

1-Phosphatidylinositol 4-Kinase↗
PubMed2006

Differential platelet deposition onto collagen in cone-and-plate and parallel plate flow chambers.

To routinely test the formation of thrombi and the effect of drugs modifying it, proper test systems are needed. Their design should rely on the laws of rheology and the physiology of laminar flow. To best model physiological or pathological shear conditions, parallel/linear and rotational type flow chambers are developed. We have compared the initial phase of platelet thrombus formation in a parallel plate flow chamber (PPC) and a cone-and-plate chamber (CPC) under von Willebrand dependent shear conditions. Blood was allowed to flow through human collagen type III surfaces at a shear rate of 1000 s(-1) for 150 s. Thrombus deposition was characterized by surface coverage, average area and height of thrombi. VWF distribution within thrombi was analyzed with confocal laser scanning microscopy. Reduced surface-specific platelet adhesion and aggregation (surface coverage and average thrombus size) were observed in CPC along with a significant increase in single platelet disappearance from the circulating blood. Our data suggest that the higher rate of platelet consumption in this device, as opposed to PPC, is limiting the adhesion to the surface. Consequently, surface-specific processes and aggregation in the flowing blood are both assessed using CPC, while comprehensive evaluation of surface-specific processes is best achieved with PPC. Therefore, the choice of chamber type as a diagnostic tool is purpose-dependent.

Collagen Type III↗
PubMed2006

Leukemic lymphoblasts, a novel expression site of coagulation factor XIII subunit A.

Blood coagulation factor XIII (FXIII) is a protransglutaminase circulating as a tetramer formed by two types of subunits (A2B2). The intracellular dimeric form of FXIII (A2) is present in platelets, megakaryocytes, monocytes and macrophages and has been detected in mono- and megakaryocytic leukemias. The aim of our study was to investigate FXIII-A expression in newly diagnosed B cell acute lymphoblastic leukemia (ALL) samples. We examined 47 de novo ALL cases of B cell origin by triple color labeling with flow cytometry. FXIII-A was detected by a FITC conjugated monoclonal antibody combined with CD34 and CD45 staining. In selected cases FXIII-A was investigated on slides prepared from blasts and visualized with a fluorescent microscope. In addition, blasts were studied by Western blot analysis and FXIII-A was measured by a highly sensitive ELISA method. By flow cytometry 19 samples of the 47 cases were found to be FXIII-A positive. Antigen concentration was 3.11 +/- 1.19 fg/blast, while normal lymphoid precursors and mature lymphocytes from B-CLL did not contain FXIII-A. In the lysate of lymphoblasts that were positive by flow cytometry, a single band (82 kDa) corresponding to FXIII-A was detected on Western blots. Confocal laser scanning microscopic examination revealed the presence of FXIII-A in the cytoplasm of these lymphoblasts. This novel expression site of FXIII-A in leukemic lymphoblasts can be utilized as a diagnostic tool and may also gain functional significance in B-lineage ALL.

Adolescent↗
PubMed2005

Hsp90 inhibitor 17-AAG reduces ErbB2 levels and inhibits proliferation of the trastuzumab resistant breast tumor cell line JIMT-1.

ErbB2, a member of the EGF receptor family of tyrosine kinases is overexpressed on many tumor cells of epithelial origin and is the molecular target of trastuzumab (Herceptin), the first humanized antibody used in the therapy of solid tumors. Trastuzumab, which is thought to act, at least in part, by downregulating ErbB2 expression is only effective in approximately 30-40% of ErbB2 positive breast tumors. Geldanamycin and its derivative 17-AAG are potential antitumor agents capable of downregulating client proteins of Hsp90, including ErbB2. To investigate the ability of 17-AAG to downregulate ErbB2 in trastuzumab resistant breast cancer cells and the possibility of 17-AAG and trastuzumab potentiating each other's effect, the recently established trastuzumab resistant breast cancer cell line, JIMT-1 was compared to the known trastuzumab sensitive SKBR-3 line. Baseline and stimulus-evoked dimerization and activation levels of ErbB2, and the effects of trastuzumab and 17-AAG alone and in combination on cell proliferation and apoptosis, as well as on ErbB2 expression and phosphorylation have been measured. Baseline activation and amenability to activation and downregulation by trastuzumab was much lower in the resistant line. However, 17-AAG enhanced ErbB2 homodimerization after 5-10 min of treatment in both cell lines, and decreased proliferation with an IC50 of 70 nM for SKBR-3 and 10nM for JIMT-1. Thus, 17-AAG may be a useful drug in trastuzumab resistant ErbB2 overexpressing tumors. The antiproliferative effect of 17-AAG was positively correlated with phosphorylation and downregulation of ErbB2 and was dominated by apoptosis, although, especially at higher doses, necrosis was also present. Interestingly, IC50 values for ErbB2 downregulation and phosphorylation, in the 30-40 nM range, were not significantly different for the two cell lines. This observation and the negative correlation between resting ErbB2 levels and the antiproliferative effect of 17-AAG may indicate that activation of ErbB2 to some extent could counteract the overall cytostatic effect, especially at higher levels of ErbB2 expression. The usual therapeutic dose of trastuzumab did not change the IC50 of 17-AAG on the proliferation of either cell line, but nevertheless decreased overall ErbB2 phosphorylation and at low doses of 17-AAG further decreased cell growth in the sensitive SKBR-3, thus trastuzumab may be a good combination partner to counteract undesired activating effects of 17-AAG.

Antibodies, Monoclonal↗
PubMed2005

Associations of ErbB2, beta1-integrin and lipid rafts on Herceptin (Trastuzumab) resistant and sensitive tumor cell lines.

ErbB2-mediated transmembrane signaling is a key target of novel anticancer agents such as Herceptin. Our comparison of Herceptin resistant (JIMT-1, MKN-7) and sensitive (SKBR-3, N-87) cell lines demonstrates the importance of ErbB2 association patterns involving integrins and lipid rafts. Flow cytometric FRET and confocal microscopic measurements revealed colocalization and molecular proximity between beta1-integrins and ErbB2, as well as their association with lipid rafts. A weak functional interaction between ErbB2 and beta1-integrin and the fact that ErbB2 did not co-patch with beta1-integrins upon crosslinking imply that ErbB2 and beta1-integrin define two distinct molecular association clusters from a functional point of view. Although Herceptin-sensitive cell lines expressed more ErbB2 and fewer beta1-integrin molecules on their surface than their resistant counterparts, this finding probably does not explain the Herceptin resistant phenotype due to the weak interaction between beta1-integrins and ErbB2. Our results imply that the true significance of the expression profile of proteins involved in oncogenesis can only be understood after characterizing their molecular interactions.

Antibodies, Monoclonal↗
PubMed2005

Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements.

BACKGROUND: Fluorescence resonance energy transfer applied in flow cytometry (FCET) is an excellent tool for determining supramolecular organization of biomolecules at the cell surface or inside the cell. Availability of new fluorophores and cytometers requires the establishment of fluorophore dye pairs most suitable for FCET measurements. METHODS: A gastric tumor cell line (N87) was labeled for major histocompatibility complex class I heavy chain and beta2-microglobulin with antibodies conjugated with fluorescein- and indocarbocyanine-like fluorophores and analyzed in FCET measurements on a cell-by-cell basis using three flow cytometers: FACSCalibur, FACSDiVa, and FACSArray. RESULTS: Normalized fluorescence intensity values were measured and normalized energy transfer efficiencies, spectral overlap integrals, and crucial dye- and instrument-dependent parameters were calculated for all matching pairs of seven fluorophores on the three commercial cytometers. The most crucial parameter in determining the applicability of the donor-acceptor pairs was the normalized fluorescence intensity and the least important one was the spectral overlap. CONCLUSIONS: On the basis of available laser lines, the optimal dye pair for all three cytometers is the Alexa546-Alexa647 pair, which produces high energy transfer efficiency values and has the best spectral characteristics with regard to laser excitation, detection of emission, and spectral overlap.

Cell Line, Tumor↗
PubMed2005

Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins.

BACKGROUND: The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. METHODS: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. RESULTS: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. CONCLUSIONS: We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry.

Calibration↗
PubMed2005

Signal transduction of erbB receptors in trastuzumab (Herceptin) sensitive and resistant cell lines: local stimulation using magnetic microspheres as assessed by quantitative digital microscopy.

BACKGROUND: ErbB2 (HER-2), a member of the epidermal growth factor (EGF) receptor family, is a class I transmembrane receptor tyrosine kinase. Although erbB2 has no known physiologic ligand, it can form complexes with other members of the family and undergo transactivation of its very potent kinase activity, thereby initiating downstream signaling and cell proliferation. ErbB2 is a frequent pathologic marker in ductal invasive breast carcinomas and is targeted by using a specific humanized monoclonal antibody, trastuzumab (Herceptin). The antibody is effective in only 20% to 50% of erbB2-positive tumors, and this resistance, as yet poorly understood, constitutes a major therapeutic challenge. METHODS: Magnetic microspheres coated with ligands or antibodies are widely used for separation of proteins and cells and allow localized, high intensity, and precisely timed stimulation of cells. We used EGF- and trastuzumab-covered paramagnetic microspheres, quantitative confocal laser scanning microscopy, and digital image processing to investigate the (trans)activation of and local signal propagation from erbB1 and erbB2 on trastuzumab sensitive and resistant carcinoma cell lines expressing these receptors at high levels. RESULTS: On A431 cells expressing high levels of endogenous erbB1 and transfected erbB2-mYFP (A4-erbB2-mYFP F4 cell line), EGF-coupled-microspheres activated erbB1 and transactivated erbB2-mYFP. In two other cell lines with comparable erbB2 expression but lower levels of erbB1, EGF microspheres transactivated erbB2 less efficiently. Trastuzumab in solution activated erbB2 on A4-erbB2-mYFP and the trastuzumab sensitive SKBR-3 cells, but only negligibly on the resistant JIMT-1 cells that showed a 10 times higher K(d) for the antibody. Nevertheless, pronounced erbB2 activation and tyrosine phosphorylation could be detected after stimulation with trastuzumab-coupled microspheres in all cell lines, although transactivation of erbB1 was negligible. Receptor phosphorylation was restricted to the immediate proximity of the microspheres, i.e., receptor clusters external to these locations remained inactive. CONCLUSION: ErbB1 ligand and erbB2 specific antibody attached to magnetic microspheres are efficient tools in assessing erbB activation, localized signal propagation, and erbB heterodimer formation. Trastuzumab coupled to microspheres is more efficient at accessing erbB2 and activating it than trastuzumab in solution.

Animals↗
PubMed2005

Computer program for analyzing donor photobleaching FRET image series.

BACKGROUND: The photobleaching fluorescence resonance energy transfer (pbFRET) technique is a spectroscopic method to measure proximity relations between fluorescently labeled macromolecules using digital imaging microscopy. To calculate the energy transfer values one has to determine the bleaching time constants in pixel-by-pixel fashion from the image series recorded on the donor-only and donor and acceptor double-labeled samples. Because of the large number of pixels and the time-consuming calculations, this procedure should be assisted by powerful image data processing software. There is no commercially available software that is able to fulfill these requirements. METHODS: New evaluation software was developed to analyze pbFRET data for Windows platform in National Instrument LabVIEW 6.1. This development environment contains a mathematical virtual instrument package, in which the Levenberg-Marquardt routine is also included. As a reference experiment, FRET efficiency between the two chains (beta2-microglobulin and heavy chain) of major histocompatibility complex (MHC) class I glycoproteins and FRET between MHC I and MHC II molecules were determined in the plasma membrane of JY, human B lymphoma cells. RESULTS: The bleaching time constants calculated on pixel-by-pixel basis can be displayed as a color-coded map or as a histogram from raw image format. CONCLUSION: In this report we introduce a new version of pbFRET analysis and data processing software that is able to generate a full analysis pattern of donor photobleaching image series under various conditions. .

Algorithms↗
PubMed2005

Biphasic calcium response of platelet-derived growth factor stimulated glioblastoma cells is a function of cell confluence.

BACKGROUND: Previous reports have linked the spiking or two-phased character of calcium transients evoked by platelet-derived growth factor (PDGF) to the position of cells in the cell cycle without regard to cell-cell contact and communication. Because cell confluence can regulate growth factor receptor expression and dephosphorylation, we investigated the effect of cell culture confluence and cell cycle on calcium responses of PDGF-BB-stimulated A172 glioblastoma cells. METHODS: Digital imaging cytometry was used to correlate the peak and duration of calcium response with bromodeoxyuridine positivity and DNA content and with culture confluence on a cell-by-cell basis. RESULTS: In serum-starved cultures, complete two-phase calcium signals and shorter, lower spikes occurred independent of cell cycle phase. However, the confluence of cell culture seemed essential for inducing a complete response because cells in sparse cultures exhibited mostly short spikes with lower peaks or no transients at all. CONCLUSION: Because cell confluence, by virtue of cell-cell contacts, is assumed to be an important regulator of proliferation, one is tempted to speculate that in transformed cells the ability to produce stronger growth signals upon reaching confluence and facing contact inhibition could provide a proliferative advantage.

Becaplermin↗
PubMed2005

A novel homozygous mutation (1619delC) in GPIIb gene associated with Glanzmann thrombasthenia, the decay of GPIIb-mRNA and the synthesis of a truncated GPIIb unable to form complex with GPIIIa.

The absence of agonist-induced platelet aggregation and the lack of fibrinogen receptor (GPIIb/IIIa) on the platelet surface demonstrated that the severe hemorrhagic complications of a child of Romany descent were caused by Glanzmann thrombasthenia. DNA sequencing revealed a novel homozygous deletion of a cytosine (1619delC) in the GPIIb gene causing a frameshift and predicting a novel stop codon at position 533 following 24 altered amino acids. Both parents possessed the same deletion in heterozygous form. The amount of GPIIb mRNA in the patient's platelets was 0.06% of the amount measured in control platelets. Neither GPIIb nor its truncated form could be detected in the platelets of the patient by Western blotting, while a small amount of GPIIIa was demonstrated. Quantitative flowcytometric analysis showed an elevated number of vitronectin receptors, a component of which is GPIIIa, on the patient's platelets. The surface expression of vitronectin receptor on thrombasthenic, but not on normal platelets was further increased by activation with thrombin receptor agonist peptide. BHK cells transfected with wild type GPIIIa andmutated GPIIb failed to express any mature GPIIb or pro-GPIIb. Immunoprecipitation with a polyclonal antibody recognizing both GPIIb and GPIIIa recovered a 60 kDa truncated form of GPIIb. This band was absent when immunoprecipitation was carried out with an antibody recognizing GPIIIa, suggesting that the truncated protein, lacking calf-1, calf-2 domains and major part of the thigh domain, is unable to form complex with GPIIIa.

Animals↗
PubMed2004

Commissural propriospinal connections between the lateral aspects of laminae III-IV in the lumbar spinal cord of rats.

It has been established that there is a strong functional link between sensory neural circuits on the two sides of the spinal cord. In one of our recent studies we provided a morphological confirmation of this functional phenomenon, presenting evidence for the presence of a direct commissural connection between the lateral aspects of the dorsal horn on the two sides of the lumbar spinal cord. By using a combination of neural tracing and immunocytochemical detection of neural markers like vesicular glutamate transporters, glutamic acid decarboxylase, glycine transporter, and met-enkephalin (which are characteristic of various subsets of excitatory and inhibitory neurons), we investigated here the distribution, synaptic relations, and neurochemical characteristics of the commissural axon terminals. We found that the cells of origin of commissural fibers in the lateral aspect of the dorsal horn were confined to laminae III-IV and projected to the corresponding area of the contralateral gray matter. Most of the commissural axon terminals established synaptic contacts with dendrites. Axospinous or axosomatic synaptic contacts were found in limited numbers. We demonstrated that interactions among commissural neurons also exist. More than three-fourths of the labeled axon terminals were immunostained for glutamic acid decarboxylase and/or glycine transporter, but none of them showed positive immunoreaction for met-enkephalin and vesicular glutamate transporters. The results indicate that there is a substantial reciprocal commissural synaptic interaction between the lateral aspects of laminae III-IV on the two sides of the lumbar spinal cord and that this pathway may transmit both inhibitory and excitatory signals to their postsynaptic targets.

Animals↗
PubMed2004

Molecular modeling of nearly full-length ErbB2 receptor.

Members of the epidermal growth factor receptor family play important roles in various cellular processes, both in physiological and in pathological conditions. Dimerization and autophosphorylation of these receptor tyrosine kinases are key events of signal transduction. Details of the molecular events of the signaling are not entirely known. To facilitate the understanding of receptor structure and function at the molecular level, a molecular model was built for the nearly full-length ErbB2 dimer. Modeling was based on the x-ray or nuclear-magnetic resonance structures of extracellular, transmembrane, and intracellular domains. The extracellular domain was positioned above the cell membrane based on the distance determined from experimentally measured fluorescence resonance energy transfer. Favorable dimerization interactions are predicted for the extracellular, transmembrane, and protein kinase domains in the model of a nearly full-length dimer of ErbB2, which may act in a coordinated fashion in ErbB2 homodimerization, and also in heterodimers of ErbB2 with other members of the ErbB family.

Cell Line, Tumor↗
PubMed2004

IL-2 and IL-15 receptor alpha-subunits are coexpressed in a supramolecular receptor cluster in lipid rafts of T cells.

The private alpha-chains of IL-2 and IL-15 receptors (IL-2R and IL-15R) share the signaling beta- and gamma(c)-subunits, resulting in both common and contrasting roles of IL-2 and IL-15 in T cell function. Knowledge of the cytokine-dependent subunit assembly is indispensable for understanding the paradox of distinct signaling capacities. By using fluorescence resonance energy transfer and confocal microscopy, we have shown that IL-2R alpha, IL-15R alpha, IL-2/15R beta and gamma(c)-subunits, as well as MHC class I and II glycoproteins formed supramolecular receptor clusters in lipid rafts of the T lymphoma line Kit 225 FT7.10. Fluorescence crosscorrelation microscopy demonstrated the comobility of IL-15R alpha with IL-2R alpha and MHC class I. A model was generated for subunit switching between IL-2R alpha and IL-15R alpha upon the binding of the appropriate cytokine resulting in the formation of high-affinity heterotrimeric receptors. This model suggests a direct role for the alpha-subunits, to which no definite function has been assigned so far, in tuning cellular responses to IL-2 or IL-15. In addition, both alpha-chains were at least partially homodimerized/oligomerized, which could be the basis of distinct signaling pathways by the two cytokines.

Antibodies, Monoclonal↗
PubMed2004

Expression of hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 in axon terminals of peptidergic nociceptive primary sensory neurons in the superficial spinal dorsal horn of rats.

Hyperpolarization-activated cyclic nucleotide-gated cation channel proteins (HCN1-4), which are potentially able to modulate membrane excitability, are abundantly expressed by neurons in spinal dorsal root ganglia (DRG). In the present experiment, we investigated whether HCN2 protein is confined exclusively to the perikarya of DRG neurons or is transported from the somata to the central axons of DRG neurons that terminate in the spinal dorsal horn. Using immunohistochemical methods, we have demonstrated that laminae I-IIo of the superficial spinal dorsal horn of the adult rat spinal cord show a strong punctate immunoreactivity for HCN2. Dorsal rhizotomy resulted in a complete loss of immunostaining in the dorsal horn, suggesting that HCN2 is confined to axon terminals of primary afferents. In double labelling immunohistochemical studies, we have also shown that HCN2 widely co-localizes with calcitonin gene-related peptide, but is almost completely segregated from isolectin-B4 binding, indicating that HCN2 is primarily expressed in peptidergic nociceptive primary afferents. The expression of HCN2 in central terminals of peptidergic primary afferents was also verified with electron microscopy. Utilizing the pre-embedding nanogold method, we found that HCN2 is largely confined to axon terminals with dense-core vesicles. Within these terminals, some of the silver grains marking the accurate location of HCN2 molecules were associated with the cell membrane, and others were scattered in the axoplasm. Within the cell membrane, HCN2 was found almost exclusively in extrasynaptic locations. The results suggest that HCN2 may contribute to the modulation of membrane excitability of nociceptive primary afferent terminals in the spinal dorsal horn.

Animals↗
PubMed2003

Mammalian phosphatidylinositol 4-kinases.

Three phosphatidylinositol 4-kinase isoforms, PI4K 230, 92 and 55 have been cloned and sequenced allowing a much wider characterization than the previously employed enzymological typing into type II and III enzymes. PI4K 230 and 92 contain a highly conserved catalytic core, PI4K55 one with a much lower degree of similarity. Candidate kinase motifs, deduced from the protein kinase super family, are absolutely conserved in all isoforms. Kinase activities are described based on their sensitivity and reactivity towards wortmannin, phenylarsine oxide (PAO) and 5'-p-fluorosulfonylbenzoyladenosine (FSBA). Localization of all isoforms in the cell is reported. All enzymes contain nuclear localization and export sequence motifs (NLS and NES) leading to the expectation that they can be transferred to the nucleus. PI4K230 has been found in the nucleolus, PI4K92 in the nucleus, additionally further broadening the function of these enzymes. In the cytoplasm of neuronal cells, PI4K230 is distributed evenly on membranes that are ultra structurally cisterns of the rough endoplasmatic reticulum, outer membranes of mitochondria, multivesicular bodies, and are in close vicinity of synaptic contacts. PI4K92 is functionally characterized as a key enzyme regulating Golgi disintegration/reorganization during mitosis probably via phosphorylation by cyclin-dependent kinases on well-defined sites. PI4K55 is involved in the production of second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3) at the plasma membrane, moreover, in the endocytotic pathway in the cytoplasm.

1-Phosphatidylinositol 4-Kinase↗
PubMed2002

Lipid rafts and the local density of ErbB proteins influence the biological role of homo- and heteroassociations of ErbB2.

The ErbB family of transmembrane receptor tyrosine kinases plays an important role in the pathogenesis of many cancers. The four members of the family, ErbB1-4, form various homo- and heterodimers during the course of signal transduction. A second hierarchical level of molecular associations involving 10(2)-10(3) molecules, termed large-scale clustering, has also been identified, but the regulatory factors and biological consequences of such structures have not been systematically evaluated. In this report, we describe the states of association of ErbB2 and their relationship to local ErbB3 density and lipid rafts based on quantitative fluorescence microscopy of SKBR-3 breast cancer cells. Clusters of ErbB2 colocalized with lipid rafts identified by the GM1-binding B subunit of cholera toxin. Pixel-by-pixel analysis of fluorescence resonance energy transfer between labeled antibodies indicated that the homoassociation (homodimerization) of ErbB2 was proportional to the local density of ErbB2 and inversely proportional to that of ErbB3 and of the raft-specific lipid GM1. Crosslinking lipid rafts with the B subunit of cholera toxin caused dissociation of the rafts and ErbB2 clusters, an effect that was independent of the cytoskeletal anchoring of ErbB2. Crosslinking also decreased ErbB2-ErbB3 heteroassociation and the EGF- and heregulin-induced tyrosine phosphorylation of Shc. When cells were treated with the anti-ErbB2 monoclonal antibody 4D5 (parent murine version of Trastuzumab used in the immunotherapy of breast cancer), internalization of the antibody was inhibited by crosslinking of lipid rafts, but the antiproliferative activity of 4D5 was retained and even enhanced. We conclude that local densities of ErbB2 and ErbB3, as well as the lipid environment profoundly influence the association properties and biological function of ErbB2.

Adaptor Proteins, Signal Transducing↗