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). These signaling pathways are activated by the specific G protein associated with the receptor. Activation of a Gs or Gi coupled receptor 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 (Figure 1).
Cisbio Bioassays has developed assay kits capable of monitoring the activation of Gs, Gi and Gq coupled receptors, using their new generation Lumi4-Tb™ TR-FRET Cryptate. This chemistry allows for the detection of two events in one well using two different acceptors, a green dye (λ = 520 nm) and a red dye (λ = 665 nm). This same chemistry is used in Cisbio’s Tag-lite® technology.
IP-One and cAMP experiments were performed on a HTS microplate reader from BMG LABTECH using Simultaneous Dual Emission detection. With this unique feature, the plate is read only once for dual emission assays, thereby decreasing time and variability.
The IP-One and cAMP HTplexTM assay is a competitive immunoassay that uses two antibodies labelled with Lumi4-TbTM (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. As cAMP or IP1 is produced upon GPCR activation, the tracer green-cAMP or the tracer red-IP1 will be uncoupled from the Tb-cryptate antibody, thereby causing a decrease in TR-FRET signal. Specific emission signals are inversely proportional to the concentration of cAMP and IP1 in a standard or in a cell lysate.
Materials & Methods
- IP-One and cAMP reagents from Cisbio Bioassays
- White 384-well format microplate, Greiner
- Multidetection microplate reader from BMG LABTECH
CHO-V2R cells (stable transfection with the vasopressin-receptor) are cultivated in F12 medium then diluted to obtain a concentration of 1,000,000 cells/mL (viability: 96.3%). Then 30 μL are distributed in each well (giving 30,000 cells/well). The plate is incubated at 37°C overnight. The cell supernatant is aspirated (the cells collapse to the well bottom) and immediately replaced with 10 μL of stimulation buffer.
For standard curve: add 20 μL of diluted standards, 5 μL cAMP-green dye/IP1-red dye, and 5 μL cAMP-Cryptate/ IP1-Cryptate, then incubate for 1 hr at RT. For vasopressin dose-response: add 10 μL stimulation buffer, 10 μL vasopressin (14 dilutions: from 0 to 10-4 M), 1 hr stimulation at 37°C. Then add 5 μL cAMP-green dye/IP1-red dye, 5 μL cAMP-Cryptate/ IP1-Cryptate and incubate for 1 hr. For Z’ calculation: add 10 μL stimulation buffer (basal) or 10 μL vasopressin 10 μM, 1 hr stimulation at 37°C. Then add 5 μL cAMP-green dye/IP1-red dye, 5 μL cAMP-Cryptate/IP1-Cryptate and incubate for 1 hr.
Specific HTRF compatible filter sets/optic modules, one for red and one for green.Integration start = 60 μs, integration time = 400 μs. Flash number = 300 (if speed is important, 100 flashes can be used). Plate is read twice. In case the instrument is equipped with a UV laser, it is recommended to use 7 flashes.
Cisbio has a patented ratiometric measurement that uses both the emission wavelength of the donor and acceptor (patent US 5,527,684* and foreign equivalents) to correct for well-to-well variability and signal quenching. Emissions at 620 nm (donor) are used as an internal reference while emissions at 665 nm and 520 nm (acceptor) are used as an indicator of the biological reaction being assessed. Ratios of fluorescence intensities 665/620 and 520/620 (acceptor/donor) are calculated in order to detect each single interaction.
Results & Discussion
Figure 2 shows the calibration curve using standards for cAMP and IP1. Both curves give the expected EC50 values.
This figure shows the occurrence of two signaling events - 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).
In Figure 3, both cAMP and IP1 were measured in CHO-V2R cells upon activation with vasopressin in a dose response manner.
Lastly, the robustness of the assay as measured by the Z prime calculation (Figure 4) shows that both signals are well above the reliability range for an HTS assay, giving Z’ values > 0.75.
The HTplexTM assay from Cisbio can evaluate two different GPCR signaling pathways, Gai/s and Gaq, through the measurement of their second messenger responses, cAMP and IP3 (via IP1), respectively. Herein, the HTplexTM assay was used to evaluate the dose response effect of vasopressin on CHO-V2R cells. As measured, there is an initial cAMP response at lower concentrations and a latent IP1 accumulation at higher concentrations. The concept of this application note can be extended to Cisbio’s complimentary HTRF® chemistry, Tag-lite®, which uses the same Lumi4-TbTM cryptate and the same red and green acceptors.
HTplex is a registered trademark of Cisbio.
HTRF is a registered trademark of Cisbio.
Lumi4 is a trademark of Lumiphore Inc.