Differences in interactions between transmembrane domains tune the activation of metabotropic glutamate receptors

Abstract

The metabotropic glutamate receptors (mGluRs) form a family of neuromodulatory G protein-coupled receptors that contain both a seven-helix transmembrane domain (TMD) and a large extracellular ligand-binding domain (LBD) which enables stable dimerization. While numerous studies have revealed variability across subtypes in the initial activation steps at the level of LBD dimers, an understanding of inter-TMD interaction and rearrangement remains limited. Here we use a combination of single molecule fluorescence, molecular dynamics, functional assays, and conformational sensors to reveal that distinct TMD assembly properties drive differences between mGluR subtypes. We uncover a variable region within transmembrane helix 4 (TM4) that contributes to homo- and heterodimerization in a subtype-specific manner and tunes orthosteric, allosteric and basal activation. We also confirm a critical role for a conserved inter-TM6 interface in stabilizing the active state during orthosteric or allosteric activation. Together this study shows that inter-TMD assembly and dynamic rearrangement drive mGluR function with distinct properties between subtypes.

Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2

“…Structurally, the VFT domain, CRD, and 7TM domain undergo unique dynamics during receptor activation (Cao et al, 2021; Grushevskyi et al, 2019; Liauw et al, 2021). Moreover, how each domain within mGluRs contribute to the overall receptor regulation and activation is now better understood (Goudet et al, 2004; Huang et al, 2011; Thibado et al, 2021). Thus, the three domains can be viewed as modular units that are linked to form a complex and conformationally coupled signaling machine.…”

“…Moreover, how each domain within mGluRs contribute to the overall receptor regulation and activation is now better understood (Goudet et al, 2004;Huang et al, 2011;Thibado et al, 2021).…”

Structural and compositional diversity in the kainate receptor family

“…Having established the general structural compatibility ( Figure 1 ) and extensive co-expression of KAR subunit types ( Figure 2 ), we aimed to assess the assembly, stoichiometry, and subunit arrangement of KARs composed of different pairs of subunit types, starting with di-heteromers. We employed the SiMPull method in which detergent-solubilized and fluorophore-tagged receptors are immobilized via antibodies and their subunits counted by photobleaching step analysis ( Jain et al, 2011 ; Lee et al, 2020 ; Levitz et al, 2016 ; Royal et al, 2019 ; Thibado et al, 2021 ). We first generated GluK1 and GluK2 constructs with an N-terminal hemagglutinin (HA) epitope followed by a SNAP tag ( Figure S4B ) amenable to labeling with organic dyes, and both maintained normal receptor function ( Figure S4B ; Table S2 ).…”

Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2

“…Structurally, the VFT domain, CRD, and 7TM domain undergo unique dynamics during receptor activation ( Cao et al, 2021 ; Grushevskyi et al, 2019 ; Liauw et al, 2021 ). Moreover, how each domain within mGluRs contribute to the overall receptor regulation and activation is now better understood ( Goudet et al, 2004 ; Huang et al, 2011 ; Thibado et al, 2021 ). Thus, the three domains can be viewed as modular units that are linked to form a complex and conformationally coupled signaling machine.…”