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

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.

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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

Biochemical Journal ◽

10.1042/bj2850247 ◽

1992 ◽

Vol 285 (1) ◽

pp. 247-253 ◽

Cited By ~ 61

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.

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

Biochemical Journal ◽

10.1042/bj2120465 ◽

1983 ◽

Vol 212 (2) ◽

pp. 465-472 ◽

Cited By ~ 23

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.

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Estrogen Action Via the G Protein-Coupled Receptor, GPR30: Stimulation of Adenylyl Cyclase and cAMP-Mediated Attenuation of the Epidermal Growth Factor Receptor-to-MAPK Signaling Axis

Molecular Endocrinology ◽

10.1210/mend.16.1.0758 ◽

2002 ◽

Vol 16 (1) ◽

pp. 70-84 ◽

Cited By ~ 396

Author(s):

Edward J. Filardo ◽

Jeffrey A. Quinn ◽

A. Raymond Frackelton ◽

Kirby I. Bland

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Adenylyl Cyclase ◽

G Protein ◽

Egf Receptor ◽

G Protein Coupled Receptor ◽

Estrogen Action ◽

Epidermal Growth ◽

G Protein Coupled ◽

Stimulation Of

Abstract Estrogen triggers rapid yet transient activation of the MAPKs, extracellular signal-regulated kinase (Erk)-1 and Erk-2. We have reported that this estrogen action requires the G protein-coupled receptor, GPR30, and occurs via Gβγ-subunit protein-dependent transactivation of the epidermal growth factor (EGF) receptor through the release of pro-heparan-bound EGF from the cell surface. Here we investigate the mechanism by which Erk-1/-2 activity is rapidly restored to basal levels after estrogen stimulation. Evidence is provided that attenuation of Erk-1/-2 activity by estrogen occurs via GPR30-dependent stimulation of adenylyl cyclase and cAMP-dependent signaling that results in Raf-1 inactivation. We show that 17β-E2 represses EGF-induced activation of the Raf-to-Erk pathway in human breast carcinoma cells that express GPR30, including MCF-7 and SKBR3 cells which express both or neither, ER, respectively. MDA-MB-231 cells, which express ERβ, but not ERα, and low levels of GPR30 protein, are unable to stimulate adenylyl cyclase or promote estrogen-mediated blockade of EGF-induced activation of Erk-1/-2. Pretreatment of MDA-MB-231 cells with cholera toxin, which ADP-ribosylates and activates Gαs subunit proteins, results in G protein-coupled receptor (GPCR)-independent adenylyl cyclase activity and suppression of EGF-induced Erk-1/-2 activity. Transfection of GPR30 into MDA-MB-231 cells restores their ability to stimulate adenylyl cyclase and attenuate EGF-induced activation of Erk-1/-2 by estrogen. Moreover, GPR30-dependent, cAMP-mediated attenuation of EGF-induced Erk-1/-2 activity was achieved by ER antagonists such as tamoxifen or ICI 182, 780; yet not by 17α-E2 or progesterone. Thus, our data delineate a novel mechanism, requiring GPR30 and estrogen, that acts to regulate Erk-1/-2 activity via an inhibitory signal mediated by cAMP. Coupled with our prior findings, these current data imply that estrogen balances Erk-1/-2 activity through a single GPCR via two distinct G protein-dependent signaling pathways that have opposing effects on the EGF receptor-to-MAPK pathway.

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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.

The Journal of Cell Biology ◽

10.1083/jcb.102.6.2211 ◽

1986 ◽

Vol 102 (6) ◽

pp. 2211-2222 ◽

Cited By ~ 169

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.

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Insulin induces epidermal growth factor (EGF) receptor clustering and potentiates EGF‐stimulated DNA synthesis in Swiss 3T3 cells: A mechanism for costimulation in mitogenic synergy

Immunology and Cell Biology ◽

10.1046/j.1440-1711.2000.00929.x ◽

2000 ◽

Vol 78 (4) ◽

pp. 408-414 ◽

Cited By ~ 24

Author(s):

Michael F Crouch ◽

Deborah A Davy ◽

Francis S Willard ◽

Leise A Berven

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Dna Synthesis ◽

Egf Receptor ◽

3T3 Cells ◽

Receptor Clustering ◽

Epidermal Growth ◽

Swiss 3T3

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

Cell Regulation ◽

10.1091/mbc.1.9.615 ◽

1990 ◽

Vol 1 (9) ◽

pp. 615-620 ◽

Cited By ~ 6

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.

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