Inter‐Subunit Conformational Dynamics and Allosteric Modulation of Metabotropic Glutamate Receptors

Abstract

Metabotropic glutamate receptors (mGluRs) are class C G protein‐coupled receptors (GPCRs) which function in synapses throughout the central nervous system where they respond to the excitatory neurotransmitter glutamate to initiate intracellular signaling cascades. A comprehensive understanding of mGluR activation requires a mechanistic description of the effects of both extracellular ligand binding domain(LBD)‐targeting “orthosteric” and transmembrane domain (TMD)‐targeting “allosteric” compounds, both of which have therapeutic potential. Here we use a combination of high resolution, time‐resolved functional and conformational assays to dissect the mechanisms by which allosteric compounds activate and modulate mGluRs. First, using an electrophysiological assay we find that mGluR2 can be activated by both orthosteric or allosteric ligands and that allosteric activation does not require LBD closure. We then use a single molecule fluorescence‐based subunit counting assay to find that mGluR transmembrane domains efficiently dimerize with variable efficiency to a degree that is consistently higher than that observed for class A GPCRs and is not sensitive to allosteric drugs. Next, we develop an inter‐TMD FRET assay that reveals that activation‐associated inter‐TMD rearrangement occurs independently of allosteric input from the LBDs. Using this assay, we test a panel of allosteric compounds to uncover diversity in apparent affinity, efficacy and kinetics between these compounds. Strikingly, we find that negative allosteric modulators are either neutral or induce TMD rearrangement which correlates with degree of inverse agonism. Together this data is consistent with a model where multiple (>2) intersubunit reorientations are part of the activation process and motivates further work on the role of multimeric assembly and inter‐domain coupling in the activation of class C GPCRs.Support or Funding InformationThis work is supported by an NIGMS R35 (R35 GM124731) grant to JL. JKT is supported by an NSF GRFP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.