HTS-compatible FRET-based conformational sensors clarify membrane receptor activation

Area of Science:

• Biochemistry and Molecular Biology
• Cell Biology
• Pharmacology

Background:

• Cell surface receptors are crucial drug targets, necessitating tools to study their function in live cells.
• Förster Resonance Energy Transfer (FRET) is a promising technique for monitoring receptor conformational changes but is limited by low signal-to-noise ratios.

Purpose of the Study:

• To develop an improved toolbox for creating highly sensitive biosensors for cell surface receptors.
• To overcome the limitations of traditional FRET methods for studying receptor dynamics.

Main Methods:

• A novel toolbox combining advanced labeling technologies and specific fluorophores compatible with time-resolved FRET was developed.
• A new signal quantification method was implemented to enhance data analysis.
• Biosensors were created for various G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), including mGlu, GABA_B, LH, PTH, EGF, and insulin receptors.

Main Results:

• The developed toolbox enables the creation of receptor biosensors with significantly improved signal-to-noise ratios.
• Successful development and application of biosensors for multiple important receptor families.
• Demonstrated utility for high-throughput screening and uncovering novel aspects of receptor activation.

Conclusions:

• The enhanced FRET-based toolbox provides a robust platform for developing sensitive biosensors for cell surface receptors.
• These biosensors are valuable tools for pharmacological studies, high-throughput screening, and fundamental research into receptor mechanisms.