scholarly journals Inhibition of the binding of 125I-labelled epidermal growth factor to mouse cells by a mitogen in goat mammary secretions

1983 ◽  
Vol 212 (2) ◽  
pp. 465-472 ◽  
Author(s):  
K D Brown ◽  
D M Blakeley
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Competitive Inhibitor ◽  
Inhibitory Effect ◽  
Temperature Sensitive ◽  
Egf Receptors ◽  
3T3 Cells ◽  
Epidermal Growth ◽  
Swiss 3T3

Pre-colostrum and colostrum from goats cause a marked inhibition of the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The ability of these secretions to inhibit 125I-EGF binding is closely correlated with the ability to stimulate DNA synthesis in quiescent 3T3 cell cultures, suggesting that goat mammary secretions may contain an EGF-related mitogen. However, the material in colostrum which inhibits 125I-EGF binding to Swiss 3T3 cells is a basic protein with Mr greater than 20000 and is thus quite different from mouse and human EGF. Furthermore, the colostral-mediated inhibition of 125I-EGF binding, although rapid and apparently competitive, differs from the inhibition of binding induced by native, unlabelled EGF. Thus, the inhibitory effect of colostrum is markedly decreased when the assay temperature is shifted from 37 degrees C to 4 degrees C whereas unlabelled EGF is an effective competitive inhibitor at both 37 degrees C and 4 degrees C. Incubation of cells with EGF causes a reduction in cell surface EGF receptors whereas exposure to colostrum does not induce down-regulation of the EGF receptor. Our results suggest that the colostral factor does not bind directly to EGF receptors but inhibits 125I-EGF binding by an indirect mechanism which involves a temperature-sensitive step.

scholarly journals Activation of Endogenous Thrombin Receptors Causes Clustering and Sensitization of Epidermal Growth Factor Receptors of Swiss 3t3 Cells without Transactivation

2001 ◽  
Vol 152 (2) ◽  
pp. 263-274 ◽  
Author(s):  
Michael F. Crouch ◽  
Deborah A. Davy ◽  
Francis S. Willard ◽  
Leise A. Berven
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Thrombin Receptor ◽  
3T3 Cells ◽  
Downstream Effectors ◽  
Epidermal Growth ◽  
Swiss 3T3 ◽  
G Protein Coupled ◽  
Stimulation Of

The G protein–coupled thrombin receptor can induce cellular responses in some systems by transactivating the epidermal growth factor (EGF) receptor. This is in part due to the stimulation of ectoproteases that generate EGF receptor ligands. We show here that this cannot account for the stimulation of proliferation or migration by thrombin of Swiss 3T3 cells. Thrombin has no direct effect on the activation state of the EGF receptor or of its downstream effectors. However, thrombin induces the subcellular clustering of the EGF receptor at filamentous actin–containing structures at the leading edge and actin arcs of migrating cells in association with other signaling molecules, including Shc and phospholipase Cγ1. In these thrombin-primed cells, the subsequent migratory response to EGF is potentiated. Thrombin did not potentiate the EGF-stimulated EGF receptor phosphorylation. Thus, in Swiss 3T3 cells the G protein–coupled thrombin receptor can potentiate the EGF tyrosine kinase receptor response when activated by EGF, and this appears to be due to the subcellular concentration of the receptor with downstream effectors and not to the overall ability of EGF to induce receptor transphosphorylation. Thus, the EGF receptor subcellular localization which is altered by thrombin appears to be an important determinant of the efficacy of downstream EGF receptor signaling in cell migration.

scholarly journals Epidermal growth factor increases sn-1,2-diacylglycerol levels and activates phospholipase D-catalysed phosphatidylcholine breakdown in Swiss 3T3 cells in the absence of inositol-lipid hydrolysis

1992 ◽  
Vol 285 (1) ◽  
pp. 247-253 ◽  
Author(s):  
S J Cook ◽  
M J O Wakelam
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase D ◽  
Egf Receptor ◽  
Kinase Inhibitor ◽  
3T3 Cells ◽  
Lipid Hydrolysis ◽  
Epidermal Growth ◽  
Swiss 3T3 ◽  
Pre Treatment

Addition of epidermal growth factor (EGF) to quiescent Swiss 3T3 cells resulted in a sustained increase in cellular diacylglycerol (DG) content in the absence of inositol-lipid hydrolysis. In the presence of non-cytotoxic concentrations of butan-1-ol, EGF stimulated the formation of phosphatidylbutanol, indicating that the EGF receptor was able to couple to the activation of phospholipase D (PLD). EGF-stimulated release of choline from Swiss 3T3 cells suggested that the major substrate for this PLD was phosphatidylcholine. Unlike bombesin-stimulated PLD activity, the response to EGF was not inhibited by a selective protein kinase C (PKC) inhibitor (Ro-31-8220), suggesting that it was not dependent on PKC activation. Pre-treatment of Swiss 3T3 cells with the EGF-receptor tyrosine kinase inhibitor AG18 selectively inhibited EGF-stimulated PLD activity; bombesin-stimulated PLD activity was unaffected. Butan-1-ol inhibited phorbol ester- and bombesin-stimulated DG formation suggesting a role for a coupled PLD/phosphatidate phosphohydrolase pathway; in contrast, EGF-stimulated DG formation was unaffected.

scholarly journals Inhibition of epidermal growth factor receptor biosynthesis caused by the src oncogene product, pp60v-src.

1990 ◽  
Vol 10 (3) ◽  
pp. 1254-1258 ◽  
Author(s):  
W J Wasilenko ◽  
M Nori ◽  
N Testerman ◽  
M J Weber
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Control ◽  
Growth Factor Receptor ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Permissive Temperature ◽  
Egf Receptors ◽  
Epidermal Growth

We have previously shown that an intracellular mechanism down regulates epidermal growth factor (EGF) receptor levels in rodent fibroblasts transformed by the src oncogene (W. J. Wasilenko, L. K. Shawver, and M. J. Weber, J. Cell. Physiol. 131:450-457, 1987). We now report that this down regulation is due to an inhibition of EGF receptor biosynthesis. With Rat-1 (R1) cells infected with a temperature-sensitive src mutant, we found that 125I-labeled EGF binding to cells began to decrease soon after the activation of pp60v-src by shift down to the permissive temperature for transformation. This effect of src on EGF receptors was reversible. Pulse-chase studies with [35S]methionine-labeled cells revealed that the tyrosine protein kinase activity of pp60v-src had little if any effect on EGF receptor degradation rate. By contrast, the expression of pp60v-src caused a large reduction in the apparent rate of EGF receptor biosynthesis. Northern (RNA) blot analysis demonstrated that pp60v-src also caused marked reductions in the steady-state level of EGF receptor mRNA. These data indicate that one way the expression of the src oncogene can affect the machinery of growth control is by affecting the expression of specific genes for growth factor receptors.

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 Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding.

1986 ◽  
Vol 102 (6) ◽  
pp. 2211-2222 ◽  
Author(s):  
I Zachary ◽  
J W Sinnett-Smith ◽  
E Rozengurt
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Cellular Protein ◽  
3T3 Cells ◽  
Kinase C ◽  
Epidermal Growth ◽  
Swiss 3T3

Addition of bombesin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an Mr 80,000 cellular protein (designated 80k). The effect was both concentration and time dependent; enhancement in 80k phosphorylation could be detected as early as 10 s after the addition of peptide. Recently, a rapid increase in the phosphorylation of an 80k cellular protein after treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact fibroblasts (Rozengurt, E., A. Rodriguez-Pena, and K. A. Smith, 1983, Proc. Natl. Acad. Sci. USA., 80:7244-7248; Rozengurt, E., A. Rodriguez-Pena, M. Coombs, and J. Sinnett-Smith, 1984, Proc. Natl. Acad. Sci. USA., 81:5748-5752). The 80k phosphoproteins generated in response to bombesin and to phorbol 12,13-dibutyrate were identical as judged by one- and two-dimensional PAGE and by peptide mapping after partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with phorbol 12,13-dibutyrate, which leads to the disappearance of protein kinase C activity, blocked the ability of bombesin to stimulate 80k. Bombesin also caused a rapid (1 min) inhibition of 125I-labeled epidermal growth factor (125I-EGF) binding to Swiss 3T3 cells. The inhibition was both concentration and temperature dependent and resulted from a marked decrease in the affinity of the EGF receptor for its ligand. Peptides structurally related to bombesin, including gastrin-releasing peptide, also stimulated 80k phosphorylation and inhibited 125I-EGF binding; both effects were selectively blocked by a novel bombesin antagonist. These results strongly suggest that these responses are mediated by specific high-affinity receptors that recognize the peptides of the bombesin family in Swiss 3T3 cells. While an increase in cytosolic Ca2+ concentration does not mediate the bombesin inhibition of 125I-EGF binding, the activation of protein kinase C in intact Swiss 3T3 cells by peptides of the bombesin family may lead to rapid inhibition of the binding of 125I-EGF to its cellular receptor.

Inhibition of epidermal growth factor receptor biosynthesis caused by the src oncogene product, pp60v-src

1990 ◽  
Vol 10 (3) ◽  
pp. 1254-1258
Author(s):  
W J Wasilenko ◽  
M Nori ◽  
N Testerman ◽  
M J Weber
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Control ◽  
Growth Factor Receptor ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Permissive Temperature ◽  
Egf Receptors ◽  
Epidermal Growth

We have previously shown that an intracellular mechanism down regulates epidermal growth factor (EGF) receptor levels in rodent fibroblasts transformed by the src oncogene (W. J. Wasilenko, L. K. Shawver, and M. J. Weber, J. Cell. Physiol. 131:450-457, 1987). We now report that this down regulation is due to an inhibition of EGF receptor biosynthesis. With Rat-1 (R1) cells infected with a temperature-sensitive src mutant, we found that 125I-labeled EGF binding to cells began to decrease soon after the activation of pp60v-src by shift down to the permissive temperature for transformation. This effect of src on EGF receptors was reversible. Pulse-chase studies with [35S]methionine-labeled cells revealed that the tyrosine protein kinase activity of pp60v-src had little if any effect on EGF receptor degradation rate. By contrast, the expression of pp60v-src caused a large reduction in the apparent rate of EGF receptor biosynthesis. Northern (RNA) blot analysis demonstrated that pp60v-src also caused marked reductions in the steady-state level of EGF receptor mRNA. These data indicate that one way the expression of the src oncogene can affect the machinery of growth control is by affecting the expression of specific genes for growth factor receptors.

Inhibition of epidermal growth factor binding to Swiss 3T3 cells by human platelet release-products

1983 ◽  
Vol 3 (7) ◽  
pp. 659-666 ◽  
Author(s):  
Kenneth D. Brown ◽  
Diane M. Blakeley ◽  
Moira MacDonald
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Human Platelet ◽  
Platelet Derived Growth Factor ◽  
Egf Receptors ◽  
3T3 Cells ◽  
Temperature Dependent ◽  
Cellular Receptors ◽  
Epidermal Growth ◽  
Swiss 3T3

Human platelet ionophore release-products (IRP) inhibit the binding of 125I-labelled epidermal growth factor (125I-EGF) to its receptors on Swiss 3T3 cells. The inhibition appears to be caused by platelet-derived growth factor (PDGF) in the IRP and results from a decrease in the apparent affinity of cellular receptors for 125I-EGF. However, our results indicate that PDGF does not bind directly to EGF receptors, since (1) PDGF does not down-regulate EGF receptors; (2) the PDGF-mediated inhibition of 125I-EGF binding is temperature-dependent; (3) cells which possess EGF receptors but lack PDGF receptors do not exhibit a PDGF-mediated inhibition of 125I-EGF binding.

scholarly journals Epidermal growth factor-induced phosphoinositide hydrolysis in permeabilized 3T3 cells: lack of guanosine triphosphate dependence and inhibition by tyrosine-containing peptides.

1990 ◽  
Vol 1 (9) ◽  
pp. 615-620 ◽  
Author(s):  
G F Verheijden ◽  
I Verlaan ◽  
J Schlessinger ◽  
W H Moolenaar
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
G Protein ◽  
Egf Receptor ◽  
Phosphoinositide Hydrolysis ◽  
3T3 Cells ◽  
Guanosine Triphosphate ◽  
Intact Cells ◽  
Epidermal Growth ◽  
Gamma S

The possible involvement of a stimulatory guanosine triphosphate (GTP)-binding (G) protein in epidermal growth factor (EGF)-induced phosphoinositide hydrolysis has been investigated in permeabilized NIH-3T3 cells expressing the human EGF receptor. The mitogenic phospholipid lysophosphatidate (LPA), a potent inducer of phosphoinositide hydrolysis, was used as a control stimulus. In intact cells, pertussis toxin partially inhibits the LPA-induced formation of inositol phosphates, but has no effect on the response to EGF. In cells permeabilized with streptolysin-O, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) dramatically increases the initial rate of inositol phosphate formation induced by LPA. In contrast, activation of phospholipase C (PLC) by EGF occurs in a GTP-independent manner. Guanine 5'-O-(2-thiodiphosphate) (GDP beta S) which keeps G proteins in their inactive state, blocks the stimulation by LPA and GTP gamma S, but fails to affect the EGF-induced response. Tyrosine-containing substrate peptides, when added to permeabilized cells, inhibit EGF-induced phosphoinositide hydrolysis without interfering with the response to LPA and GTP gamma S. These data suggest that the EGF receptor does not utilize an intermediary G protein to activate PLC and that receptor-mediated activation of effector systems can be inhibited by exogenous substrate peptides.

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

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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