G Protein-Coupled Receptor Signaling Analysis Using Homogenous Time-Resolved Förster Resonance Energy Transfer (HTRF®) Technology

Autor: Lenea Nørskov-Lauritsen, Alex Rojas Bie Thomsen, Hans Bräuner-Osborne
Jazyk: angličtina
Rok vydání: 2014
Předmět:
Zdroj: International Journal of Molecular Sciences, Vol 15, Iss 2, Pp 2554-2572 (2014)
Druh dokumentu: article
ISSN: 1422-0067
DOI: 10.3390/ijms15022554
Popis: Studying multidimensional signaling of G protein-coupled receptors (GPCRs) in search of new and better treatments requires flexible, reliable and sensitive assays in high throughput screening (HTS) formats. Today, more than half of the detection techniques used in HTS are based on fluorescence, because of the high sensitivity and rich signal, but quenching, optical interferences and light scattering are serious drawbacks. In the 1990s the HTRF® (Cisbio Bioassays, Codolet, France) technology based on Förster resonance energy transfer (FRET) in a time-resolved homogeneous format was developed. This improved technology diminished the traditional drawbacks. The optimized protocol described here based on HTRF® technology was used to study the activation and signaling pathways of the calcium-sensing receptor, CaSR, a GPCR responsible for maintaining calcium homeostasis. Stimulation of the CaSR by agonists activated several pathways, which were detected by measuring accumulation of the second messengers D-myo-inositol 1-phosphate (IP1) and cyclic adenosine 3',5'-monophosphate (cAMP), and by measuring the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Here we show how an optimized HTRF® platform with numerous advantages compared to previous assays provides a substantial and robust mode of investigating GPCR signaling. It is furthermore discussed how these assays can be optimized and miniaturized to meet HTS requirements and for screening compound libraries.
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