Location of the epidermal growth factor binding site on the EGF receptor. A resonance energy transfer study

Biochemistry ◽  
1990 ◽  
Vol 29 (37) ◽  
pp. 8741-8747 ◽  
Author(s):  
Kermit L. Carraway ◽  
John G. Koland ◽  
Richard A. Cerione
Keyword(s):  
Energy Transfer ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Site ◽  
Egf Receptor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Factor Binding Site ◽  
Epidermal Growth ◽  
Transfer Study

scholarly journals Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation.

1995 ◽  
Vol 129 (6) ◽  
pp. 1543-1558 ◽  
Author(s):  
T W Gadella ◽  
T M Jovin
Keyword(s):  
Energy Transfer ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
A431 Cells ◽  
High Affinity ◽  
Fret Efficiency ◽  
Epidermal Growth

The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound to the cells and the extent of oligomerization was monitored by the spatially resolved FRET efficiency as a function of the donor/acceptor ratio and treatment conditions. An average FRET efficiency of 5% was determined after a low temperature (4 degrees C) incubation with the fluorescent EGF analogs for 40 min. A subsequent elevation of the temperature for 5 min caused a substantial increase of the average FRET efficiency to 14% at 20 degrees C and 31% at 37 degrees C. In the context of a two-state (monomer/dimer) model for the EGFR, these FRET efficiencies were consistent with minimal average receptor dimerizations of 13, 36, and 69% at 4, 20, and 37 degrees C, respectively. A431 cells were pretreated with the monoclonal antibody mAb 2E9 that specifically blocks EGF binding to the predominant population of low affinity EGFR (15). The average FRET efficiency increased dramatically to 28% at 4 degrees C, indicative of a minimal receptor dimerization of 65% for the subpopulation of high affinity receptors. These results are in accordance with prior studies indicating that binding of EGF leads to a fast and temperature-dependent microclustering of EGFR, but suggest in addition that the high affinity functional subclass of receptors on quiescent A431 cells are present in a predimerized or oligomerized state. We propose that the transmission of the external ligand-binding signal to the cytoplasmic domain is effected by a concerted relative rotational rearrangement of the monomeric units comprising the dimeric receptor, thereby potentiating a mutual activation of the tyrosine kinase domains.

scholarly journals Epidermal Growth Factor Abrogates Hypoxia-Induced Apoptosis in Cultured Human Trophoblasts through Phosphorylation of BAD Serine 112

Endocrinology ◽  
2008 ◽  
Vol 149 (5) ◽  
pp. 2131-2137 ◽  
Author(s):  
Rachel G. Humphrey ◽  
Christina Sonnenberg-Hirche ◽  
Steven D. Smith ◽  
Chaobin Hu ◽  
Aaron Barton ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Fluorescence Resonance Energy Transfer ◽  
P38 Mapk ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Epidermal Growth ◽  
Fluorescence Resonance ◽  
Induced Apoptosis

We tested the hypothesis that epidermal growth factor (EGF) limits hypoxia-induced apoptosis in cultured human trophoblasts by phosphorylation of the proapoptotic protein Bcl-2-associated death promoter (BAD). Cytotrophoblasts were isolated from placentas of uncomplicated pregnancies at 38–40 wk gestation. Primary trophoblasts or transfected JEG3 trophoblast cells were cultured in less than 1 or 20% oxygen in the presence or absence of EGF and signaling pathway inhibitors. BAD, green fluorescent protein (GFP)-BAD, 14-3-3, Bcl-XL, and neoepitopes formed during apoptotic cleavage of cytokeratin 18 intermediate filaments were quantified using immunoblotting. Cultures immunostained by fluorescent antibodies were analyzed by confocal microscopy for BAD and GFP. Fluorescence resonance energy transfer was used to detect molecular interaction between endogenous BAD and GFP-BAD. We found EGF increased the phosphorylation of BADser112 under standard culture conditions. Whereas hypoxia enhanced apoptosis and increased phosphorylation of both BADser136 and BADser155, hypoxia diminished phosphorylation of BADser112, and this effect was reversible by EGF. Transfected GFP-BAD, which directly interacted with endogenous BAD by colocalization and fluorescence resonance energy transfer, enhanced hypoxia-induced apoptosis in JEG3 cells. EGF reduced apoptosis in hypoxic JEG3 cells that overexpressed GFP-BAD but not in cells overexpressing GFP-BAD that harbored a serine-to-alanine mutation at the 112 site. Coimmunoprecipitation studies showed that EGF reduced the proapoptotic interaction of BAD with Bcl-XL. The effect of EGF on phosphorylation of BADser112 was dependent on the action of p38 MAPK. We conclude that EGF signals via p38 MAPK to increase phosphorylation of BADser112 and thereby limit trophoblast apoptosis.

scholarly journals Regulation of EGFR Endocytosis by CBL During Mitosis

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 257 ◽  
Author(s):  
Ping Wee ◽  
Zhixiang Wang
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Signaling ◽  
Binding Site ◽  
Egf Receptor ◽  
Dominant Negative ◽  
Low Doses ◽  
Interphase Cells ◽  
Epidermal Growth ◽  
Mcf 7

The overactivation of epidermal growth factor (EGF) receptor (EGFR) is implicated in various cancers. Endocytosis plays an important role in EGFR-mediated cell signaling. We previously found that EGFR endocytosis during mitosis is mediated differently from interphase. While the regulation of EGFR endocytosis in interphase is well understood, little is known regarding the regulation of EGFR endocytosis during mitosis. Here, we found that contrary to interphase cells, mitotic EGFR endocytosis is more reliant on the activation of the E3 ligase CBL. By transfecting HeLa, MCF-7, and 293T cells with CBL siRNA or dominant-negative 70z-CBL, we found that at high EGF doses, CBL is required for EGFR endocytosis in mitotic cells, but not in interphase cells. In addition, the endocytosis of mutant EGFR Y1045F-YFP (mutation at the direct CBL binding site) is strongly delayed. The endocytosis of truncated EGFR Δ1044-YFP that does not bind to CBL is completely inhibited in mitosis. Moreover, EGF induces stronger ubiquitination of mitotic EGFR than interphase EGFR, and mitotic EGFR is trafficked to lysosomes for degradation. Furthermore, we showed that, different from interphase, low doses of EGF still stimulate EGFR endocytosis by non-clathrin mediated endocytosis (NCE) in mitosis. Contrary to interphase, CBL and the CBL-binding regions of EGFR are required for mitotic EGFR endocytosis at low doses. This is due to the mitotic ubiquitination of the EGFR even at low EGF doses. We conclude that mitotic EGFR endocytosis exclusively proceeds through CBL-mediated NCE.

scholarly journals The Membrane-anchoring Domain of Epidermal Growth Factor Receptor Ligands Dictates Their Ability to Operate in Juxtacrine Mode

2005 ◽  
Vol 16 (6) ◽  
pp. 2984-2998 ◽  
Author(s):  
Jianying Dong ◽  
Lee K. Opresko ◽  
William Chrisler ◽  
Galya Orr ◽  
Ryan D. Quesenberry ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Resonance Energy ◽  
Structural Basis ◽  
Cell Contact ◽  
Heparin Binding ◽  
Epidermal Growth ◽  
Membrane Anchoring

All ligands of the epidermal growth factor (EGF) receptor (EGFR) are synthesized as membrane-anchored precursors. Previous work has suggested that some ligands, such as EGF, must be proteolytically released to be active, whereas others, such as heparin-binding EGF-like growth factor (HB-EGF) can function while still anchored to the membrane (i.e., juxtacrine signaling). To explore the structural basis for these differences in ligand activity, we engineered a series of membrane-anchored ligands in which the core, receptor-binding domain of EGF was combined with different domains of both EGF and HB-EGF. We found that ligands having the N-terminal extension of EGF could not bind to the EGFR, even when released from the membrane. Ligands lacking an N-terminal extension, but possessing the membrane-anchoring domain of EGF, still required proteolytic release for activity, whereas ligands with the membrane-anchoring domain of HB-EGF could elicit full biological activity while still membrane anchored. Ligands containing the HB-EGF membrane anchor, but lacking an N-terminal extension, activated EGFR during their transit through the Golgi apparatus. However, cell-mixing experiments and fluorescence resonance energy transfer studies showed that juxtacrine signaling typically occurred in trans at the cell surface, at points of cell-cell contact. Our data suggest that the membrane-anchoring domain of ligands selectively controls their ability to participate in juxtacrine signaling and thus, only a subclass of EGFR ligands can act in a juxtacrine mode.

scholarly journals Coactivation of Rac1 and Cdc42 at Lamellipodia and Membrane Ruffles Induced by Epidermal Growth Factor

2004 ◽  
Vol 15 (3) ◽  
pp. 1003-1010 ◽  
Author(s):  
Kazuo Kurokawa ◽  
Reina E. Itoh ◽  
Hisayoshi Yoshizaki ◽  
Yusuke Ohba Takeshi Nakamura ◽  
Michiyuki Matsuda
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Morphological Changes ◽  
Critical Role ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
A431 Cells ◽  
Major Function ◽  
Membrane Ruffling ◽  
Epidermal Growth

A major function of Rho-family GTPases is to regulate the organization of the actin cytoskeleton; filopodia, lamellipodia, and stress fiber are regarded as typical phenotypes of the activated Cdc42, Rac, and Rho, respectively. Using probes based on fluorescent resonance energy transfer, we report on the spatiotemporal regulation of Rac1 and Cdc42 at lamellipodia and membrane ruffles. In epidermal growth factor (EGF)-stimulated Cos1 and A431 cells, both Rac1 and Cdc42 were activated diffusely at the plasma membrane, followed by lamellipodial protrusion and membrane ruffling. Although Rac1 activity subsided rapidly, Cdc42 activity was sustained at lamellipodia. A critical role of Cdc42 in these EGF-induced morphological changes was demonstrated as follows. First, phorbol 12-myristate 13-acetate, which activated Rac1 but not Cdc42, could not induce full-grown lamellipodia in Cos1 cells. Second, a GTPase-activating protein for Cdc42, KIAA1204/CdGAP, inhibited lamellipodial protrusion and membrane ruffling without interfering with Rac1 activation. Third, expression of the Cdc42-binding domain of N-WASP inhibited the EGF-induced morphological changes. Therefore, Rac1 and Cdc42 seem to synergistically induce lamellipodia and membrane ruffles in EGF-stimulated Cos1 cells and A431 cells.

scholarly journals Inhibition of tyrosine kinase activity of the epidermal growth factor (EGF) receptor by a truncated receptor form that binds to EGF: role for interreceptor interaction in kinase regulation.

1989 ◽  
Vol 9 (2) ◽  
pp. 671-677 ◽  
Author(s):  
A Basu ◽  
M Raghunath ◽  
S Bishayee ◽  
M Das
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Binding Site ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Kinase Domain ◽  
Tyrosine Kinase Activity ◽  
Truncated Receptor ◽  
Epidermal Growth

The tyrosine kinase activity of the epidermal growth factor (EGF) receptor is regulated by a truncated receptor of 100 kilodaltons (kDa) that contains the EGF-binding site but not the kinase domain. The inhibition of kinase is not due to competition for available EGF or for the kinase substrate-binding site. Chemical cross-linking studies suggest that the 100-kDa receptor may form a heterodimer with the intact EGF receptor. Structurally related receptor kinases, such as the platelet-derived growth factor receptor, the insulin receptor, and the Neu receptor, were not inhibited by the 100-kDa receptor. The results indicate that (i) the inhibition was specific for the EGF receptor, (ii) the kinase domain had little or no role in determining target specificity, and (iii) the regulation of kinase may be due to a specific interaction of the 100-kDa receptor with the ligand-binding domain of the EGF receptor kinase.

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