scholarly journals EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

2018 ◽  
Author(s):  
Charlotte Fournier ◽  
Adam J. M. Wollman ◽  
Isabel Llorente-Garcia ◽  
Oliver Harriman ◽  
Djamila Ouarat ◽  
...  
Keyword(s):  
Single Molecule ◽  
Egf Receptor ◽  
Receptor Activation ◽  
Carcinoma Cells ◽  
Live Cells ◽  
Egfr Expression ◽  
Egfr Activation ◽  
Before And After ◽  
Epidermal Growth ◽  
Differentiation And Proliferation

AbstractEpidermal growth factor (EGF) signalling regulates cell growth, differentiation and proliferation in epithelium and EGF receptor (EGFR) overexpression has been reported in several carcinoma types. Structural and biochemical evidence suggests EGF binding stimulates EGFR monomer-dimer transitions, activating downstream signalling. However, mechanistic details of ligand binding to functional receptors in live cells remain contentious. We report real time single-molecule TIRF of human epithelial carcinoma cells with negligible native EGFR expression, transfected with GFP-tagged EGFR, before and after receptor activation with TMR-labelled EGF ligand. Fluorescently labelled EGFR and EGF are simultaneously tracked to 40nm precision to explore stoichiometry and spatiotemporal dynamics upon EGF binding. Using inhibitors that block binding to EGFR directly, or indirectly through HER2, our results indicate that pre-activated EGFR consists of preformed homoclusters, while larger heteroclusters including HER2 form upon activation. The relative stoichiometry of EGFR to EGF after binding peaks at 2, indicating negative cooperativity of EGFR activation.

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

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.

scholarly journals Targeting the EGF Receptor for Ovarian Cancer Therapy

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Reema Zeineldin ◽  
Carolyn Y. Muller ◽  
M. Sharon Stack ◽  
Laurie G. Hudson
Keyword(s):  
Ovarian Cancer ◽  
Targeted Therapies ◽  
Egf Receptor ◽  
Receptor Activation ◽  
Molecular Heterogeneity ◽  
Cancer Pathogenesis ◽  
The Us ◽  
The Status ◽  
Advanced Stages ◽  
Epidermal Growth

Ovarian carcinoma is the leading cause of death from gynecologic malignancy in the US. Factors such as the molecular heterogeneity of ovarian tumors and frequent diagnosis at advanced stages hamper effective disease treatment. There is growing emphasis on the identification and development of targeted therapies to disrupt molecular pathways in cancer. The epidermal growth factor (EGF) receptor is one such protein target with potential utility in the management of ovarian cancer. This paper will discuss contributions of EGF receptor activation to ovarian cancer pathogenesis and the status of EGF receptor inhibitors and EGF receptor targeted therapies in ovarian cancer treatment.

Neutrophil elastase induces mucin production by ligand-dependent epidermal growth factor receptor activation

2002 ◽  
Vol 283 (3) ◽  
pp. L531-L540 ◽  
Author(s):  
Kazuhiro Kohri ◽  
Iris F. Ueki ◽  
Jay A. Nadel
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Neutrophil Elastase ◽  
Growth Factor Receptor ◽  
Receptor Activation ◽  
Cell Culture Supernatant ◽  
Mucin Production ◽  
Egfr Activation ◽  
Epidermal Growth

Neutrophil products are implicated in hypersecretory airway diseases. To determine the mechanisms linking a proteolytic effect of human neutrophil elastase (HNE) and mucin overproduction, we examined the effects of HNE on MUC5AC mucin production in human airway epithelial (NCI-H292) cells. Stimulation with HNE for 5–30 min induced MUC5AC production 24 h later, which was prevented by HNE serine active site inhibitors, implicating a proteolytic effect of HNE. MUC5AC induction was preceded by epidermal growth factor receptor (EGFR) tyrosine phosphorylation and was prevented by selective EGFR tyrosine kinase inhibitors, implicating EGFR activation. HNE-induced MUC5AC production was inhibited by a neutralizing transforming growth factor-α (TGF-α, an EGFR ligand) antibody and by a neutralizing EGFR antibody but not by oxygen free radical scavengers, further implicating TGF-α and ligand-dependent EGFR activation in the response. HNE decreased pro-TGF-α in NCI-H292 cells and increased TGF-α in cell culture supernatant. From these results, we conclude that HNE-induced MUC5AC mucin production occurs via its proteolytic activation of an EGFR signaling cascade involving TGF-α.

scholarly journals Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging

2011 ◽  
Vol 192 (3) ◽  
pp. 463-480 ◽  
Author(s):  
Rinshi S. Kasai ◽  
Kenichi G. N. Suzuki ◽  
Eric R. Prossnitz ◽  
Ikuko Koyama-Honda ◽  
Chieko Nakada ◽  
...  
Keyword(s):  
Equilibrium Constant ◽  
Single Molecule ◽  
Dynamic Equilibrium ◽  
Dissociation Rate ◽  
Formyl Peptide Receptor ◽  
Live Cells ◽  
Imaging Method ◽  
Full Characterization ◽  
Before And After ◽  
Dissociation Rate Constants

Receptor dimerization is important for many signaling pathways. However, the monomer–dimer equilibrium has never been fully characterized for any receptor with a 2D equilibrium constant as well as association/dissociation rate constants (termed super-quantification). Here, we determined the dynamic equilibrium for the N-formyl peptide receptor (FPR), a chemoattractant G protein–coupled receptor (GPCR), in live cells at 37°C by developing a single fluorescent-molecule imaging method. Both before and after liganding, the dimer–monomer 2D equilibrium is unchanged, giving an equilibrium constant of 3.6 copies/µm2, with a dissociation and 2D association rate constant of 11.0 s−1 and 3.1 copies/µm2s−1, respectively. At physiological expression levels of ∼2.1 receptor copies/µm2 (∼6,000 copies/cell), monomers continually convert into dimers every 150 ms, dimers dissociate into monomers in 91 ms, and at any moment, 2,500 and 3,500 receptor molecules participate in transient dimers and monomers, respectively. Not only do FPR dimers fall apart rapidly, but FPR monomers also convert into dimers very quickly.

Eicosanoids enhance epidermal growth factor receptor activation and proliferation in glomerular epithelial cells

1992 ◽  
Vol 262 (4) ◽  
pp. F639-F646 ◽  
Author(s):  
A. V. Cybulsky ◽  
P. R. Goodyer ◽  
M. D. Cyr ◽  
A. J. McTavish
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Epithelial Cells ◽  
Egf Receptor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Receptor Activation ◽  
Scatchard Analysis ◽  
Glomerular Epithelial Cells ◽  
Epidermal Growth

Proliferation of glomerular epithelial cells (GEC) and release of prostaglandins (PG) and thromboxane (Tx) A2 may occur in glomerular injury. We studied the relationship of eicosanoids to epidermal growth factor (EGF)-induced proliferation of rat GEC in culture. After 48 h of serum-deprivation, EGF stimulated [3H]thymidine incorporation ninefold above serum-deprived cells. Inhibition of cyclooxygenase with indomethacin or of Txsynthase with OKY-046 decreased the proliferative effect of EGF by 50 and 38%, respectively. The effect of indomethacin was reversed by addition of PGE2. Synthesis of PGE2, PGF2 alpha, and TxA2 by serum-deprived GEC was not enhanced by EGF. Scatchard analysis of 125I-EGF binding to GEC demonstrated two populations of EGF receptors; the high-affinity site had a dissociation constant (Kd) of 444 pM and 24,864 receptors/cell. EGF receptor autophosphorylation (reflecting receptor activation) was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of GEC membrane proteins with anti-phosphotyrosine antibody. EGF increased phosphorylation of a protein of approximately 170 kDa, which comigrated with proteins immunoprecipitated from [35S]methionine-labeled GEC with antibodies to EGF receptor. Indomethacin and OKY-046 decreased the EGF-dependent phosphorylation of the 170-kDa protein, and this decrease was overcome by addition of PGE2. Indomethacin and OKY-046 did not, however, reduce 125I-EGF binding. Thus, in GEC, the basal synthesis of eicosanoids enhanced EGF-induced proliferation. This effect appears to be due to enhancement of EGF receptor activation.

scholarly journals Apical Epidermal Growth Factor Receptor Signaling: Regulation of Stretch-dependent Exocytosis in Bladder Umbrella Cells

2007 ◽  
Vol 18 (4) ◽  
pp. 1312-1323 ◽  
Author(s):  
Elena M. Balestreire ◽  
Gerard Apodaca
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Physiological Role ◽  
Receptor Activation ◽  
Mitogen Activated Protein Kinase ◽  
Apical Surface ◽  
Mechanical Stimuli ◽  
Epidermal Growth ◽  
Umbrella Cells

The apical surface of polarized epithelial cells receives input from mediators, growth factors, and mechanical stimuli. How these stimuli are coordinated to regulate complex cellular functions such as polarized membrane traffic is not understood. We analyzed the requirement for growth factor signaling and mechanical stimuli in umbrella cells, which line the mucosal surface of the bladder and dynamically insert and remove apical membrane in response to stretch. We observed that stretch-stimulated exocytosis required apical epidermal growth factor (EGF) receptor activation and that activation occurred in an autocrine manner downstream of heparin-binding EGF-like growth factor precursor cleavage. Long-term changes in apical exocytosis depended on protein synthesis, which occurred upon EGF receptor-dependent activation of mitogen-activated protein kinase signaling. Our results indicate a novel physiological role for the EGF receptor that couples upstream mechanical stimuli to downstream apical EGF receptor activation that may regulate apical surface area changes during bladder filling.

Determining the geometry of oligomers of the human epidermal growth factor family on cells with <10 nm resolution

2015 ◽  
Vol 43 (3) ◽  
pp. 309-314 ◽  
Author(s):  
Sarah R. Needham ◽  
Laura C. Zanetti-Domingues ◽  
Kathrin M. Scherer ◽  
Michael Hirsch ◽  
Daniel J. Rolfe ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Spatial Resolution ◽  
Single Molecule ◽  
Egf Receptor ◽  
Limited Range ◽  
Length Scales ◽  
Human Epidermal Growth Factor ◽  
Epidermal Growth ◽  
Macromolecular Organization

There is a limited range of methods available to characterize macromolecular organization in cells on length scales from 5–50 nm. We review methods currently available and show the latest results from a new single-molecule localization-based method, fluorophore localization imaging with photobleaching (FLImP), using the epidermal growth factor (EGF) receptor (EGFR) as an example system. Our measurements show that FLImP is capable of achieving spatial resolution in the order of 6 nm.

Norepinephrine modulates the zonally different hepatocyte proliferation through the regulation of transglutaminase activity

2010 ◽  
Vol 299 (1) ◽  
pp. G106-G114 ◽  
Author(s):  
Yosuke Ohtake ◽  
Tomonori Kobayashi ◽  
Akiko Maruko ◽  
Nao Oh-ishi ◽  
Fumihiko Yamamoto ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Egf Receptor ◽  
Transglutaminase 2 ◽  
Hepatocyte Proliferation ◽  
Scatchard Analysis ◽  
Binding Studies ◽  
Perfusion Technique ◽  
Egfr Activation ◽  
Epidermal Growth ◽  
Collagenase Perfusion

A neurotransmitter, norepinephrine (NE), amplifies the mitogenic effect of epidermal growth factor (EGF) in the liver by acting on the α1-adrenergic receptor coupled with G protein, Gαh. However, the molecular mechanism is not well understood. Gαh is known as a transglutaminase 2 (TG2), a cross-linking enzyme implicated in hepatocyte proliferation. We investigated the effect of NE on EGF-induced cell proliferation and TG2 activity using hepatocytes isolated in periportal and perivenous regions of the liver, which differ in proliferative capacity. Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated by the digitonin-collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, EGF receptor (EGFR) dimerization and phosphorylation, and TG2 activity were measured. NE enhanced EGF-induced DNA synthesis, EGF-induced EGFR dimerization, and its phosphorylation in PVH but not in PPH. [3H]NE binding studies indicated that PVH was found to have a greater affinity and number of receptors than PPH. Furthermore, NE treatment decreased TG2 activity and increased phospholipase C activity in PVH although TG2 level showed no change. These results suggest that NE-induced amplification of EGF-induced DNA synthesis especially in PVH is caused by upregulation of EGFR activation through the switching of function from TG2 to Gαh.

Heparin-binding epidermal growth factor and Src family kinases in proliferation of renal epithelial cells

2008 ◽  
Vol 294 (3) ◽  
pp. F459-F468 ◽  
Author(s):  
Shougang Zhuang ◽  
Gilbert R. Kinsey ◽  
Kyle Rasbach ◽  
Rick G. Schnellmann
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Receptor Activation ◽  
Heparin Binding ◽  
Oxidant Injury ◽  
Mmp Inhibitor ◽  
Epidermal Growth ◽  
Renal Proximal Tubular Cells ◽  
Renal Proximal Tubular

Our recent studies have shown that proliferation of renal proximal tubular cells (RPTC) in the absence of growth factors requires activation of the epidermal growth factor (EGF) receptor. We sought to identify the endogenous EGF receptor ligand and investigate the mechanism(s) by which RPTC proliferate in different models. RPTC expressed both pro- and cleaved forms of heparin-binding epidermal growth factor (HB-EGF) and several metalloproteinases (MMP-2, -3, -9, and ADAM10, ADAM17) that have been reported to cleave HB-EGF. Treatment of RPTC with CRM 197, an inhibitor of HB-EGF binding to the EGF receptor, or downregulation of HB-EGF with small interfering RNA inhibited RPTC proliferation following plating. Furthermore, GM6001 (pan-MMP inhibitor), tumor-necrosis factor protease inhibitor-1 (TAPI-1; MMP and ADAM17 inhibitor), and GW280264X (ADAM10 and -17 inhibitor), but not GI254023X (ADAM10 inhibitor), attenuated the proliferation after plating. Although EGF receptor activation is required for RPTC proliferation after oxidant injury, CRM197, GM6001, and TAPI-1 did not block this response. In contrast, inhibition of Src with PP1 blocked EGF receptor activation and RPTC proliferation after oxidant injury. In addition, PP1 treatment attenuated HB-EGF-enhanced RPTC proliferation. We suggest that RPTC proliferation after plating is mediated by HB-EGF produced through an autocrine/paracrine mechanism and RPTC proliferation following oxidant injury is mediated by Src without involvement of HB-EGF.

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