PROJECT SUMMARY G protein-coupled receptors (GPCRs) are finely tuned signaling molecules that are central to diverse neurophysiological processes and serve as major drug targets for neurological and psychiatric disorders. A major form of GPCR regulation occurs through the action of multifunctional β-arrestins (β-arrs) which bind to activated receptors to drive functional desensitization, control receptor trafficking, and initiate G protein-independent signaling cascades. Despite progress, our mechanistic understanding of GPCR/β-arr coupling is limited and based on a small number of prototypical family A GPCRs. It is critical to improve our understanding of GPCR/β- arr coupling by unraveling the biophysical basis and biological consequences of variability between different GPCR subfamilies and subtypes, as well as between different pharmacological ligands. The metabotropic glutamate receptors (mGluRs) form an eight-member family of family C GPCRs with a unique architecture consisting of large extracellular, ligand binding domains that mediate constitutive dimerization. Due to their roles in synaptic neuromodulation, mGluRs have emerged as drug targets for neurodevelopmental, neurodegenerative, and psychiatric disoders, as well as cancers. However, it has been difficult to harness mGluRs therapeutically because of a lack of understanding of their basic signaling and regulatory properties. Deciphering the mechanisms of mGluR regulation is particularly challenging since this subfamily is dramatically expanded by heterodimerization and is targeted by a broad panel of orthosteric and allosteric ligands with distinct properties. Until recently, mGluR/β-arr coupling has been poorly characterized, but we recently found that a subset of mGluRs is capable of robust β-arr coupling while others are highly resistant, providing another dimension of molecular diversity to this GPCR family. We will build on our recent findings with a battery of structural, biophysical, and cell-based measurements, to understand the underlying mechanisms and physiological consequences of mGluR/β-arr coupling. In aim 1, we will develop and harness a single molecule imaging assay to define the determinants and stoichiometry of mGluR/β-arr complex formation and then use electron microscopy (negative stain, cryo-EM) to solve high resolution structural snapshots of mGluR/β-arr complexes. In aim 2, we will use in vitro and live cell biophysical assays to define the basis of mGluR C-terminal domain and transmembrane core coupling to β-arrs across subtypes and probe the effects of distinct ligand types and heterodimerization on mGluR/β-arr coupling. In aim 3, we will investigate the trafficking and functional consequences of mGluR/β-arr coupling using high- resolution optical and proximity proteomics techniques in both cell lines and cortical neurons, with a focus on presynaptic signaling and trafficking. Together, this project will provide a full picture of the regulation of mGluRs by...