Background

Seven-transmembrane receptors (7TMR), also known as G-protein-coupled receptors, are the most important transmembrane protein superfamily involved in signal transduction. They are the target of 30 to 50% of clinically approved drugs (Overington et al., 2006). Gα_q/15-coupled 7TMRs activate phospholipase C β (PLCβ) and produce D-myo-inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 trigger a release of intracellular Ca⁺⁺ storages and is later degraded into IP2, IP1 and ultimately D-myo-inositol (Figure 1).


Figure 1. The Gα_q signaling pathway. Upon agonist binding at a given Gα_q-coupled 7TMR, the α subunit dissociates from the βγ dimer and activates the PLCβ, thus initiating the cleavage of PIP2 into IP3 and DAG. IP3 is then degraded into IP2, IP1, and myo-inositol. Accumulation of IP1 is achieved by using a stimulation buffer containing LiCl to inhibit IP1 degradation into myo-inositol and further recycling.

Gα_q/15 activation is commonly measured by dectecting Ca⁺⁺ flux using fluorescent calcium dye indicators. However, these indicators require either the use of an Atto-Fluor microscope with very low throughput, or investment in an expensive fluorescent imaging plate reader (FLIPR, Arkin et al., 2004). Another Gα_q/15 activity quantification method relies on the detection of [³H]-inositol-phosphates ([³H]-IPs) following incubation of cells with [³H]-inositol. This highly reliable assay is based on the accumulation of [³H]-IPs after stimulation with a 7TMR agonist (Thomsen and Behan, 2007). However, both the FLIPR calcium flux assay and the [³H]-IPs detection method have important drawbacks. On the one hand, the Ca⁺⁺ flux is transient and rapid, the assay does not permit the detection of constitutive activity (and thus, inverse agonism), and the readout can be interfered with by fluorescent compounds (Garbison et al., 2004). On the other hand, [³H]-IPs detection assays are time-consuming, very expensive, and generate radioactive waste that needs to be disposed of properly. A non-radioactive assay based on the detection of accumulated IP1 using a homogenous time-resolved fluorescence assay has been developed by Cisbio (Trinquet et al., 2011). This assay is based on a competition between endogenous IP1 and d2-labeled IP1 binding to an Eu cryptate-conjugated IP1 antibody (Figure 2, Liu et al., 2008).

Several protocols have been developed for the quantification of IP1 using Cisbio’s IP-One assay, but they only apply for the screening of different compounds at a single receptor. Traditional protocols for IP-One assay using transient or stable receptor expression are designed in a 2-step protocol. First, cells are plated into petri-dishes and transfected for 24 to 48 h to achieve a suitable cell-surface receptor expression (if transient expression is preferred). Next, cells are detached using trypsin and re-suspended in stimulation buffer before being distributed and stimulated into the assay 384-well plate (Shehata et al., 2016; St-Pierre et al., 2018). Other variations of this protocol using stable cell lines are reported and involve a plating step into the assay plate 24 h before stimulation (Cassutt et al., 2007; Zhang et al., 2010).


Figure 2. IP-One assay principle. Endogenous IP1 produced after 7TMR activation enter into competition with the exogenous d2-labeled IP1 for binding to the exogenous Eu-cryptate anti-IP1 antibody. When d2-IP1 is bound to the Eu-cryptate anti-IP1 antibody, the close proximity of the two fluorescent dyes allows for energy transfer during the readout (represented by the orange arrow). The more IP1 is produced by 7TMR activation, the less d2-IP1 is bound to the Eu-cryptate anti-IP1 antibody and the less energy transfer can be recorded during readout.

In order to avoid the transfection of receptors and mutants into petri dishes and then the transfer of cells into the assay plates, and to avoid unnecessary exposition to trypsin that can lead to non-functional surface receptors, we have developed a single step transfection and assay protocol. This protocol uses reverse transfection of the receptor’s DNA and direct plating of cells in a cell culture-treated and assay-suitable 384-well plate. After 48-h incubation, adherent cells are then washed and used in the IP-One assay (Gusach et al., 2019; Luginina et al., 2019). This protocol can also be used for receptors that are not coupled to Gα_q by co-transfection of the Gα_15/16 G protein-coding plasmid along with the receptor’s plasmid as Gα_15/16 are promiscuous G proteins able to couple to non-Gα_q-coupled 7TMRs (New and Wong, 2004; Robas and Fidock, 2005).