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

Abstract

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.

Dopamine D2 and serotonin 5‐HT1A receptor interaction in the context of the effects of antipsychotics – in vitro studies

“…; Norskov‐Lauritsen et al . ). A phospho‐ERK assay that measured the level of phosphorylation of ERK1/2 was adopted in this study.…”

“…The TR‐FRET between the K‐anti‐cAMP and cAMP‐d2 is disrupted by cAMP in cell lysates, allowing detection of the second messenger (Norskov‐Lauritsen et al . ). In brief, in the case of cAMP detection, cells were seeded in 384‐well plates at a density of 1 × 10 4 cells/well in 5 μL of F‐12 medium supplemented with 4% FBS, 250 μM IBMX (a phosphodiesterase inhibitor responsible for cAMP accumulation by inhibiting its phosphodiesterase‐driven degradation to 5′‐AMP) and 1 μM forskolin.…”

“…The detection kit included a lanthanide cryptate-labeled anti-cAMP antibody (K-anti-cAMP) and d2 dyelabeled cAMP (cAMP-d2), which were used as tracers in a timeresolved fluorescence energy transfer (TR-FRET) detection system. The TR-FRET between the K-anti-cAMP and cAMP-d2 is disrupted by cAMP in cell lysates, allowing detection of the second messenger (Norskov-Lauritsen et al 2014). In brief, in the case of cAMP detection, cells were seeded in 384-well plates at a density of 1 9 10 4 cells/well in 5 lL of F-12 medium supplemented with 4% FBS, 250 lM IBMX (a phosphodiesterase inhibitor responsible for cAMP accumulation by inhibiting its phosphodiesterase-driven degradation to 5 0 -AMP) and 1 lM forskolin.…”

“…Gq protein activation upon ligand stimulation generates the second messenger D-myo-inositol 1,4,5-trisphosphate (IP 3 ) via the enzyme phospholipase C. IP 3 triggers calcium release within the cell and is rapidly metabolized into D-myoinositol bisphosphate (IP 2 ), D-myo-inositol 1-phosphate (IP 1 ) and eventually D-myo-inositol (Norskov-Lauritsen et al 2014). The assay used in this study measures the accumulation of IP 1 .…”

Solvent-accessibility of discrete residue positions in the polypeptide hormone glucagon by 19F-NMR observation of 4-fluorophenylalanine

“…We applied cAMP assays (Norskov-Lauritsen et al 2014) to test [4F-Phe6,22]-glucagon for activity. The binding curves demonstrated that [4F-Phe6,22]-glucagon had similar activity as glucagon (Fig.…”