scholarly journals Epidermal Growth Factor Receptor Activation Remodels the Plasma Membrane Lipid Environment To Induce Nanocluster Formation

2010 ◽  
Vol 30 (15) ◽  
pp. 3795-3804 ◽  
Author(s):  
Nicholas Ariotti ◽  
Hong Liang ◽  
Yufei Xu ◽  
Yueqiang Zhang ◽  
Yoshiya Yonekubo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Molecular Mechanisms ◽  
Receptor Activation ◽  
Membrane Lipid ◽  
Dependent Manner ◽  
Epidermal Growth ◽  
Lipid Environment ◽  
Lateral Segregation

ABSTRACT Signal transduction is regulated by the lateral segregation of proteins into nanodomains on the plasma membrane. However, the molecular mechanisms that regulate the lateral segregation of cell surface receptors, such as receptor tyrosine kinases, upon ligand binding are unresolved. Here we used high-resolution spatial mapping to investigate the plasma membrane nanoscale organization of the epidermal growth factor (EGF) receptor (EGFR). Our data demonstrate that in serum-starved cells, the EGFR exists in preformed, cholesterol-dependent, actin-independent nanoclusters. Following stimulation with EGF, the number and size of EGFR nanoclusters increase in a time-dependent manner. Our data show that the formation of EGFR nanoclusters requires receptor tyrosine kinase activity. Critically, we show for the first time that production of phosphatidic acid by phospholipase D2 (PLD2) is essential for ligand-induced EGFR nanocluster formation. In accordance with its crucial role in regulating EGFR nanocluster formation, we demonstrate that modulating PLD2 activity tunes the degree of EGFR nanocluster formation and mitogen-activated protein kinase signal output. Together, these data show that EGFR activation drives the formation of signaling domains by regulating the production of critical second-messenger lipids and modifying the local membrane lipid environment.

scholarly journals Eps15 Is Recruited to the Plasma Membrane upon Epidermal Growth Factor Receptor Activation and Localizes to Components of the Endocytic Pathway during Receptor Internalization

1999 ◽  
Vol 10 (2) ◽  
pp. 417-434 ◽  
Author(s):  
Maria Rosaria Torrisi ◽  
Lavinia Vittoria Lotti ◽  
Francesca Belleudi ◽  
Roberto Gradini ◽  
Anna Elisabetta Salcini ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Receptor Activation ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Epidermal Growth

Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.

scholarly journals Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

1996 ◽  
Vol 16 (5) ◽  
pp. 1946-1954 ◽  
Author(s):  
L V Lotti ◽  
L Lanfrancone ◽  
E Migliaccio ◽  
C Zompetta ◽  
G Pelicci ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Rough Endoplasmic Reticulum ◽  
Receptor Tyrosine Kinase ◽  
Receptor Activation ◽  
Kinase Activation ◽  
Epidermal Growth

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein.

scholarly journals Epidermal growth factor receptor activation is localized within low-buoyant density, non-caveolar membrane domains

1999 ◽  
Vol 337 (3) ◽  
pp. 591-597 ◽  
Author(s):  
Mark G. WAUGH ◽  
Durward LAWSON ◽  
J. Justin HSUAN
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Receptor Activation ◽  
Cell Fractionation ◽  
Membrane Domains ◽  
Egf Receptors ◽  
Cell Extracts ◽  
Epidermal Growth

Increasing evidence for the organization of cell-surface proteins and lipids into different detergent-insoluble rafts led us to investigate epidermal growth factor (EGF) receptor activation in the plasma membranes of A431 carcinoma cells, using a combination of cell fractionation and immunoprecipitation techniques. Density-gradient centrifugation of sodium carbonate cell extracts revealed that the vast majority of both stimulated and unstimulated EGF receptors were concentrated in a caveolin-rich light membrane (CLM) fraction, with the biochemical characteristics of detergent-insoluble glycolipid-rich domains (DIGs). However, ultrastructural analysis of the CLM fraction revealed that it contained a heterogeneous collection of vesicles, some with sizes greater than that expected for individual caveolae. Experiments with detergent-solubilized cells and isolated CLMs indicated that, in contrast with caveolin, EGF receptors were unlikely to be localized to DIG domains. Furthermore, immunoisolation of caveolin from CLMs revealed that EGF receptor activation occurs in a compartment distinct from caveolae. Similarly, using an anti-(EGF receptor) antibody, the bulk of the cellular caveolin was not co-immunoprecipitated from CLMs, thereby confirming that these two proteins reside in separate membrane domains. The deduction that caveolar signalling and EGF receptor activation occur in separable rafts argues for a multiplicity of signal transduction compartments within the plasma membrane. In addition, by demonstrating that EGF receptor activation is compartmentalized within low-density, non-caveolar regions of the plasma membrane, it is also shown that the co-localization of proteins in a CLM fraction is insufficient to prove caveolar localization.

scholarly journals Cholesterol Depletion from the Plasma Membrane Triggers Ligand-independent Activation of the Epidermal Growth Factor Receptor

2002 ◽  
Vol 277 (51) ◽  
pp. 49631-49637 ◽  
Author(s):  
Xu Chen ◽  
Marilyn D. Resh
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Growth Factor Receptor ◽  
Receptor Activation ◽  
Cholesterol Depletion ◽  
Mapk Activation ◽  
Epidermal Growth

We recently demonstrated that depletion of plasma membrane cholesterol with methyl-β-cyclodextrin (MβCD) caused activation of MAPK (Chen, X., and Resh, M. D. (2001)J. Biol. Chem. 276, 34617–34623). MAPK activation was phosphatidylinositol 3-kinase (PI3K)-dependent and involved increased tyrosine phosphorylation of the p85 subunit of PI3K. We next determined whether MβCD treatment induced tyrosine phosphorylation of other cellular proteins. Here we report that cholesterol depletion of serum-starved COS-1 cells with MβCD or filipin caused an increase in Tyr(P) levels of a 180-kDa protein that was identified as the epidermal growth factor receptor (EGFR). Cross-linking experiments showed that MβCD induced dimerization of EGFR, indicative of receptor activation. Reagents that block release of membrane-bound EGFR ligands did not affect MβCD-induced tyrosine phosphorylation of EGFR, indicating that MβCD activation of EGFR is ligand-independent. Moreover, MβCD treatment resulted in increased tyrosine phosphorylation of EGFR downstream targets and Ras activation. Incubation of cells with the specific EGFR inhibitor AG4178 blocked MβCD-induced phosphorylation of EGFR, SHC, phospholipase C-γ, and Gab-1 as well as MAPK activation. We conclude that cholesterol depletion from the plasma membrane by MβCD causes ligand-independent activation of EGFR, resulting in MAPK activation by PI3K and Ras-dependent mechanisms. Moreover, these studies reveal a novel mode of action of MβCD, in addition to its ability to disrupt membrane rafts.

scholarly journals Spatial Localization of m-Calpain to the Plasma Membrane by Phosphoinositide Biphosphate Binding during Epidermal Growth Factor Receptor-Mediated Activation

2006 ◽  
Vol 26 (14) ◽  
pp. 5481-5496 ◽  
Author(s):  
Hanshuang Shao ◽  
Jeff Chou ◽  
Catherine J. Baty ◽  
Nancy A. Burke ◽  
Simon C. Watkins ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Cell Body ◽  
Wound Repair ◽  
Egf Receptor ◽  
Binding Capacity ◽  
Dependent Manner ◽  
Epidermal Growth

ABSTRACT Calpain activity is required for de-adhesion of the cell body and rear to enable productive locomotion of adherent cells during wound repair and tumor invasion. Growth factors activate m-calpain (calpain 2, CAPN2) via ERK/mitogen-activated protein kinases, but only when these kinases are localized to the plasma membrane. We thus hypothesized that m-calpain is activated by epidermal growth factor (EGF) only when it is juxtaposed to the plasma membrane secondary to specific docking. Osmotic disruption of NR6 fibroblasts expressing the EGF receptor demonstrated m-calpain being complexed with the substratum-adherent membrane with this increasing in an EGF-dependent manner. m-Calpain colocalized with phosphoinositide biphosphate (PIP2) with exogenous phospholipase C removal of phosphoinositides, specifically, PI(4,5)P2 but not PI(4)P1 or PIP3, releasing the bound m-calpain. Downregulation of phosphoinositide production by 1-butanol resulted in diminished PIP2 in the plasma membrane and eliminated EGF-induced calpain activation. This PIP2-binding capacity resided in domain III of calpain, which presents a putative C2-like domain. This active conformation of this domain appears to be partially masked in the holoenzyme as both activation of m-calpain by phosphorylation at serine 50 and expression of constitutively active phosphorylation mimic glutamic acid-increased m-calpain binding to the membrane, consistent with blockade of this cascade diminishing membrane association. Importantly, we found that m-calpain was enriched toward the rear of locomoting cells, which was more pronounced in the plasma membrane footprints; EGF further enhanced this enrichment, in line with earlier reports of loss of PIP2 in lamellipodia of motile cells. These data support a model of m-calpain binding to PIP2 concurrent with and likely to enable ERK activation and provides a mechanism by which cell de-adhesion is directed to the cell body and tail as phospholipase C-γ hydrolyzes PIP2 in the protruding lamellipodia.

Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblasts and neuronal cells

2005 ◽  
Vol 33 (4) ◽  
pp. 631-634 ◽  
Author(s):  
K. Kurokawa ◽  
T. Nakamura ◽  
K. Aoki ◽  
M. Matsuda
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Function ◽  
Leading Edge ◽  
Dependent Manner ◽  
Rhoa Activity ◽  
Rho Family Gtpases ◽  
Epidermal Growth ◽  
Rho Family

Rho-family GTPases regulate various aspects of cell function by controlling cytoskeletal changes; however, their spatial regulation within the cells remains largely unknown. To understand this regulation, we have studied the spatiotemporal activity of Rho-family GTPases in migrating cells and growth factor-stimulated cells by using probes based on the principle of fluorescence resonance energy transfer. In migrating fibroblasts and epithelial cells, the level of RhoA activity is high both at the contractile tail and at the leading edge, whereas Rac1 and Cdc42 activities are high only at the leading edge. In cells stimulated with epidermal growth factor or nerve growth factor, activities of Rac1 and Cdc42 were transiently elevated in a broad area of the plasma membrane, followed by a localized activation at nascent lamellipodia. In contrast, on epidermal growth factor stimulation, RhoA activity decreased diffusely at the plasma membrane. Notably, RhoA activity persisted at the tip of growth factor-induced membrane ruffles and, in agreement with this finding, RhoA is required for membrane ruffling. These observations suggest that the activities of Rho-family GTPases are elaborately regulated in a time- and space-dependent manner to control cytoskeletal changes and that the basic mechanism of controlling cell shape via Rho-family GTPases is common to various cell types.

scholarly journals Heterogeneity of signal transduction at the subcellular level: microsphere-based focal EGF receptor activation and stimulation of Shc translocation

2001 ◽  
Vol 114 (13) ◽  
pp. 2437-2447
Author(s):  
Roland Brock ◽  
Thomas M. Jovin
Keyword(s):  
Signal Transduction ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Chinese Hamster Ovary Cells ◽  
Adaptor Protein ◽  
Receptor Activation ◽  
Laser Scanning Microscopy ◽  
Dependent Manner ◽  
Epidermal Growth ◽  
Subcellular Level

Epidermal growth factor receptor (EGFR, erbB1) activation and translocation of the Shc adaptor protein to activated receptors were analyzed at the subcellular level by dual-label immunofluorescence and confocal laser scanning microscopy in conjunction with a new microsphere-based protocol. In the Quantitative Microsphere Recruitment Assay (QMRA) introduced here, epidermal growth factor-coated 1 μm diameter microspheres were distributed over the surface of adherent tissue culture cells expressing the receptor. High-resolution confocal microscopy of a fusion construct of the receptor and the green fluorescent protein expressed in Chinese hamster ovary cells demonstrated that engulfment and internalization of the microspheres occurred rapidly within minutes, and in a receptor activation-dependent manner. In human epidermoid carcinoma A431 cells, receptor activation and Shc translocation persisted over the 20-minute time course of the experiments. However, at the subcellular level the positive correlation of receptor activation and Shc translocation observed at 5-8 minutes dissipated, indicating a time-dependent decoupling of the two events and variation in the kinetics of signal transduction for different subcellular locations.

scholarly journals Akt–PDK1 Complex Mediates Epidermal Growth Factor-induced Membrane Protrusion through Ral Activation

2007 ◽  
Vol 18 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Hisayoshi Yoshizaki ◽  
Naoki Mochizuki ◽  
Yukiko Gotoh ◽  
Michiyuki Matsuda
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Time Course ◽  
Resonance Energy ◽  
Dependent Manner ◽  
Akt Inhibitor ◽  
Epidermal Growth ◽  
Ral Gtpase

We studied the spatiotemporal regulation of Akt (also called protein kinase B), phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] by using probes based on the principle of fluorescence resonance energy transfer. On epidermal growth factor (EGF) stimulation, the amount of PtdIns(3,4,5)P3 was increased diffusely in the plasma membrane, whereas that of PtdIns(3,4)P2 was increased more in the nascent lamellipodia than in the plasma membrane of the central region. The distribution and time course of Akt activation were similar to that of increased PtdIns(3,4)P2 levels, which were most prominent in the nascent lamellipodia. Moreover, we found that upon EGF stimulation 3-phosphoinositide–dependent protein kinase-1 (PDK1) was also recruited to nascent lamellipodia in an Akt-dependent manner. Because PDK1 is known to activate Ral GTPase and because Ral is required for EGF-induced lamellipodial protrusion, we speculated that the PDK1–Akt complex may be indispensable for the induction of lamellipodia. In agreement with this idea, EGF-induced lamellipodia formation was promoted by the overexpression of Akt and inhibited by an Akt inhibitor or a Ral-binding domain of Sec5. These results identified the Akt–PDK1 complex as an upstream positive regulator of Ral GTPase in the induction of lamellipodial protrusion.

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