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

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. Although 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.