Fig. 3: PAMs produce LBD-dependent and independent conformational and functional effects.

a Schematic showing inter-LBD FRET sensor where LBD closure and reorientation from relaxed to active dimer states upon agonist exposure decrease FRET efficiency. b Bar graph showing normalized mGluR3 inter-LBD FRET decrease following application of saturating VU6023326 (10 µM; VU602; n = 6; p < 0.0001), glutamate (n = 19) or both (n = 5; p = 0.5802). c Bar graph showing the inhibition of VU602 inter-LBD FRET responses by competitive antagonist LY341495 (5 µM; LY34; n = 4) or NAM MNI-137 (60 µM; n = 5). d Schematic showing inter-TMD FRET sensor where agonist application receptor increases the FRET efficiency as TMD domains undergo activation-associated rearrangements. e Bar graph showing normalized mGluR3 inter-TMD FRET increase following application of saturating PAM VU6023326 (10 µM; VU602; n = 6; p < 0.0001), glutamate (n = 10) or both (n = 4; p < 0.0001). f Bar graph showing the inhibition of VU602 inter-TMD FRET responses by saturating competitive antagonist LY341495 (10 µM; LY34; n = 5) or NAM MN1-137 (60 µM; n = 5; p < 0.0001). g Representative GIRK current trace for mGluR3 showing the lack of response to PAM (VU602) when applied in the presence of competitive antagonist. h Same as in (g) but for mGluR2, which maintains a clear PAM response in the presence of competitive antagonist. i Bar graph summarizing inhibition of PAM-evoked GIRK currents when applied in the presence of competitive antagonist LY341495 (5 µM) or NAM MNI137 (10 µM) for mGluR3 (LY341495, n = 10 cells; MNI137, n = 5 cells; p = 0.391) and mGluR2 (LY341495, n = 7 cells; MNI137, n = 5 cells; p = 0.0001). Values are normalized to the response to saturating glutamate. Dotted line shows the response to PAM alone for each receptor. j Bar graph showing inhibition of PAM-induced mGluR3 internalization by competitive antagonist LY341495 (20 µM; n = 14 experimental days) or NAM MNI137 (10 µM; n = 7 experimental days; p < 0.0001). k Representative images showing mGluR3 internalization in the presence of antagonist+PAM but not NAM + PAM conditions. Red arrows highlight clusters of internalized receptors. Scale bar = 10 µm. l Schematic summarizing the effects of competitive antagonist on PAM-induced functional effects for mGluR3 and mGluR2. Bar-plots represent mean ± SEM for n = separate preparations for FRET experiments (b, c, e, f), n = cells for electrophysiology experiments (i), and n = biological replicates (separate days) for imaging experiments (j). For (b, e, i): One-way ANOVA with Dunnet’s multiple comparisons against.