2Cisbio Bioassays, Bagnols-sur-Cèze, France
Full Text (PDF)
The G protein–coupled receptor (GPCR) superfamily has almost 1000 members and thus represents the largest protein family in the human genome. GPCRs carry information within cells via two major signaling pathways: regulation of cAMP levels and increases in intracellular Ca2+ triggered by inositol 1,4,5-triphosphate (IP3). Activation of these pathways via Gs-or Gi-coupled receptors results in the increase or decrease of cAMP levels, respectively, while activation of a Gq-coupled receptor activates phospholipase C (PLC) and triggers the inositol phosphate (IP) cascade.
Cisbio Bioassays’ new generation Lumi4-Tb TR-FRET cryptate chemistry allows for the detection of cAMP and IP1 levels in one well by using two different acceptors, a green dye (λ = 520 nm) and a red dye (λ = 665 nm). The experiments described here where performed on the PHERAstar Plus HTS microplate reader using Simultaneous Dual Emission detection. This unique feature allows the plate to be read once for dual emission assays, thereby decreasing time and variability.
The HTplex assay from CisbioThe HTplex assay uses two antibodies labeled with Lumi4-Tb (anti-cAMP Cryptate and anti-IP1 Cryptate as donors) and two acceptors (cAMP-green dye and IP1-red dye). In the inactivate state, a high TR-FRET signal is seen for both the red and green emissions (Figure 1). CHO cells were stimulated with 10 µM vasopression. Upon GPCR activation, cAMP and IP1 were produced, thereby decoupling the tracer green-cAMP and the tracer red-IP1 from the Tb-cryptate antibody. This leads to a decrease in the TR-FRET signal. Hence specific emission signals are inversely proportional to the concentration of cAMP and IP.
Results & Discussion
Figure 2 shows an initial cAMP (Gs) response at lower concentrations of vasopressin presumably through activation of endogenous V1 receptors, and a delayed IP3 (Gq) response at higher concentrations through activation of the transfected V2 receptor (as measured by IP1 accumulation). EC50 for cAMP and IP1 were 15 × 10-6 M and 23 × 10-6 M, respectively, and both signals were well above the reliability range for an HTS assay, giving Z' values > 0.75. The HTPlex assay from Cisbio was successfully employed with BMG LABTECH's PHERAstar Plus HTS microplate reader.
