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•Application Note: HitHunter IP3 Assay For GPCR Screening Using The PHERAstar

G protein coupled receptor (GPCR) activation regulates cell signaling via several second messengers, including 3'-5'-cyclic AMP (cAMP), inositol phospholipids and calcium. The quantitation of accumulation of these second messengers is used to pharmacologically characterize both the action of GPCR ligands and to identify novel compounds in high-throughput screening (HTS). GPCRs coupling to Gas and Gai proteins activate or inhibit, respectively, adenylate cyclase, subsequently changing intracellular cAMP levels. GPCRs coupling to Gaq or Gao proteins activate phosphoinositol phospholipase C-ß, an enzyme that hydrolyzes phosphatidylinositol-4,5- bisphosphate (PIP2) to forming sn-1,2-diacylglycerol and inositol- 1,4,5- trisphosphate (IP3). IP3 binds and opens endoplasmic IP3 gated calcium channel, resulting in the release of bound calcium into the cytosol. Metabolic products of IP3 also modulate cell signaling, such as inositol 1,3,4,5-tetrakisphosphate (Ins P4), which synergistically facilitates IP3, mediated calcium release.

DiscoveRx has developed a homogeneous assay based on fluorescence polarization (FP), to measure IP3 levels generated by GPCR activation. This application note, describes the use of IP3 assay along with PHERAstar multimode HTS plate reader.

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•Application Note: HitHunter IP3 Assay For GPCR Screening Using The PHERAstar

SOURCE: BMG LABTECH, GmbH