Constitutive activity of the metabotropic glutamate receptor 2 explored with a whole-cell label-free biosensor

Abstract

Label-free cellular assays using a biosensor provide new opportunities for studying G protein-coupled receptor (GPCR) signaling. As opposed to conventional in vitro assays, integrated receptor-mediated cellular responses are determined in real-time rather than a single downstream signaling pathway. In this study, we examined the potential of a label-free whole cell impedance-based biosensor system (i.e. xCELLigence) to study the pharmacology of one GPCR in particular, the mGlu 2 receptor. This receptor is a target for the treatment of several psychiatric diseases such as schizophrenia and depression.After optimization of assay conditions to prevent interference of endogenous glutamate in the culture medium, detailed pharmacological assessments were performed. Concentration-response curves showed a concentration-dependent increase in impedance for agonists and positive allosteric modulators, whereas receptor inhibition by an antagonist or negative allosteric modulator resulted in a concentration-dependent decrease in cellular impedance. Interestingly, constitutive receptor activity was observed that was decreased by LY341495, which therefore behaved as an inverse agonist here, a property that was heretofore unappreciated. This was confirmed by concentration-dependent modulation of LY341495 potency and efficacy by a allosteric modulators.In summary, the use of the xCELLigence system to study mGlu 2 receptor pharmacology was validated. This is the first class C GPCR to be characterized extensively by such method, opening new avenues to study receptor pharmacology including inverse agonism and demonstrating its value for future drug discovery efforts of mGlu receptors as well as other GPCRs.

Chloride ions stabilize the glutamate-induced active state of the metabotropic glutamate receptor 3

“…We have recorded concentrations ranging from 0.6 to more than 5 µM. These glutamate concentrations are in line with those measured in another recent publication (Doornbos et al, 2018). Thus, Clions are pure positive allosteric modulators of the mGlu3 receptor.…”

Computational Drug Design Applied to the Study of Metabotropic Glutamate Receptors

“…They modeled the VFT domain and went on to show that Cl − ions stabilize the glutamate-induced active state of the extracellular domain of the mGlu 3 receptor and the large basal activity that had for many years been seen was due to chloride mediated positive allosteric modulation effect on low levels of residual glutamate concentrations. This is consistent with other recent reports of mGlu receptor activation being sensitive to residual levels of glutamate [99].…”

In vitro profiling of orphan G protein coupled receptor (GPCR) constitutive activity

“…We reasoned that the difference in luciferase expression obtained comparing cells expressing the reporter with or without expression of the G protein chimeras could represent an index of reporter activation by endogenously expressed G i/o ‐coupled receptors. As a positive control, we co‐transfected GRM2 because of its reported high constitutive activity (Doornbos et al, 2018) (Figure 2b,c). We found that expression of G q ‐based chimeras only produced a negligible amount of NFAT reporter induction (0.1‐fold increase on average) (Figure 2b), whereas we observed an average of 83‐fold increase in CRE‐induced luciferase expression using the G s ‐based chimeras (Figure 2c).…”