Tuning of Metabotropic Glutamate Receptor Assembly and Activation by Interactions between Transmembrane Domains

Abstract

Metabotropic glutamate receptors (mGluRs) are class C G protein-coupled receptors (GPCRs) that respond to the excitatory neurotransmitter glutamate and modulate neuronal function throughout the central nervous system. In addition to the seven-transmembrane domain (TMD) conserved across all GPCRs, mGluRs also contain a large extracellular ligand-binding domain (LBD) which facilitates dimer formation. Previous work established that ligand-induced reorientation of LBDs in mGluR dimers is a key step in activation. However, how the motions of the LBDs are transmitted to the TMDs and how the TMDs themselves interact and move in this process is unclear. Understanding inter-TMD dynamics is necessary for fully understanding both glutamate and orthosteric agonist-mediated activation and the mechanisms of allosteric drugs that bind the TMD. Here we use a combination of structural, functional and conformational assays in either live cells or isolated single proteins to dissect the role of inter-TMD interactions in the activation process. Our recent publication (Gutzeit et al., eLife, 2019) shows that the TMD contributes directly to mGluR dimerization and that TMD dimerization efficiency varies across mGluR subtypes. We also developed an inter-TMD FRET assay which demonstrates that TMDs reorient in response to allosteric drugs in the absence of LBDs. These findings motivate our current work in which we use a single molecule fluorescence-based subunit-counting assay which reveals an interface between TMDs that determines the relative dimerization strength of mGluR subtypes. Next, we use calcium imaging to determine the functional role of this interface and find that inter-TMD interactions at this interface control both orthosteric and allosteric activation of mGluRs. We further examine the conformational changes between TMDs using FRET assays from multiple structural perspectives. Together, this work demonstrates that the TMD independently influences dimerization and activation of mGluRs.