scholarly journals Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells.

1988 ◽  
Vol 8 (3) ◽  
pp. 1345-1351 ◽  
Author(s):  
E Sturani ◽  
R Zippel ◽  
L Toschi ◽  
L Morello ◽  
P M Comoglio ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Human Fibroblasts ◽  
Atp Depletion ◽  
Egf Receptors ◽  
A431 Cells ◽  
Intact Cells ◽  
Receptor Phosphorylation ◽  
Epidermal Growth

We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.

Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells

1988 ◽  
Vol 8 (3) ◽  
pp. 1345-1351
Author(s):  
E Sturani ◽  
R Zippel ◽  
L Toschi ◽  
L Morello ◽  
P M Comoglio ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Human Fibroblasts ◽  
Atp Depletion ◽  
Egf Receptors ◽  
A431 Cells ◽  
Intact Cells ◽  
Receptor Phosphorylation ◽  
Epidermal Growth

We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.

scholarly journals Localization of the epidermal growth factor (EGF) receptor within the endosome of EGF-stimulated epidermoid carcinoma (A431) cells.

1986 ◽  
Vol 102 (2) ◽  
pp. 500-509 ◽  
Author(s):  
K Miller ◽  
J Beardmore ◽  
H Kanety ◽  
J Schlessinger ◽  
C R Hopkins
Keyword(s):  
Acid Phosphatase ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Epidermoid Carcinoma ◽  
Egf Receptors ◽  
A431 Cells ◽  
Thin Sections ◽  
Receptor Complexes ◽  
Epidermal Growth

We have followed the internalization pathway of both epidermal growth factor (EGF) and its receptor in human epidermoid carcinoma (A431) cells. Using EGF conjugated with horseradish peroxidase and anti-receptor monoclonal antibodies (TL5 and EGFR1) coupled either directly or indirectly to colloidal gold we have identified an extensive elaboration of endosomal compartments, consisting of a peripheral branching network of tubular cisternae connected to vacuolar elements that contain small vesicles and a pericentriolar compartment consisting of a tubular cisternal network connected to multivesicular bodies. Immunocytochemistry on frozen thin sections using receptor-specific antibody-gold revealed that at 4 degrees C in the presence of EGF, receptors were mainly on the plasma membrane and, to a lesser extent, within some elements of both the peripheral and pericentriolar endosomal compartments. Upon warming to 37 degrees C there was an EGF-dependent redistribution of most binding sites, first to the peripheral endosome compartment and then to the pericentriolar compartment and lysosomes. Upon warming only to 20 degrees C the ligand-receptor complex accumulated in the pericentriolar compartment. Acid phosphatase cytochemistry identifies hydrolytic activity only within secondary lysosomes and trans cisternae of the Golgi stacks. Together these observations suggest that the prelysosomal endosome compartment extends to the pericentriolar complex and that the transfer of EGF receptor complexes to the acid phosphatase-positive lysosome involves a discontinuous, temperature-dependent step.

Epidermal growth factor receptor cytoplasmic domain mutations trigger ligand-independent transformation

1990 ◽  
Vol 10 (6) ◽  
pp. 3048-3055
Author(s):  
S Massoglia ◽  
A Gray ◽  
T J Dull ◽  
S Munemitsu ◽  
H J Kun ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Growth Factor Receptor ◽  
Cytoplasmic Domain ◽  
Control Mechanisms ◽  
Egf Receptors ◽  
Intact Cells ◽  
Epidermal Growth

The transforming gene product of avian erythroblastosis virus, v-erbB, is derived from the epidermal growth factor (EGF) receptor but has lost its extracellular ligand-binding domain and was mutated in its cytoplasmic portion, which is thought to be responsible for biological signal generation. We have repaired the deletion of extracellular EGF-binding sequences and investigated the functional consequences of cytoplasmic erbB mutations. Within the resulting EGF receptors, the autophosphorylation activities of the cytoplasmic domains of v-erbB-H and v-erbB-ES4 were fully ligand dependent in intact cells. However, the mitogenic and transforming signaling activities of an EGF receptor carrying v-erbB-ES4 (but not v-erbB-H) cytoplasmic sequences remained ligand independent, whereas those of a receptor with a v-erbB-H cytoplasmic domain were regulated by EGF or transforming growth factor alpha. Thus, structural alterations in the cytoplasmic domain of growth factor receptor tyrosine kinases may induce constitutive signaling activity without autophosphorylation. These findings provide new insight into the mechanism of receptor-mediated signal transduction and suggest a novel alternative for subversion of cellular control mechanisms and proto-oncogene activation.

scholarly journals Epidermal growth factor receptors associated to cytoskeletal elements of epidermoid carcinoma (A431) cells.

1986 ◽  
Vol 103 (1) ◽  
pp. 87-94 ◽  
Author(s):  
F A Wiegant ◽  
F J Blok ◽  
L H Defize ◽  
W A Linnemans ◽  
A J Verkley ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Immunogold Labeling ◽  
Egf Receptors ◽  
A431 Cells ◽  
Gold Particles ◽  
Epidermal Growth ◽  
Cytoskeletal Elements ◽  
Triton X

The structural interaction of the epidermal growth factor (EGF) receptor and the cytoskeleton of A431 cells has been studied using a monoclonal anti-EGF receptor antibody. This has been done with immunogold labeling using a variety of electron microscopical preparation procedures and EGF binding studies. By providing an image of the membrane-associated cytoskeleton, the dry cleavage method reveals a preferential localization of EGF receptors superimposed upon cytoskeletal filaments. The colocalization of gold particles with cytoskeletal filaments is not affected when pre-labeled cells are extracted with the non-ionic detergent Triton X-100, as visualized by dry cleavage. Using surface replication, this treatment results in visualization of the cytoskeleton. In these latter preparations, it is also observed that EGF receptor-coupled gold particles remain associated with cytoskeletal elements. Moreover, Triton extraction performed before immunogold labeling of EGF receptors demonstrates that isolated cytoskeletons contained binding sites for anti-EGF receptor antibodies. Using stereo micrographs of replica's obtained from these isolated cytoskeletons, it is shown that gold-labeled EGF receptors are exclusively present on the cortical membrane-associated region of the cytoskeleton and not on more intracellular-located filaments. Scatchard analysis of EGF binding to cells fixed with glutaraldehyde and treated with Triton X-100 before and after EGF binding indicates that a high affinity EGF binding site is associated with the Triton X-100 insoluble cytoskeleton.

scholarly journals Activity of the epidermal-growth-factor receptor and phospholipase C-γ 1 in heat-stressed fibroblasts and A-431 cells

1992 ◽  
Vol 286 (2) ◽  
pp. 541-547 ◽  
Author(s):  
S M Liu ◽  
G Carpenter
Keyword(s):  
Growth Factor ◽  
Heat Shock ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Egf Receptor ◽  
Inositol Phosphates ◽  
Human Fibroblasts ◽  
Egf Receptors ◽  
Epidermal Growth ◽  
Phospholipase C Gamma

A variety of changes in the functions of specific plasma-membrane components have been reported in cells exposed to a heat shock. In this study, we examined the consequences of heat stress on epidermal-growth-factor (EGF)-induced receptor autophosphorylation and receptor-mediated tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), a cellular substrate. Although the tyrosine kinase activity of the EGF receptor is rapidly inactivated at 45 degrees C in vitro [Carpenter, King & Cohen (1979) J. Biol. Chem. 254, 4884-4891], EGF stimulates autophosphorylation of its receptor in both A-431 cells and human fibroblasts after a prolonged heat shock. Phosphoamino acid analysis of the receptor reveals an EGF-induced increase in phosphotyrosine and phosphoserine at 46 degrees C. EGF also stimulates the phosphorylation of phospholipase C-gamma 1 and induces the formation of inositol phosphates under heat-shock conditions. 125I-EGF binding and internalization in A-431 cells is not decreased during incubations at 46 degrees C for up to 90 min. EGF-induced dimerization of EGF receptors on the cell surface is preserved during heat shock. Though EGF-receptor-mediated endocytosis is not inhibited by elevated temperature, the degradation of internalized 125I-EGF is dramatically decreased. These results indicate that, aside from ligand degradation, the EGF-mediated pathway of signal transduction through phospholipase C-gamma 1 remains remarkably intact during conditions of extreme cellular stress.

scholarly journals Interactions between the receptors for platelet-derived growth factor and epidermal growth factor.

1983 ◽  
Vol 96 (3) ◽  
pp. 679-683 ◽  
Author(s):  
D F Bowen-Pope ◽  
P E Dicorleto ◽  
R Ross
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Human Fibroblasts ◽  
Insulin Binding ◽  
Platelet Derived Growth Factor ◽  
Egf Receptors ◽  
3T3 Cells ◽  
Low Concentrations ◽  
Epidermal Growth

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.

scholarly journals Inhibition of the apparent affinity of the epidermal growth factor receptor caused by phorbol diesters correlates with phosphorylation of threonine-654 but not other sites on the receptor

1986 ◽  
Vol 233 (2) ◽  
pp. 435-441 ◽  
Author(s):  
R J Davis ◽  
M P Czech
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Affinity Binding ◽  
Egf Receptors ◽  
A431 Cells ◽  
High Affinity Binding ◽  
Phosphorylation State ◽  
High Affinity ◽  
Epidermal Growth

Addition of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) to A431 human epidermoid carcinoma cells causes a marked increase in the phosphorylation state of the epidermal growth factor (EGF) receptor with a concomitant inhibition of both the high-affinity binding of 125I-EGF and the receptor tyrosine kinase activity. It was found in the present studies that the diuretic drug amiloride has no effect on the action of PMA to inhibit the binding of 125I-EGF. However, amiloride was observed to inhibit markedly the effect of PMA to cause a 3-fold increase in the phosphorylation state of the EGF receptors. In the presence of PMA and amiloride, the increase in the phosphorylation state of the EGF receptors was found to be only 1.2-fold over controls. Analysis of the EGF receptor phosphorylation sites by phosphopeptide mapping by reverse-phase h.p.l.c. demonstrated that PMA increases the phosphorylation state of the EGF receptor at many sites. One of these sites has been identified as a C-kinase substrate, threonine-654. In the presence of amiloride, PMA causes phosphorylation of threonine-654 to the same stoichiometry as that observed in the absence of amiloride. However, the marked increase in the phosphorylation state of the EGF receptor at other sites caused by PMA is abolished in the presence of amiloride. We conclude that the extensive phosphorylation of the EGF receptor at several sites caused by the addition of PMA to A431 cells is not required for the action of PMA to inhibit the high-affinity binding of 125I-EGF. The results indicate that the phosphorylation state of threonine-654 may play a role in this process.

scholarly journals Retention of epidermal growth factor receptors in the endoplasmic reticulum of adenovirus-infected cells

1992 ◽  
Vol 282 (1) ◽  
pp. 115-121 ◽  
Author(s):  
G F Verheijden ◽  
W H Moolenaar ◽  
H L Ploegh
Keyword(s):  
Endoplasmic Reticulum ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Intracellular Transport ◽  
Egf Receptor ◽  
Egf Receptors ◽  
A431 Cells ◽  
Infected Cells ◽  
Epidermal Growth ◽  
Endoglycosidase H

The epidermal growth factor (EGF) receptor is down-regulated during early infection with adenovirus, and this has been attributed to accelerated internalization and degradation of the receptor in the absence of ligand (Carlin, Tollefson, Brady, Hoffman & Wold (1989) Cell 57, 135-144]. Using pulse-chase analysis, we show that loss of functional EGF receptors after infection of human KB and A431 cells with adenovirus type 5 is accompanied by accumulation of a receptor precursor that remains fully sensitive to endoglycosidase H, indicative of retention in the endoplasmic reticulum. A truncated receptor, normally secreted by A431 cells, also accumulates intracellularly as an endoglycosidase H-sensitive precursor. In no case is the block in intracellular transport of EGF receptors complete. We conclude that both stimulation of EGF receptor internalization and degradation and inhibition of intracellular transport of newly synthesized EGF receptors from the endoplasmic reticulum towards the cell surface contribute to EGF receptor down-regulation in adenovirus-infected cells.

scholarly journals Epidermal growth factor receptor cytoplasmic domain mutations trigger ligand-independent transformation.

1990 ◽  
Vol 10 (6) ◽  
pp. 3048-3055 ◽  
Author(s):  
S Massoglia ◽  
A Gray ◽  
T J Dull ◽  
S Munemitsu ◽  
H J Kun ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Growth Factor Receptor ◽  
Cytoplasmic Domain ◽  
Control Mechanisms ◽  
Egf Receptors ◽  
Intact Cells ◽  
Epidermal Growth

The transforming gene product of avian erythroblastosis virus, v-erbB, is derived from the epidermal growth factor (EGF) receptor but has lost its extracellular ligand-binding domain and was mutated in its cytoplasmic portion, which is thought to be responsible for biological signal generation. We have repaired the deletion of extracellular EGF-binding sequences and investigated the functional consequences of cytoplasmic erbB mutations. Within the resulting EGF receptors, the autophosphorylation activities of the cytoplasmic domains of v-erbB-H and v-erbB-ES4 were fully ligand dependent in intact cells. However, the mitogenic and transforming signaling activities of an EGF receptor carrying v-erbB-ES4 (but not v-erbB-H) cytoplasmic sequences remained ligand independent, whereas those of a receptor with a v-erbB-H cytoplasmic domain were regulated by EGF or transforming growth factor alpha. Thus, structural alterations in the cytoplasmic domain of growth factor receptor tyrosine kinases may induce constitutive signaling activity without autophosphorylation. These findings provide new insight into the mechanism of receptor-mediated signal transduction and suggest a novel alternative for subversion of cellular control mechanisms and proto-oncogene activation.

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