HTS-compatible FRET-based conformational sensors clarify membrane receptor activation.
Pauline SchollerDavid Moreno-DelgadoNathalie Lecat-GuilletEtienne DoumazaneCarine MonnierFabienne Charrier-SavourninLudovic FabreCédric ChouvetStéphanie SoldevilaLaurent LamarqueGeoffrey DonsimoniThomas RouxJurriaan M ZwierEric TrinquetPhilippe RondardJean-Philippe PinPublished in: Nature chemical biology (2017)
Cell surface receptors represent a vast majority of drug targets. Efforts have been conducted to develop biosensors reporting their conformational changes in live cells for pharmacological and functional studies. Although Förster resonance energy transfer (FRET) appears to be an ideal approach, its use is limited by the low signal-to-noise ratio. Here we report a toolbox composed of a combination of labeling technologies, specific fluorophores compatible with time-resolved FRET and a novel method to quantify signals. This approach enables the development of receptor biosensors with a large signal-to-noise ratio. We illustrate the usefulness of this toolbox through the development of biosensors for various G-protein-coupled receptors and receptor tyrosine kinases. These receptors include mGlu, GABAB, LH, PTH, EGF and insulin receptors among others. These biosensors can be used for high-throughput studies and also revealed new information on the activation process of these receptors in their cellular environment.
Keyphrases
- energy transfer
- quantum dots
- single molecule
- high throughput
- cell surface
- air pollution
- molecular dynamics simulations
- single cell
- molecular dynamics
- induced apoptosis
- emergency department
- metabolic syndrome
- living cells
- label free
- oxidative stress
- adipose tissue
- cell cycle arrest
- case control
- signaling pathway
- insulin resistance
- glycemic control