Quantification of mGluR-Mediated Signaling

Phospholipase C activation was quantified by measuring the inositol monophosphate accumulation in HEK293 cells transiently expressing the mGlu receptors and a chimeric Gqi₉ protein 24 h after transfection with Lipofectamine 2000, as previously described^{35 (link)}. Cells were incubated with the indicated ligands and 10 mM LiCl for 30 min, and the IP1 accumulated was quantified using the IP One HTRF assay kit from Cisbio according to the manufacturer instruction in 384-well plates^{49 (link)}. The amount of cAMP was determined using the Glosensor cAMP assay (Promega Corporation, Madison, USA), as previously described^{26 (link)}. HEK293 cells were co-transfected with the indicated mGluR plasmids, the pGloSensor-22F plasmid and EAAC1 encoding plasmid. The day after, cells were starved for 2 h in serum-free medium and then incubated in Krebs buffer with 450 μg ml⁻¹ luciferin (Sigma-Aldrich) for 30 min, followed by a 30 min incubation with the nanobody at the indicated concentration. The luminescence peak signal was measured on a Mithras microplate reader at 28 °C during 8 min until luminescence signal was stable. Then, forskolin (1 µM) and the indicated concentration of mGlu2 agonist LY379268 were added and luminescence was measured for 30 min.