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

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
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.

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

2015 ◽  
Vol 112 (14) ◽  
pp. 4334-4339 ◽  
Author(s):  
Karol Kaszuba ◽  
Michał Grzybek ◽  
Adam Orłowski ◽  
Reinis Danne ◽  
Tomasz Róg ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Lipid Bilayers ◽  
Growth Factor Receptor ◽  
Md Simulations ◽  
Full Length ◽  
Membrane Receptors ◽  
Crystallographic Structure ◽  
Epidermal Growth

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.

scholarly journals Validation of a Phosphoprotein Array Assay for Characterization of Human Tyrosine Kinase Receptor Downstream Signaling in Breast Cancer

2009 ◽  
Vol 55 (7) ◽  
pp. 1327-1336 ◽  
Author(s):  
Fadila Chergui ◽  
Anne-Sophie Chrétien ◽  
Sanae Bouali ◽  
Carole Ramacci ◽  
Marie Rouyer ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Western Blot ◽  
Growth Factor Receptor ◽  
Antibody Detection ◽  
Tyrosine Kinase Receptor ◽  
Downstream Signaling ◽  
Epidermal Growth

Abstract Background: Human epidermal growth factor receptor (HER) downstream signaling kinases have important effects on tumor response to anti-HER monoclonal antibodies and tyrosine kinase inhibitors. We validated an assay that uses phosphoprotein arrays for measurement of HER downstream signaling functionality in breast carcinomas. Methods: Using the Bio-Plex® phosphoprotein array (BPA), we performed multiplex immunoanalysis to investigate the expression of phosphorylated epidermal growth factor receptor and phosphorylated HER downstream signaling proteins (phosphorylated protein kinase B, phosphorylated glycogen synthase kinase −3β, phosphorylated P70 ribosomal protein S6 kinase, and phosphorylated extracellular signal regulated kinase 42/44) in 49 frozen specimens of ductal infiltrating breast carcinoma taken at diagnosis. BPA was cross-validated with Western blot analysis. Sample size, homogenicity, tumor content, protein extraction, and monoclonal antibody detection were in accordance with optimized standard operating procedures. Results: Linear regression showed significant quantitative correlations between BPA and Western blot, with regression coefficient values of 0.71–0.87 (P < 0.001). BPA intra- and interassay CVs were <17% and 15%, respectively. Compared to limits of detection established by using the mean + 3SD of 10 blanks, large variations of phosphoprotein expression, up to several hundred-fold, were observed among the 49 tumor specimens. Conclusions: Our results validate the use of the multiplex phosphoprotein array assay in human clinical tumor specimens. Further prospective evaluation is warranted to investigate the use of HER downstream signaling phosphoproteins as predictive and/or surrogate markers for clinical response to anti-HER targeted therapy. .

Active and inactive conformations of the epidermal growth factor receptor

2004 ◽  
Vol 32 (5) ◽  
pp. 742-745 ◽  
Author(s):  
K.M. Ferguson
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Structural Studies ◽  
Her Family ◽  
Extracellular Region ◽  
Epidermal Growth

The members of the EGFR (epidermal growth factor receptor) family of RTKs (receptor tyrosine kinases), also known as the ErbB or HER family, have been implicated in many human cancers. Structural studies of the EGFR extracellular region (sEGFR) have led to the proposal of a novel mechanism for ligand-induced receptor dimerization. In this model EGF binding induces a dramatic conformational change in EGFR, exposing a dimerization site that is normally occluded in the inactivated conformation, and thus promoting the formation of an entirely receptor-mediated dimer. It is well established that antibodies against the extracellular region of EGFR that prevent ligand binding and/or receptor signalling can inhibit tumour growth in vivo. At least five such anti-EGFR antibodies are currently in clinical trials and one, C225/cetuximab (Erbitux™), was recently approved in the U.S. and Europe for use in advanced colorectal cancers. Recent structural studies of ErbB2 in complex with anti-ErbB2 antibodies (trastuzumab/Herceptin™ and pertuzumab/Omnitarg™) have provided significant insights into how these drugs function. There have been no such studies for similar EGFR-targeted drugs to date. The implications of this model for the possible mechanisms of antibody-mediated inhibition of EGFR are discussed.

scholarly journals Epidermal Growth Factor Receptor Dimerization and Activation Require Ligand-Induced Conformational Changes in the Dimer Interface

2005 ◽  
Vol 25 (17) ◽  
pp. 7734-7742 ◽  
Author(s):  
Jessica P. Dawson ◽  
Mitchell B. Berger ◽  
Chun-Chi Lin ◽  
Joseph Schlessinger ◽  
Mark A. Lemmon ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Conformational Changes ◽  
Growth Factor Receptor ◽  
Intramolecular Interactions ◽  
Receptor Dimerization ◽  
Dimer Interface ◽  
Extracellular Region ◽  
Epidermal Growth

ABSTRACT Structural studies have shown that ligand-induced epidermal growth factor receptor (EGFR) dimerization involves major domain rearrangements that expose a critical dimerization arm. However, simply exposing this arm is not sufficient for receptor dimerization, suggesting that additional ligand-induced dimer contacts are required. To map these contributions to the dimer interface, we individually mutated each contact suggested by crystallographic studies and analyzed the effects on receptor dimerization, activation, and ligand binding. We find that domain II contributes >90% of the driving energy for dimerization of the extracellular region, with domain IV adding little. Within domain II, the dimerization arm forms much of the dimer interface, as expected. However, a loop from the sixth disulfide-bonded module (immediately C-terminal to the dimerization arm) also makes a critical contribution. Specific ligand-induced conformational changes in domain II are required for this loop to contribute to receptor dimerization, and we identify a set of ligand-induced intramolecular interactions that appear to be important in driving these changes, effectively “buttressing” the dimer interface. Our data also suggest that similar conformational changes may determine the specificity of ErbB receptor homo- versus heterodimerization.

scholarly journals Tyrosine Phosphorylation of the Human Glutathione S-Transferase P1 by Epidermal Growth Factor Receptor

2009 ◽  
Vol 284 (25) ◽  
pp. 16979-16989 ◽  
Author(s):  
Tatsunori Okamura ◽  
Simendra Singh ◽  
John Buolamwini ◽  
Timothy Haystead ◽  
Henry Friedman ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Growth Factor Receptor ◽  
Glutathione S Transferase ◽  
Epidermal Growth

scholarly journals A truncated, secreted form of the epidermal growth factor receptor is encoded by an alternatively spliced transcript in normal rat tissue

1990 ◽  
Vol 10 (6) ◽  
pp. 2973-2982
Author(s):  
L A Petch ◽  
J Harris ◽  
V W Raymond ◽  
A Blasband ◽  
D C Lee ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Epithelial Cells ◽  
Blot Analysis ◽  
Growth Factor Receptor ◽  
Terminal Sequence ◽  
External Domain ◽  
Normal Rat ◽  
Epidermal Growth

Two independent cDNA clones corresponding to a 2.7-kilobase (kb) epidermal growth factor receptor (EGF-R) mRNA were isolated from a rat liver cDNA library. Sequence analysis revealed 100% homology in the external domain when compared with the full-length rat EGF-R nucleotide sequence and 80 to 90% similarity relative to the human EGF-R. However, the 3'-terminal sequence of these clones did not match EGF-R or any other known sequence(s) and was distinct from the 3' end of the 2.8-kb mRNA, which encodes a truncated EGF-R in A431 cells. The deduced amino acid sequence revealed an open reading frame which is homologous to the external domain of the EGF-R but which terminates prior to the transmembrane region. Southern blot analysis of rat genomic DNA indicated that the 3'-terminal sequence of this transcript is derived from the EGF-R gene. Analysis of a genomic clone containing the 3' end of the 2.7-kb transcript revealed that this sequence is present as a discrete exon in the mid-region of the receptor gene in proximity to the exon encoding the transmembrane domain. Introduction of an expression vector containing the truncated EGF-R cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 95-kilodalton protein which was detected in conditioned media, by using antisera directed against the EGF-R. A similarly sized protein was also detected in the media of WB cells, a continuous, nontransformed line of rat hepatic epithelial cells. Northern (RNA blot) analysis established that the truncated receptor is encoded by a 2.7-kb transcript found in normal rat liver. Furthermore, Northern analysis of rat poly(A)+ RNA showed that the 2.7-kb EGF-R transcript is expressed at differing levels in various fetal and adult tissues. These data indicate that alternative splicing of the EGF-R primary transcript yields a 2.7-kb mRNA which codes for a truncated form of the receptor. This receptor is secreted by rat hepatic epithelial cells in culture, which suggests that it may be secreted by normal rat cells or tissues and perhaps serve an as yet unknown physiological function.

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