Glutamate is the main excitatory neurotransmitter in the brain and mediates its actions by both ionotropic (e.g. NMDA and AMPA) and metabotropic glutamate receptors (e.g. mGluRs). The Groups 2 and 3 mGluRs, which pre-synaptically inhibit glutamate release, are important for synaptic plasticity, modulating neuronal excitation, learning and memory. Our current understanding of the structural organization and dynamics of these and other mGluRs, as well as most other GPCRs, relies mainly on studies using recombinant and highly engineered systems in vitro. Here, we combine CRISPR-mediated protein tagging, proteomics and a rapid immunoaffinity purification method to isolate endogenous mGluR2-containing assemblies from mouse brain and visualize them via cryo-EM. Analysis of the particle sets reveals the molecular structures of at least 11 distinct endogenous receptor assemblies that span active and inactive states, homomeric and heteromeric dimers, and G protein-coupled and uncoupled species. We find that mGluR2 homodimers and mGluR2/3 heterodimers are the major endogenous mGluR2-containing species present in the brain, with the mGluR2/3 heterodimers detected only in active state complexes. Reconstructing the comprehensive activation trajectory for endogenous mGluRs reveals endogenous ternary complexes comprising mGluR2 homodimers and mGluR2/3 heterodimers with a single GαoA heterotrimer. These complexes isolated from brain exhibit significant differences from structures reported previously using recombinant systems. Our work illuminates the endogenous conformational, proteomic and compositional landscape of the heterogenous mGluR2 complexes in the brain, thereby providing a structural framework for targeting mGluR2 therapeutically.
