Visualization of epidermal growth factor (EGF) receptor aggregation in plasma membranes by fluorescence resonance energy transfer. Correlation of receptor activation with aggregation.
AuthorsCarraway KL, Koland JG, Cerione RA
JournalJ Biol Chem
PubMed ID2498317
'Fluorescence resonance energy transfer between epidermal growth factor (EGF) molecules, labeled with fluorescent reporter groups, was used as a monitor for EGF receptor-receptor interactions in plasma membranes isolated from human epidermoid A431 cells. Epidermal growth factor molecules labeled at the amino terminus with fluorescein isothiocyanate served as donor molecules in ... More
The interaction of epidermal growth factor with its receptor in A431 cell membranes: a stopped-flow fluorescence anisotropy study.
AuthorsRousseau DL, Staros JV, Beechem JM
JournalBiochemistry
PubMed ID7578056
'We describe a quantitative examination of the interaction of epidermal growth factor (EGF) with the EGF receptor using A431 cell membrane vesicles as a receptor source. Using T-format steady-state fluorescence anisotropy detection coupled with stopped-flow mixing, we measured the association and EGF-induced dissociation kinetics of fluorescein 5-isothiocyanate-labeled mEGF (FITC-EGF) with ... More
Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin.
AuthorsZhao M, Dick A, Forrester JV, McCaig CD
JournalMol Biol Cell
PubMed ID10198071
'Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. ... More
Heterogeneity of epidermal growth factor binding kinetics on individual cells.
AuthorsChung JC, Sciaky N, Gross DJ
JournalBiophys J
PubMed ID9251825
'Binding of fluorescein-conjugated epidermal growth factor (EGF) to individual A431 cells at 4 degrees C is measured by a quantitative fluorescence imaging technique. After background fluorescence and cell autofluorescence photobleaching corrections, the kinetic data are fit to simple models of one monovalent site and two independent monovalent sites, both of ... More
AuthorsMitra S, Lee JS, Cantrell M, Van den Berg CL,
JournalJ Biol Chem
PubMed ID21357683
Membrane-bound receptors induce biochemical signals to remodel the actin cytoskeleton and mediate cell motility. In association with receptor tyrosine kinases, several downstream mitogen-induced kinases facilitate cell migration. Here, we show a role for c-Jun N-terminal kinase 2 (JNK2) in promoting mammary cancer cell migration through inhibition of epidermal growth factor ... More
Roles of BLOC-1 and adaptor protein-3 complexes in cargo sorting to synaptic vesicles.
AuthorsNewell-Litwa K, Salazar G, Smith Y, Faundez V,
JournalMol Biol Cell
PubMed ID19144828
Neuronal lysosomes and their biogenesis mechanisms are primarily thought to clear metabolites and proteins whose abnormal accumulation leads to neurodegenerative disease pathology. However, it remains unknown whether lysosomal sorting mechanisms regulate the levels of membrane proteins within synaptic vesicles. Using high-resolution deconvolution microscopy, we identified early endosomal compartments where both ... More
Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data.
A method is developed for determining ligand-cell association parameters from a model-free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated ... More
Calibration of flow cytometric fluorescence standards using the isoparametric analysis of ligand binding.
AuthorsChatelier RC, Ashcroft RG
JournalCytometry
PubMed ID3123175
To extract ligand-cell binding parameters from flow cytometric fluorescence data, one needs a method of converting the measured cellular fluorescence to the actual number of bound molecules producing that fluorescence. Bead standards containing a known number of fluorophores do not necessarily provide the correct conversion factor. We therefore present a ... More
Comparison of epidermal growth factor (EGF) receptor-receptor interactions in intact A431 cells and isolated plasma membranes. Large scale receptor micro-aggregation is not detected during EGF-stimulated early events.
AuthorsCarraway KL, Cerione RA
JournalJ Biol Chem
PubMed ID2026602
We have used resonance energy transfer to monitor epidermal growth factor (EGF) receptor micro-aggregation at the surface of intact human epidermoid carcinoma (A431) cells. EGF molecules labeled with fluorescein isothiocyanate and eosin isothiocyanate were demonstrated to bind tightly to cellsurface receptors, to elicit immediate changes in cytosolic free [Ca2+], and ... More
Fluorescent-labeled growth factor molecules serve as probes for receptor binding and endocytosis.
AuthorsCarraway KL, Cerione RA
JournalBiochemistry
PubMed ID8218281
In this report, we describe the applicability of fluorescein-labeled EGF (FITC-EGF) in monitoring the interaction between EGF and its cellular receptor in real time. This work takes advantage of previous studies that demonstrated that EGF may be labeled at its amino terminus with FITC without significant deleterious effects on the ... More
Inhibition of epidermal growth factor receptor aggregation by an antibody directed against the epidermal growth factor receptor extracellular domain.
AuthorsCarraway KL, Cerione RA
JournalJ Biol Chem
PubMed ID8226925
We have examined the perturbation of epidermal growth factor (EGF) receptor-receptor interactions by a monoclonal antibody (13A9) that binds to the receptor extracellular domain. While 13A9 did not inhibit EGF binding, it inhibited energy transfer between fluorescent-labeled EGF molecules bound to receptors in membranes from human A431 cells by 70-100%. ... More
Peroxisome proliferator-activated receptor gamma control of dendritic cell function contributes to development of CD4+ T cell anergy.
AuthorsKlotz L, Dani I, Edenhofer F, Nolden L, Evert B, Paul B, Kolanus W, Klockgether T, Knolle P, Diehl L
JournalJ Immunol
PubMed ID17277116
There is increasing evidence that dendritic cell (DC) immunogenicity is not only positively regulated by ligands of pattern recognition receptors, but also negatively by signals that prevent DC activation and full functional maturation. Depending on their activation status, DCs can induce either immunity or tolerance. In this study, we provide ... More
Temperature-dependent lateral and transverse distribution of the epidermal growth factor receptor in A431 plasma membranes.
AuthorsAzevedeo JR, Johnson DA
JournalJ Membr Biol
PubMed ID2077131
To elucidate further the structure and molecular dynamics of the epidermal growth factor receptor, temperature-dependent aggregation and extracellular protrusion of the epidermal growth factor receptor in isolated plasma membranes from A431 cells were examined by fluorescence energy-transfer techniques. Epidermal growth factor was labeled at the amino terminus with either fluorescein ... More
A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity.
AuthorsBosch M, Benito A, Ribó M, Puig T, Beaumelle B, Vilanova M
JournalBiochemistry
PubMed ID14979713
Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all ... More
Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation.
AuthorsGadella TW, Jovin TM
JournalJ Cell Biol
PubMed ID7790353
The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound ... More
Differential roles of syntaxin 7 and syntaxin 8 in endosomal trafficking.
AuthorsPrekeris R, Yang B, Oorschot V, Klumperman J, Scheller RH
JournalMol Biol Cell
PubMed ID10564279
To understand molecular mechanisms that regulate the intricate and dynamic organization of the endosomal compartment, it is important to establish the morphology, molecular composition, and functions of the different organelles involved in endosomal trafficking. Syntaxins and vesicle-associated membrane protein (VAMP) families, also known as soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein ... More
Electrical stimulation directly induces pre-angiogenic responses in vascular endothelial cells by signaling through VEGF receptors.
Authors
JournalJ Cell Sci
PubMed ID14679307
Characterizing molecular diffusion in the lens capsule.