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Proximity biotinylation of A431 cells by EGFR-FabID

ABSTRACT: A431 cells were pretreated with DMSO or gefitinib and unstimulated or stimulated with EGF. Then AGIA-FabID or EGFR-FabID were added to the cells in three biological replicates. Cell lysates were subjected to methanol-chloroform precipitation and digested with trypsin. Biotinylated peptides were purified by Tamavidin 2-REV and identified by LC-MS/MS analysis.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD039450 | JPOST Repository | Tue Nov 14 00:00:00 GMT 2023

REPOSITORIES: jPOST

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Proximity extracellular protein-protein interaction analysis of EGFR using AirID-conjugated fragment of antigen binding.

Yamada Kohdai K Shioya Ryouhei R Nishino Kohei K Furihata Hirotake H Hijikata Atsushi A Kaneko Mika K MK Kato Yukinari Y Shirai Tsuyoshi T Kosako Hidetaka H Sawasaki Tatsuya T

Nature communications 20231214 1

Receptor proteins, such as epidermal growth factor receptor (EGFR), interact with other proteins in the extracellular region of the cell membrane to drive intracellular signalling. Therefore, analysis of extracellular protein-protein interactions (exPPIs) is important for understanding the biological function of receptor proteins. Here, we present an approach using a proximity biotinylation enzyme (AirID) fusion fragment of antigen binding (FabID) to analyse the proximity exPPIs of EGFR. AirID w ...[more]

PMID: 38097606

Similar Datasets

Proximity biotinylation of NCI-H226 cells by EGFR-FabID

Project description:NCI-H226 cells were pretreated with DMSO or gefitinib and unstimulated or stimulated with EGF. Then AGIA-FabID or EGFR-FabID were added to the cells in three biological replicates. Cell lysates were subjected to methanol-chloroform precipitation and digested with trypsin. Biotinylated peptides were purified by Tamavidin 2-REV and identified by LC-MS/MS analysis.

2023-11-14 | PXD039451 | JPOST Repository

Proximity biotinylation of A431 cells with gefitinib alone treatment by EGFR-FabID

Project description:A431cells were pretreated with DMSO or gefitinib. Then AGIA-FabID or EGFR-FabID were added to the cells in three biological replicates. Cell lysates were subjected to methanol-chloroform precipitation and digested with trypsin. Biotinylated peptides were purified by Tamavidin 2-REV and identified by LC-MS/MS analysis.

2023-11-14 | PXD043526 | JPOST Repository

Proximity biotinylation of NCI-H226 cells with gefitinib alone treatment by EGFR-FabID

Project description:NCI-H226 cells were pretreated with DMSO or gefitinib. Then AGIA-FabID or EGFR-FabID were added to the cells in three biological replicates. Cell lysates were subjected to methanol-chloroform precipitation and digested with trypsin. Biotinylated peptides were purified by Tamavidin 2-REV and identified by LC-MS/MS analysis.

2023-11-14 | PXD043527 | JPOST Repository

Proximity biotinylation of Expi293F cells overexpressing EGFR by EGFR-FabID

Project description:Expi293F cells were transfected with pcDNA3.1-EGFR and treated with AGIA-FabID or EGFR-FabID in three biological replicates. Cell lysates were subjected to methanol-chloroform precipitation and digested with trypsin. Biotinylated peptides were purified by Tamavidin 2-REV and identified by LC-MS/MS analysis.

2023-11-14 | PXD039449 | JPOST Repository

Proximity biotinylation of Expi293F cells stably expressing EGFR by EGFR-FabID

Project description:Expi293F cells stably expressing EGFR were treated with AGIA-FabID or EGFR-FabID in three biological replicates. Cell lysates were subjected to methanol-chloroform precipitation and digested with trypsin. Biotinylated peptides were purified by Tamavidin 2-REV and identified by LC-MS/MS analysis.

2023-11-14 | PXD043525 | JPOST Repository

Transcription profiling of Human Endometrial Cancer Cells treated with EGF or gefitinib to investigate gene regulation of EGFR Isoforms.

Project description:Microarrays were used to analyze differential gene expression and to help determine the efficacy of Iressa (gefitinib), a tyrosine kinase inhibitor, on endometrial cancer cells. Type I Ishikawa H and type II Hec50co endometrial carcinoma cells both express EGFR and sEGFR, but differ markedly in their responsiveness to the EGFR inhibitor gefitinib. This difference is paralleled by differences in the expression of sEGFR and EGFR, as well as in their transcriptional response following treatment with either EGFor gefitinib. The small cluster of differently regulated genes reported here in these type I vs. type II endometrial cancer-derived cell lines may identify candidate biomarkers useful for predicting sensitivity to EGFR blockade. Type I (Ishikawa H cells) and type II (Hec50co) derived endometrial carcinomas, were dosed with either EGF(epidermal growth factor) or Iressa (gefitinib) for 12 or 24 hours and gene expression was examined.

2010-03-23 | E-GEOD-20854 | biostudies-arrayexpress

The target genes of EGFR activity in glioma cells

Project description:As a first step towards identifying the target genes of EGFR activity in glioma cells, genome-wide expression analyses were performed using the Affymetrix GeneChip Human Genome U133A array. To accomplish this, mRNA expression levels of these genes were measured in the glioblastoma cell lines, U87 and U178, engineered with EGFR by retrovirus transduction (termed U87-EGFR and U178-EGFR respectively), with or without 20 ng/mL EGF treatment for 3 h. U87 and U178 cells engineered to express EGFR were stimulated with or without EGF. The experiment was replicated twice for each U87 and U178 cells.

2011-11-05 | E-GEOD-33442 | biostudies-arrayexpress

Effect of EGFR activation on mouse chondrocytes

Project description:We examined the change of chondrocyte transcriptome after EGFR activation by TGF-α and inhibition by gefitinib. Data analysis showed that EGFR activation down-regulates cartilage ECM biosynthesis and promotes ECM degradation. Gefitinib can reverse the effects of EGFR on chondrocytes.

2019-04-24 | GSE113271 | GEO

Flotillin-2 regulates EGFR activation, degradation, and cancer growth

Project description:Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in a variety of cancers. The ubiquitin ligase Cbl regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. We used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes in the absence and presence of EGF stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by the EGFR inhibitor erlotinib. CRISPR knockout of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and trafficking to the early endosome. FLOT2 interacts with both Cbl and EGFR. Downregulation of EGFR upon FLOT2 loss is Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Stable overexpression of FLOT2 in HeLa cells decreased EGF-stimulated EGFR phosphorylation and ubiquitination. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induces EGFR-dependent proliferation and anchorage-independent cell growth. Lastly, FLOT2 knockout increases tumor formation and tumor volume in nude mice and NSG mice, respectively. These data demonstrate that FLOT2 negatively regulates EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation. FLOT2 negatively regulates proliferation in vitro and in vivo.

2023-03-11 | PXD031988 | Pride

Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR

Project description:Ligand binding to the EGF receptor (EGFR) initiates signal transduction and endocytosis of the receptor. The mechanisms of endocytosis and regulation of signaling by endocytosis are poorly understood. Here, we combined peroxidase-catalyzed proximity labeling, isobaric peptide tagging and quantitative mass-spectrometry to define the dynamics of the proximity proteome of EGFR in response to ligand activation. Using this approach, we identified a network of signaling proteins, which remain associated with the receptor during its endocytosis and trafficking through the endosomal system. We showed that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, was enriched early/sorting endosome proximity proteome of EGFR and localized in early an sorting endosomes, and demonstrated that TFG regulates endosomal sorting of EGFR. This study provides a large-scale resource of time-dependent nanoscale environment of activated EGFR, thus opening avenues to discovering new regulatory mechanisms of membrane trafficking of receptor tyrosine kinases. EGFR-APEX2 Experiment Datasets: TMT 1-3: HEK293T cells (0, 1, 10, 30 and 60 minutes EGF treatment) TMT 4: HCT116 cells (0, 2, 4, 6 , 8 and 10 minutes EGF treatment) TMT 5: HEK293T cells (0, 2, 4, 6 , 8 and 10 minutes EGF treatment) TMT 6: HEK293T cells (0 and 10 minutes EGF treatment) TMT 7: HCT116 cells (0 and 10 minutes EGF treatment)

2022-06-04 | PXD030072 | Pride

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