Binding of single-mutant epidermal growth factor (EGF) ligands alter the stability of the EGF receptor dimer and promote growth signaling

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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Estrogen induces epidermal growth factor (EGF) receptor and its ligands in human fallopian tube: involvement of EGF but not transforming growth factor-alpha in estrogen-induced tubal cell growth in vitro.

Endocrinology ◽

10.1210/endo.136.5.7720660 ◽

1995 ◽

Vol 136 (5) ◽

pp. 2110-2119 ◽

Cited By ~ 9

Author(s):

K Adachi ◽

H Kurachi ◽

H Homma ◽

H Adachi ◽

T Imai ◽

...

Keyword(s):

Growth Factor ◽

Cell Growth ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Transforming Growth Factor ◽

Transforming Growth Factor Alpha ◽

Human Fallopian Tube ◽

Epidermal Growth ◽

Factor Alpha

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Epidermal Growth Factor Receptor Distribution during Chemotactic Responses

Molecular Biology of the Cell ◽

10.1091/mbc.11.11.3873 ◽

2000 ◽

Vol 11 (11) ◽

pp. 3873-3883 ◽

Cited By ~ 63

Author(s):

Maryse Bailly ◽

Jeffrey Wyckoff ◽

Boumediene Bouzahzah ◽

Ross Hammerman ◽

Vonetta Sylvestre ◽

...

Keyword(s):

Plasma Membrane ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Fluorescent Protein ◽

Growth Factor Receptor ◽

Spatial Gradient ◽

Spatial Gradients ◽

Epidermal Growth ◽

Green Fluorescent

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.

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Activation of the Epidermal Growth Factor (EGF) Receptor Induces Formation of EGF Receptor- and Grb2-Containing Clathrin-Coated Pits

Molecular and Cellular Biology ◽

10.1128/mcb.26.2.389-401.2006 ◽

2006 ◽

Vol 26 (2) ◽

pp. 389-401 ◽

Cited By ~ 81

Author(s):

Lene E. Johannessen ◽

Nina Marie Pedersen ◽

Ketil Winther Pedersen ◽

Inger Helene Madshus ◽

Espen Stang

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Point Mutations ◽

Adaptor Protein ◽

Mitogen Activated Protein Kinase ◽

Coated Pits ◽

Α Subunit ◽

Sh3 Domains ◽

Epidermal Growth

ABSTRACT In HeLa cells depleted of adaptor protein 2 complex (AP2) by small interfering RNA (siRNA) to the μ2 or α subunit or by transient overexpression of an AP2 sequestering mutant of Eps15, endocytosis of the transferrin receptor (TfR) was strongly inhibited. However, epidermal growth factor (EGF)-induced endocytosis of the EGF receptor (EGFR) was inhibited only in cells where the α subunit had been knocked down. By immunoelectron microscopy, we found that in AP2-depleted cells, the number of clathrin-coated pits was strongly reduced. When such cells were incubated with EGF, new coated pits were formed. These contained EGF, EGFR, clathrin, and Grb2 but not the TfR. The induced coated pits contained the α subunit, but labeling density was reduced compared to control cells. Induction of clathrin-coated pits required EGFR kinase activity. Overexpression of Grb2 with inactivating point mutations in N- or C-terminal SH3 domains or in both SH3 domains inhibited EGF-induced formation of coated pits efficiently, even though Grb2 SH3 mutations did not block activation of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K). Our data demonstrate that EGFR-induced signaling and Grb2 are essential for formation of clathrin-coated pits accommodating the EGFR, while activation of MAPK and PI3K is not required.

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Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1575-1581.1994 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1575-1581

Author(s):

G J Pronk ◽

A M de Vries-Smits ◽

L Buday ◽

J Downward ◽

J A Maassen ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Insulin Receptor ◽

Tyrosine Phosphorylation ◽

Egf Receptor ◽

Growth Factor Receptor ◽

Nucleotide Exchange ◽

Similar Time ◽

Epidermal Growth

Shc proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have studied the role of Shc in insulin- and EGF-induced activation of p21ras in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21ras. Analysis of Shc immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in Shc tyrosine phosphorylation and (ii) Shc antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of Shc and the presence of Grb2 and mSOS in Shc immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, Shc could be detected as well. Furthermore, Shc immune complexes contained guanine nucleotide exchange activity toward p21ras in vitro. From these results, we conclude that after insulin and EGF treatment, Shc associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21ras. In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with Shc was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor. These results indicate that the association of Grb2-nSOS with Shc may be an important event in insulin-induced, mSOS-mediated activation of p21ras.

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