N-Glycosylation as determinant of epidermal growth factor receptor conformation in membranes

The epidermal growth factor receptor (EGFR) regulates several critical cellular processes and is an important target for cancer therapy. In lieu of a crystallographic structure of the complete receptor, atomistic molecular dynamics (MD) simulations have recently shown that they can excel in studies of the full-length receptor. Here we present atomistic MD simulations of the monomeric N-glycosylated human EGFR in biomimetic lipid bilayers that are, in parallel, also used for the reconstitution of full-length receptors. This combination enabled us to experimentally validate our simulations, using ligand binding assays and antibodies to monitor the conformational properties of the receptor reconstituted into membranes. We find that N-glycosylation is a critical determinant of EGFR conformation, and specifically the orientation of the EGFR ectodomain relative to the membrane. In the absence of a structure for full-length, posttranslationally modified membrane receptors, our approach offers new means to structurally define and experimentally validate functional properties of cell surface receptors in biomimetic membrane environments.

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Conformational landscape of the epidermal growth factor receptor kinase reveals a mutant specific allosteric pocket

Chemical Science ◽

10.1039/c8sc01262h ◽

2018 ◽

Vol 9 (23) ◽

pp. 5212-5222 ◽

Cited By ~ 16

Author(s):

Srinivasaraghavan Kannan ◽

Gireedhar Venkatachalam ◽

Hong Hwa Lim ◽

Uttam Surana ◽

Chandra Verma

Keyword(s):

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Growth Factor Receptor ◽

Md Simulations ◽

Receptor Kinase ◽

Epidermal Growth ◽

Conformational Landscape

An oncogenic mutant-specific druggable allosteric pocket captured by MD simulations.

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Epidermal Growth Factor Receptor and c-erbB-2 Contents in Unresectable (UICC R1 or R2) Gastric Cancer

The International Journal of Biological Markers ◽

10.1177/172460080301800308 ◽

2003 ◽

Vol 18 (3) ◽

pp. 200-206 ◽

Cited By ~ 7

Author(s):

I. García ◽

J.M. Del Casar ◽

M.D. Corte ◽

M.T. Allende ◽

J.L. García-Muñiz ◽

...

Keyword(s):

Gastric Cancer ◽

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Radioligand Binding ◽

Prognostic Significance ◽

Growth Factor Receptor ◽

Membrane Receptors ◽

Gastric Carcinomas ◽

Epidermal Growth

Background Epidermal growth factor receptor (EGFR) and c-erbB-2 are membrane receptors expressed in a variety of solid human cancers and directly correlated with poor prognosis. The objective of this work was to evaluate the EGFR and c-erbB-2 levels in non-resectable gastric carcinomas, their possible relationship with a variety of clinicopathological tumor parameters, and their prognostic significance. Methods This was a prospective analysis of 65 patients with unresectable gastric carcinomas (UICC R1 or R2), who underwent palliative surgery and were followed up for a median period of 13 months. Membranous EGFR levels were examined by radioligand binding assays and cytosolic c-erbB-2 levels by means of an immunoenzymatic assay. Results There was a wide variability in EGFR (80.3-2910 fmol/mg of protein) and c-erbB-2 (0.4-10071 NHU/mg of protein) levels in neoplastic tissues from patients with unresectable gastric carcinomas. Median c-erbB2 was significantly higher in tumors of the intestinal type than in tumors of the diffuse type (p=0.035) and in R2 than in R1 tumors (p=0.016). Statistical analysis showed that there was no relationship between tumor c-erbB-2 or EGFR content and any other patient or tumor characteristics. However, high levels of EGFR were significantly associated with a shorter overall survival (p=0.01). Conclusion Our data suggest a role of both transmembrane proteins in the progression of gastric cancer. EGFR and c-erbB-2 contents in unresectable gastric cancer could be utilized as appropriate biological markers for selecting candidates for treatment based on EGFR and/or c-erbB-2 inhibition.

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Construction of Epidermal Growth Factor Receptor Peptide Magnetic Nanovesicles with Lipid Bilayers for Enhanced Capture of Liver Cancer Circulating Tumor Cells

Analytical Chemistry ◽

10.1021/acs.analchem.6b01443 ◽

2016 ◽

Vol 88 (18) ◽

pp. 8997-9003 ◽

Cited By ~ 12

Author(s):

Jian Ding ◽

Kai Wang ◽

Wen-Jie Tang ◽

Dan Li ◽

You-Zhen Wei ◽

...

Keyword(s):

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Liver Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Lipid Bilayers ◽

Growth Factor Receptor ◽

Epidermal Growth

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Analysis of the Role of the C-Terminal Tail in the Regulation of the Epidermal Growth Factor Receptor

Molecular and Cellular Biology ◽

10.1128/mcb.00248-15 ◽

2015 ◽

Vol 35 (17) ◽

pp. 3083-3102 ◽

Cited By ~ 42

Author(s):

Erika Kovacs ◽

Rahul Das ◽

Qi Wang ◽

Timothy S. Collier ◽

Aaron Cantor ◽

...

Keyword(s):

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Lipid Bilayers ◽

Growth Factor Receptor ◽

Kinase Domain ◽

Activation Loop ◽

Epidermal Growth ◽

Peculiar Phenomenon

The ∼230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and found that the first ∼80 residues of the tail are inhibitory, as demonstrated previously. The entire ∼80-residue span is important for autoinhibition and needs to be released from both kinases that form the dimer. These results are interpreted in terms of crystal structures of the inactive kinase domain, including two new ones presented here. Deletions in the remaining portion of the tail do not affect autophosphorylation, except for a six-residue segment spanning Tyr 1086 that is critical for activation loop phosphorylation. Phosphorylation of the two tails in the dimer is asymmetric, with the activator tail being phosphorylated somewhat more strongly. Unexpectedly, we found that reconstitution of the transmembrane and cytoplasmic domains of EGFR in vesicles leads to a peculiar phenomenon in which kinase domains appear to be trapped between stacks of lipid bilayers. This artifactual trapping of kinases between membranes enhances an intrinsic functional asymmetry in the two tails in a dimer.

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Localization to the Cortical Cytoskeleton Is Necessary for Nf2/Merlin-Dependent Epidermal Growth Factor Receptor Silencing

Molecular and Cellular Biology ◽

10.1128/mcb.01139-07 ◽

2007 ◽

Vol 28 (4) ◽

pp. 1274-1284 ◽

Cited By ~ 64

Author(s):

Banumathi K. Cole ◽

Marcello Curto ◽

Annie W. Chan ◽

Andrea I. McClatchey

Keyword(s):

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Growth Factor Receptor ◽

Membrane Receptors ◽

Cortical Actin ◽

Membrane Compartment ◽

Epidermal Growth ◽

Cortical Cytoskeleton ◽

Cell Cell

ABSTRACT Merlin, the product of the NF2 tumor suppressor gene, is closely related to the ERM (ezrin, radixin, moesin) proteins, which provide anchorage between membrane proteins and the underlying cortical cytoskeleton; all four proteins are members of the band 4.1 superfamily. Despite their similarity, the subcellular distributions and functional properties of merlin and the ERM proteins are largely distinct. Upon cell-cell contact merlin prevents internalization of and signaling from the epidermal growth factor receptor (EGFR) by sequestering it into an insoluble membrane compartment. Here we show that the extreme amino (N) terminus directs merlin biochemically to an insoluble membrane compartment and physically to the cortical actin network, with a marked concentration along cell-cell boundaries. This insoluble-membrane distribution is required for the growth-suppressing function of merlin and for the functional association of merlin with EGFR and other membrane receptors. Our data support a model whereby locally activated merlin sequesters membrane receptors such as EGFR at the cortical network, contributing to the long-held observation that the cortical actin cytoskeleton can control the lateral mobility of and signaling from certain membrane receptors.

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A Premature Termination of Human Epidermal Growth Factor Receptor Transcription inEscherichia coli

The Scientific World JOURNAL ◽

10.1155/2014/830923 ◽

2014 ◽

Vol 2014 ◽

pp. 1-6 ◽

Cited By ~ 1

Author(s):

Jihene Elloumi-Mseddi ◽

Karim Jellali ◽

Antonio Villalobo ◽

Sami Aifa

Keyword(s):

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Western Blot ◽

Blot Analysis ◽

Growth Factor Receptor ◽

Full Length ◽

Glutathione S Transferase ◽

Extracellular Region ◽

Epidermal Growth

Our success in producing an active epidermal growth factor receptor (EGFR) tyrosine kinase inEscherichia coliencouraged us to express the full-length receptor in the same host. Despite its large size, we were successful at producing the full-length EGFR protein fused to glutathione S-transferase (GST) that was detected by Western blot analysis. Moreover, we obtained a majoritarian truncated GST-EGFR form detectable by gel electrophoresis and Western blot. This truncated protein was purified and confirmed by MALDI-TOF/TOF analysis to belong to the N-terminal extracellular region of the EGFR fused to GST. Northern blot analysis showed two transcripts suggesting the occurrence of a transcriptional arrest.

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