Abstract
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.
Highlights
G-protein-coupled receptors form an extremely diverse family of membrane signaling proteins that play central roles in most physiological processes and serve as the most frequent class of drug targets in biology (Lagerstrom and Schioth, 2008)
Our observations lead to a model of Metabotropic glutamate receptors (mGluRs) gating at the level of the transmembrane domains (TMDs) that accounts for the complex effects of allosteric drugs and motivates further work aimed at unraveling the heterogeneity of G-protein-coupled receptors (GPCRs)-targeting drugs
A comprehensive understanding of class C GPCR activation and signaling requires a mechanistic description of the effects of both ligand binding domains (LBDs)-targeting ‘orthosteric’ and TMD-targeting ‘allosteric’ compounds (Figure 1A)
Summary
G-protein-coupled receptors form an extremely diverse family of membrane signaling proteins that play central roles in most physiological processes and serve as the most frequent class of drug targets in biology (Lagerstrom and Schioth, 2008). Metabotropic glutamate receptors (mGluRs) form a important family of GPCRs in the brain, where they work in concert with ionotropic iGluRs to control glutamatergic transmission (Reiner and Levitz, 2018) Based on their central roles in basic synaptic neurobiology along with compelling preclinical and clinical evidence, mGluRs serve as potential drug targets for a wide range of neurological and psychiatric diseases (Nicoletti et al, 2011). We use a battery of electrophysiological and imaging-based assays to show that positive allosteric modulators of mGluR2 serve directly as agonists which can drive activation by reorienting TMD dimers independently of allosteric input from the LBDs. Using a single-molecule subunit counting assay, we find that this inter-TMD reorientation is underscored by a unique, high propensity for mGluR2 TMD dimerization that is not seen in other group I and II mGluR subtypes or in canonical class A GPCRs. Using a new inter-TMD FRET assay we find that mGluR2 PAMs show variable apparent affinity, efficacy, kinetics, and reversibility of mGluR2 modulation which should inform future applications and drug development. Our observations lead to a model of mGluR gating at the level of the TMD that accounts for the complex effects of allosteric drugs and motivates further work aimed at unraveling the heterogeneity of GPCR-targeting drugs
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