Structures of human mGlu2 and mGlu7 homo- and heterodimers

Abstract

The metabotropic glutamate receptors (mGlus) are involved in modulation of synaptic transmission and neuronal excitability in the central nervous system 1 . These receptors likely exist as both homo-and heterodimers with unique pharmacological and functional properties 2-4 . Here we report four cryo-electron microscopy structures of the human mGlus, including inactive mGlu2 and mGlu7 homodimers, agonist/PAM-bound mGlu2 homodimer, and inactive mGlu2-7 heterodimer. A subtype-dependent dimerization mode of mGlus was observed, as a unique dimer interface mediated by helix IV important to limit receptor activity exists in the inactive mGlu2 structure only. The structures provide molecular details of inter-and intrasubunit conformational changes that are required for receptor activation, which distinguish class C G-proteincoupled receptors (GPCRs) from classes A and B receptors. Furthermore, the mGlu2-7 heterodimer structure and functional studies suggest that the mGlu7 subunit plays a dominant role in controlling dimeric association and G protein activation in the heterodimer. These insights into mGlu homo-and heterodimers highlight the complex landscape of mGlu dimerization and activation.The mGlus contain a large extracellular domain (ECD) composed of the Venus flytrap (VFT) domain that binds agonist and a cysteine-rich domain (CRD) connected to a seven-helical transmembrane domain (TMD) responsible for G protein coupling 4 . It has been acknowledged that homodimerization is mandatory for the function of the mGlus, making them complex allosteric proteins with two subunits influencing each other 3 . In addition, there is increasing evidence suggesting that different mGlu subunits can associate to form multiple types of heterodimers, adding complexity of function modulation in this receptor family 2,4 . However, conformational difference between the mGlu homo-and heterodimerization and how these dimers control mGlu function remain unclear. Thus, we performed single-particle

Reconstitution of purified membrane protein dimers in lipid nanodiscs with defined stoichiometry and orientation using a split GFP tether

“…These DNA tethers can yield parallel heterodimers and larger oligomers but require separate purification and labeling steps for each component, as well as minimal-cysteine receptor constructs to allow maleimide-mediated conjugation of the DNA strands. An approach similar to our method was reported using FKBP (the 12-kDa FK506 binding protein) and FKBP12/rapamycin binding–fused receptors dimerized during expression with rapamycin and reconstituted into nanodiscs ( 24 ). These structured domains could be a viable alternative to our method if poorly behaved sGFP1–10 hampers expression, although without a dimer-specific purification handle such as the GFPnb resin, this method requires tandem affinity purification.…”

Metabotropic Glutamate Receptors at Ribbon Synapses in the Retina and Cochlea

“…By this means, mGluR C-termini and interacting proteins form signalling complexes that represent the molecular basis for a dynamic regulation of receptor function. A further diversity in receptor characteristics is achieved by the formation of homo- and heterodimeric receptors [ 43 , 44 , 45 , 46 , 47 , 48 , 49 ].…”

Agonist and antagonist TRUPATH assays for G protein-coupled receptors

“…Additionally, the “non-optimal” G and GFP2-G plasmids are available for TRUPATH users investigating specific heterotrimeric G protein combinations. To date, numerous groups have successfully employed TRUPATH in their studies to interrogate GPCR structure and function ( Nagai et al., 2020 ; Kim et al., 2020 ; Knight et al., 2021 ; Du et al., 2021 ; Pryce et al., 2021 ; Gao et al., 2021 ; Chakraborty et al., 2021a , 2021b ; Chao et al., 2021 ; Von Moo et al., 2021 ; Lu et al., 2021 ; Lin et al., 2021 ; Cao et al., 2021 ; Yang et al., 2021 ; Yan et al., 2021 ).

Figure 1 An overview of the TRUPATH platform G subunits fused to RLuc8 and G subunits fused to GFP2 produce a BRET signal (calculated as the ratio of GFP2 to RLuc8 counts) in the presence of RLuc8 substrate that is both spatially- and orientationally-dependent.

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