Phosphoinositide hydrolysis assay

Phosphoinositide (PI) hydrolysis assays measuring inositol phosphates (IP) were performed using the scintillation proximity assay (Bourdon et al., 2006; Huang et al., 2009). The MRGPRX2 WT construct was developed from the TANGO system as described above and mutant constructs were directly subcloned as viral transgenes to pCDNA3.1. The day before transfection, HEK293T cells were split to yield approximately 9×10⁶ cells/15-cm plate next day. The following day cells were transfected with 5μg DNA per 15-cm dish with TransIT-2020. On the day before the assay, transfected cells were seeded into 96-well poly-lysine coated plates at a density of 40–50,000 cells/well in 100μL inositol-free DMEM (Caisson Labs, DML13) containing 1% dialyzed FBS. After 6 hr, an additional 100μL of label media was added containing 1μCi/well (final concentration) of [³H]-myo-inositol (PerkinElmer, NET1177001MC) in inositol-free DMEM containing 1% dialyzed FBS and plates were incubated overnight for 16–18 hr. The next day, label media was removed and cells were washed twice with 60μL of drug buffer (1 x HBSS, 20 mM HEPES, 0.1% BSA, 0.01% ascorbic acid, pH 7.4), then 60μL of drug buffer was added per well. Afterward, 30μL of drug (3X) was added per well and incubated at 37°C for 1 hr. To capture IP accumulation, lithium chloride (10μL/well, 15 mM final concentration) was added 30 min before lysis. The assay was terminated by replacement of the incubation medium with 40μL of 50 mM formic acid. After overnight incubation at 4°C, 10μL of lysates were added to 96-well flexible, clear microplates (PerkinElmer, 1450–401C) containing 75μL of 0.2 mg/well RNA binding yttrium silicate beads (PerkinElmer), and incubated for 1 hr on a shaker. Afterward, plates were centrifuged at 300 x g for 1 min, and radioactivity was measured using a Wallac MicroBeta Trilux plate reader (PerkinElmer). Data were plotted as counts per minute (CPM) as a function of drug concentration, and analyzed using “log(agonist) vs. response (three parameters)” in GraphPad Prism 8.0.