Metabotropic glutamate receptors (mGluRs) are class C, synaptic G-protein-coupled receptors (GPCRs) that contain large extracellular ligand binding domains (LBDs) and form constitutive dimers. Despite the existence of a detailed picture of inter-LBD conformational dynamics and structural snapshots of both isolated domains and full-length receptors, it remains unclear how mGluR activation proceeds at the level of the transmembrane domains (TMDs) and how TMD-targeting allosteric drugs exert their effects. Here, we use time-resolved functional and conformational assays to dissect the mechanisms by which allosteric drugs activate and modulate mGluR2. Single-molecule subunit counting and inter-TMD fluorescence resonance energy transfer measurements in living cells reveal LBD-independent conformational rearrangements between TMD dimers during receptor modulation. Using these assays along with functional readouts, we uncover heterogeneity in the magnitude, direction, and the timing of the action of both positive and negative allosteric drugs. Together our experiments lead to a three-state model of TMD activation, which provides a framework for understanding how inter-subunit rearrangements drive class C GPCR activation.

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