Fig. 1: Orthosteric agonists drive activation and internalization of mGluRs with correlated efficacy.
a Schematic showing mGluR-mediated GIRK activation through Gαi/o activation and Gβγ release. b Representative whole-cell patch clamp current trace showing agonist-evoked mGluR3-mediated GIRK potassium currents showing the relative maximum agonist efficacies. Gray dashed line represents peak glutamate current. c Bar plot quantifying the maximum mGluR3-mediated current normalized to the glutamate (100 µM; n = 11 cells) condition for DCG-IV (100 µM; n = 6 cells; p = 0.003) and LY379268 (1 µM; LY37; n = 7 cells; p = 0.007). d Schematic showing surface labeling assay to quantify ligand-evoked receptor internalization. Cells are incubated with agonist for 60 min followed by labelling of SNAP-tagged surface receptors with a membrane-impermeable fluorophore. e Quantification of the % internalization of mGluR3 following treatment with each agonist (same concentrations as panel c) (Glu: n = 6 experimental days, 10 averaged images per day; DCG-IV: n = 5, p = 0.025; LY37: n = 6; p = 0.509). f Representative widefield fluorescence images depicting internalization of SNAP-mGluR3 labeled with BG-Alexa-546 prior to agonist treatment for 30 min. Red arrows highlight endocytosed receptors. Scale Bar = 5 µm. g Scatter plot showing maximum GIRK activation versus internalization for a range of agonists normalized to saturating glutamate for three mGluR subtypes (mGluR3, mGluR7, mGluR8). Linear regression was applied to the entire data set and constrained to go through the glutamate reference point (data points are presented as mean values ± SEM). Bar-plots represent mean ± SEM For (c), (e): One-way ANOVA with Dunnet’s multiple comparisons test against WT. P-values are reported in the specific panel legend.
