The Tethering Arm of the EGF Receptor Is Required for Negative Cooperativity and Signal Transduction

Integrins mediate cell adhesion, migration, and a variety of signal transduction events. These integrin actions can overlap or even synergize with those of growth factors. We examined for mechanisms of collaboration or synergy between integrins and growth factors involving MAP kinases, which regulate many cellular functions. In cooperation with integrins, the growth factors EGF, PDGF-BB, and basic FGF each produced a marked, transient activation of the ERK (extracellular signal-regulated kinase) class of MAP kinase, but only if the integrins were both aggregated and occupied by ligand. Transmembrane accumulation of total tyrosine-phosphorylated proteins, as well as nonsynergistic MAP kinase activation, could be induced by simple integrin aggregation, whereas enhanced transient accumulation of the EGF-receptor substrate eps8 required integrin aggregation and occupancy, as well as EGF treatment. Each type of growth factor receptor was itself induced to aggregate transiently by integrin ligand-coated beads in a process requiring both aggregation and occupancy of integrin receptors, but not the presence of growth factor ligand. Synergism was also observed between integrins and growth factors for triggering tyrosine phosphorylation of EGF, PDGF, and FGF receptors. This collaborative response also required both integrin aggregation and occupancy. These studies identify mechanisms in the signal transduction response to integrins and growth factors that require various combinations of integrin aggregation and ligands for integrin or growth factor receptors, providing opportunities for collaboration between these major regulatory systems.

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Abstract 486: Artificial miRNA Based Adenoviral Adam17 Silencing Reveals Essential Role of This Metalloprotease For EGF Receptor Transactivation In Primary Cultured Vascular Smooth Muscle Cells

Hypertension ◽

10.1161/hyp.60.suppl_1.a486 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Katherine Elliott ◽

Allison Bourne ◽

Takehiko Takayanagi ◽

Akira Takaguri ◽

Kunie Eguchi ◽

...

Keyword(s):

Signal Transduction ◽

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Gene Silencing ◽

Vascular Smooth Muscle Cells ◽

Egf Receptor ◽

Muscle Cells ◽

Vascular Cells ◽

Receptor Transactivation

siRNA mediated gene silencing has been recently utilized as a powerful molecular tool to study the functional significance of a specific protein. However, due to the transient nature of silencing and insufficient transfection efficiency, this approach can be problematic in primary cell culture. To overcome such weakness of the siRNA based silencing and in order to establish reliable gene silencing in vascular cells, we devised an adenoviral-encoded miRNA based gene silencing system. Here we report the results of silencing ADAM17 in cultured rat vascular smooth muscle cells (VSMCs) and its functional consequences in angiotensin II (AngII) signal transduction. Four distinct miRNA sequences targeting rat ADAM17 were chosen based on recommendations from Invitrogen’s Block-iT RNAi Designer algorithm. The miRNA sequences were inserted into a mammalian expression vector, pcDNA 6.2-GW/EmGFP-miR, and the effective silencing by these vectors was confirmed in HEK cells expressing HA-tagged rat ADAM17. The 4 cassettes carrying the miRNAs were inserted into pAd/CMV/V5-DEST and adenoviral solutions were obtained. Greater than 95% silencing of ADAM17 was achieved when VSMC were infected with 100-200 moi of the ADAM17 miRNA encoding adeonvirus for 72 h with enhancement of infection by fugene6. Relatively linear time and concentration dependencies were observed between 1 to 3 days and 10 to 100 moi of the infection. A miR-ADAM17 (100 moi) but not miR-control (100 moi) completely inhibited 100 nM AngII-induced HB-EGF shedding in VSMCs as assessed by a reporter assay. A miR-ADAM17 but not miR-control also inhibited AngII-induced EGF receptor transactivation and subsequent ERK1/2 activation in VSMCs as assessed by immunoblotting with phospho-selective antibodies. In conclusion, ADAM17 was found to be a major sheddase for HB-EGF contributing to the growth promoting signals induced by AngII in VSMCs. An artificial miRNA-base adenoviral approach appears to be a reliable gene-silencing strategy for signal transduction research in primary cultured vascular cells.

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Evidence for epidermal growth factor (EGF)-induced intermolecular autophosphorylation of the EGF receptors in living cells

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4035-4044.1990 ◽

1990 ◽

Vol 10 (8) ◽

pp. 4035-4044

Author(s):

A M Honegger ◽

A Schmidt ◽

A Ullrich ◽

J Schlessinger

Keyword(s):

Signal Transduction ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Phosphorylation ◽

Egf Receptor ◽

Living Cells ◽

Egf Receptors ◽

Receptor Molecule ◽

Epidermal Growth ◽

Negative Mutant

In response to epidermal growth factor (EGF) stimulation, the intrinsic protein tyrosine kinase of EGF receptor is activated, leading to tyrosine phosphorylation of several cellular substrate proteins, including the EGF receptor molecule itself. To test the mechanism of EGF receptor autophosphorylation in living cells, we established transfected cell lines coexpressing a kinase-negative point mutant of EGF receptor (K721A) with an active EGF receptor mutant lacking 63 amino acids from its carboxy terminus. The addition of EGF to these cells caused tyrosine phosphorylation of the kinase-negative mutant by the active receptor molecule, demonstrating EGF receptor cross-phosphorylation in living cells. After internalization the kinase-negative mutant and CD63 have separate trafficking pathways. This limits their association and the extent of cross-phosphorylation of K721A by CD63. The coexpression of the kinase-negative mutant together with active EGF receptors in the same cells suppressed the mitogenic response toward EGF as compared with that in cells that express active receptors alone. The presence of the kinase-negative mutant functions as a negative dominant mutation suppressing the response of active EGF receptors, probably by interfering with EGF-induced signal transduction. It appears, therefore, that crucial events of signal transduction occur before K721A and active EGF receptors are separated by their different endocytic itineraries.

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Healing of Gastrointestinal Mucosa: Involvement of Polyamines

Physiology ◽

10.1152/physiologyonline.1999.14.1.12 ◽

1999 ◽

Vol 14 (1) ◽

pp. 12-17 ◽

Cited By ~ 1

Author(s):

Leonard R. Johnson ◽

Shirley A. McCormack

Keyword(s):

Signal Transduction ◽

Cell Migration ◽

Growth Factor ◽

Egf Receptor ◽

Receptor Function ◽

Gastrointestinal Mucosa ◽

Cellular Content ◽

Mucosal Lesions ◽

Signal Transduction Proteins ◽

Cell Migration And Proliferation

Polyamines are involved in the processes of cell migration and proliferation that result in the repair of mucosal lesions. Depletion of polyamines dramatically alters the arrangement of the cytoskeleton, EGF receptor function, the activities of signal transduction proteins, the levels of several protooncogenes, and the expression and cellular content of at least one growth factor involved in these processes.

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The TGF-α precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction

Cell ◽

10.1016/0092-8674(89)90252-3 ◽

1989 ◽

Vol 56 (3) ◽

pp. 495-506 ◽

Cited By ~ 302

Author(s):

Sharon T. Wong ◽

Lisa F. Winchell ◽

Bryan K. McCune ◽

H.Shelton Earp ◽

Joaquin Teixido ◽

...

Keyword(s):

Signal Transduction ◽

Cell Surface ◽

Egf Receptor

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EGF signaling in bowel carcinoma cells utilizes higher order architectures of EGFR and HER2

10.1101/2020.07.11.198572 ◽

2020 ◽

Author(s):

Adam J. M. Wollman ◽

Charlotte Fournier ◽

Isabel Llorente-Garcia ◽

Oliver Harriman ◽

Alex L. Hargreaves ◽

...

Keyword(s):

Single Molecule ◽

Normal Cell ◽

Egf Receptor ◽

Carcinoma Cells ◽

Time Dependent ◽

Negative Cooperativity ◽

Kinetics Modelling ◽

Preferential Binding ◽

Epidermal Growth ◽

Egf Signaling

AbstractEpidermal growth factor (EGF) signaling regulates normal cell development, however EGF receptor (EGFR) overexpression is reported in several carcinomas. Despite structural and biochemical evidence that EGF-EGFR ligation activates signaling through monomer-dimer transitions, live cell mechanistic details remain contentious. We report single-molecule multispectral TIRF of human epithelial carcinoma cells transfected with fluorescent EGFR, and of CHO-K1 cells containing fluorescent EGFR and HER2, enabling super-resolved localization to quantify receptor architectures and spatiotemporal dynamics upon EGF ligation. Using inhibitors that block binding to EGFR, and time-dependent kinetics modelling, we find that pre-activated EGFR consist predominantly of preformed clusters that contain a mixture of EGFR and HER2, whose stoichiometry increases following EGF activation. Although complicated by EGFR internalization and recycling, our observation of an EGFR:EGF stoichiometry >1 for plasma membrane colocalized EGFR/EGF foci soon after activation may indicate preferential binding of EGF ligand to EGFR monomers, negative cooperativity and preferential ligated-unligated dimerization of monomers.

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Regulation of ROS signal transduction by NADPH oxidase 4 localization

The Journal of Cell Biology ◽

10.1083/jcb.200709049 ◽

2008 ◽

Vol 181 (7) ◽

pp. 1129-1139 ◽

Cited By ~ 337

Author(s):

Kai Chen ◽

Michael T. Kirber ◽

Hui Xiao ◽

Yu Yang ◽

John F. Keaney

Keyword(s):

Signal Transduction ◽

Intracellular Signaling ◽

Egf Receptor ◽

Tyrosine Phosphatase ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Diverse Range ◽

Nadph Oxidase 4 ◽

Er Targeting ◽

Ptp 1B ◽

Egf Signaling

Reactive oxygen species (ROS) function as intracellular signaling molecules in a diverse range of biological processes. However, it is unclear how freely diffusible ROS dictate specific cellular responses. In this study, we demonstrate that nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (Nox4), a major Nox isoform expressed in nonphagocytic cells, including vascular endothelium, is localized to the endoplasmic reticulum (ER). ER localization of Nox4 is critical for the regulation of protein tyrosine phosphatase (PTP) 1B, also an ER resident, through redox-mediated signaling. Nox4-mediated oxidation and inactivation of PTP1B in the ER serves as a regulatory switch for epidermal growth factor (EGF) receptor trafficking and specifically acts to terminate EGF signaling. Consistent with this notion, PTP1B oxidation could also be modulated by ER targeting of antioxidant enzymes but not their untargeted counterparts. These data indicate that the specificity of intracellular ROS-mediated signal transduction may be modulated by the localization of Nox isoforms within specific subcellular compartments.

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Role of Rab5 in EGF receptor-mediated signal transduction

European Journal of Cell Biology ◽

10.1078/0171-9335-00381 ◽

2004 ◽

Vol 83 (6) ◽

pp. 305-314 ◽

Cited By ~ 54

Author(s):

M. Alejandro Barbieri ◽

Sebastian Fernandez-Pol ◽

Christine Hunker ◽

Bruce H. Horazdovsky ◽

Philip D. Stahl

Keyword(s):

Signal Transduction ◽

Egf Receptor

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Signal transduction by epidermal growth factor and heregulin via the kinase-deficient ErbB3 protein

Biochemical Journal ◽

10.1042/bj3340189 ◽

1998 ◽

Vol 334 (1) ◽

pp. 189-195 ◽

Cited By ~ 71

Author(s):

Hong-Hee KIM ◽

Ulka VIJAPURKAR ◽

Nathan J. HELLYER ◽

Dolores BRAVO ◽

John G. KOLAND

Keyword(s):

Signal Transduction ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Egf Receptor ◽

Tyrosine Kinase Activity ◽

Epidermal Growth ◽

Protein Tyrosine Kinase Activity

The role of protein tyrosine kinase activity in ErbB3-mediated signal transduction was investigated. ErbB3 was phosphorylated in vivo in response to either heregulin (HRG) in cells expressing both ErbB3 and ErbB2, or epidermal growth factor (EGF) in cells expressing both ErbB3 and EGF receptor. A recombinant receptor protein (ErbB3-K/M, in which K/M stands for Lys → Met amino acid substitution) containing an inactivating mutation in the putative ATP-binding site was also phosphorylated in response to HRG and EGF. Both the wild-type ErbB3 and mutant ErbB3-K/M proteins transduced signals to phosphatidylinositol 3-kinase, Shc and mitogen-activated protein kinases. Separate kinase-inactivating mutations in the EGF receptor and ErbB2 proteins abolished ErbB3 phosphorylation and signal transduction activated by EGF and HRG respectively. Hence the protein tyrosine kinase activity necessary for growth factor signalling via the ErbB3 protein seems to be provided by coexpressed EGF and ErbB2 receptor proteins.

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