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Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay
Published on: October 20, 2018
Visualization and quantitation of epidermal growth factor receptor homodimerization and activation with a proximity
Keiichi Ota1, Taishi Harada1,2, Kohei Otsubo1
1Research Institute for Diseases of The Chest, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan.
Objectives:
Activation of the epidermal growth factor receptor (EGFR) results from receptor homodimerization and autophosphorylation and confers sensitivity to tyrosine kinase inhibitors in some tumors. However, the visual detection and quantitation of activated EGFR in the clinical setting has not been established.
Materials And Methods:
A proximity ligation assay (PLA) was applied to detect EGFR homodimers in non-small cell lung cancer (NSCLC) cell lines and tissue specimens.
Results:
PLA signals corresponding to EGFR homodimers were higher in NSCLC cell lines and tissue specimens positive for activating EGFR mutations than in those wild type (WT) for EGFR. Stimulation with EGF in NSCLC cells WT for EGFR or forced overexpression of EGFR in Ba/F3 cells resulted in marked EGFR homodimerization. The extent of EGFR homodimerization appeared related to that of EGFR autophosphorylation in NSCLC cells WT for EGFR.
Conclusion:
PLA may provide a new tool for detection and quantitation of EGFR homodimers in NSCLC and other tumors.
Insights
A new proximity ligation assay (PLA) can detect epidermal growth factor receptor (EGFR) homodimers in non-small cell lung cancer (NSCLC). This method shows promise for quantifying activated EGFR in tumors.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Epidermal growth factor receptor (EGFR) activation involves homodimerization and autophosphorylation.
- EGFR activation is crucial for tyrosine kinase inhibitor sensitivity in certain cancers.
- Clinical methods for visualizing and quantifying activated EGFR are lacking.
Purpose of the Study:
- To establish a method for detecting and quantifying EGFR homodimers in clinical samples.
- To assess the utility of proximity ligation assay (PLA) for visualizing activated EGFR.
Main Methods:
- Proximity ligation assay (PLA) was employed to detect EGFR homodimers.
- Assays were performed on non-small cell lung cancer (NSCLC) cell lines and patient tissue specimens.
Main Results:
- PLA signals for EGFR homodimers were elevated in NSCLC with activating EGFR mutations compared to wild-type.
- EGF stimulation and EGFR overexpression induced significant EGFR homodimerization.
- EGFR homodimerization correlated with EGFR autophosphorylation in wild-type NSCLC cells.
Conclusions:
- Proximity ligation assay (PLA) offers a novel approach for detecting and quantifying EGFR homodimers.
- PLA has potential applications in NSCLC and other malignancies involving EGFR activation.
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