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Cell-Based Sensors for the Detection of EGF and EGF-Stimulated Ca2+ Signaling
Euiyeon Lee1,2, Keshab Lal Shrestha1, Seonhye Kang1
1Department of Biomedical Engineering, Dongguk University, Seoul 04620, Republic of Korea.
Abstract:
Epidermal growth factor (EGF)-mediated activation of EGF receptors (EGFRs) has become an important target in drug development due to the implication of EGFR-mediated cellular signaling in cancer development. While various in vitro approaches are developed for monitoring EGF-EGFR interactions, they have several limitations. Herein, we describe a live cell-based sensor system that can be used to monitor the interaction of EGF and EGFR as well as the subsequent signaling events. The design of the EGF-detecting sensor cells is based on the split-intein-mediated conditional protein trans-cleavage reaction (CPC). CPC is triggered by the presence of the target (EGF) to activate a signal peptide that translocates the fluorescent cargo to the target cellular location (mitochondria). The developed sensor cell demonstrated excellent sensitivity with a fast response time. It was also successfully used to detect an agonist and antagonist of EGFR (transforming growth factor-α and Cetuximab, respectively), demonstrating excellent specificity and capability of screening the analytes based on their function. The usage of sensor cells was then expanded from merely detecting the presence of target to monitoring the target-mediated signaling cascade, by exploiting previously developed Ca2+-detecting sensor cells. These sensor cells provide a useful platform for monitoring EGF-EGFR interaction, for screening EGFR effectors, and for studying downstream cellular signaling cascades.
Insights
A novel live cell-based sensor system monitors epidermal growth factor (EGF) and EGF receptor (EGFR) interactions and signaling. This system offers a sensitive and specific platform for drug development and cancer research.
Area of Science:
- Biotechnology
- Molecular Biology
- Cellular Signaling
Background:
- Epidermal growth factor (EGF) receptor (EGFR) signaling is crucial in cancer development, making it a key drug development target.
- Existing in vitro methods for monitoring EGF-EGFR interactions have limitations.
- Live cell-based assays are needed to effectively track these interactions and downstream signaling.
Purpose of the Study:
- To develop a live cell-based sensor system for monitoring EGF-EGFR interactions.
- To assess the system's sensitivity, specificity, and response time.
- To demonstrate its utility in screening EGFR modulators and studying signaling cascades.
Main Methods:
- Engineered sensor cells utilizing split-intein-mediated conditional protein trans-cleavage (CPC).
- CPC activation by EGF triggers signal peptide translocation of fluorescent cargo to mitochondria.
- Integration with calcium (Ca2+) detecting sensor cells to monitor signaling cascades.
Main Results:
- The developed sensor cells exhibited high sensitivity and rapid response times.
- Successfully detected EGFR agonist (transforming growth factor-α) and antagonist (Cetuximab), confirming specificity.
- Demonstrated capability for functional screening of EGFR modulators and monitoring downstream signaling.
Conclusions:
- The live cell-based sensor system provides a robust platform for monitoring EGF-EGFR interactions.
- It is effective for screening EGFR-targeting drugs and investigating cellular signaling pathways.
- This technology advances research in cancer biology and therapeutic development.
