Configurational diversity of metabotropic glutamate receptor complexes with beta-arrestins

Beta-arrestins (β-arrs) are cytosolic proteins which mediate G protein-coupled receptor (GPCR) desensitization, endocytosis, and signaling. Despite the widespread physiological roles of β-arr coupling, the molecular basis of GPCR/β-arr interaction has been studied primarily in monomeric family A GPCRs. Here we develop a single molecule pulldown (SiMPull) assay that reports on both relative GPCR:β-arr interaction strength and stoichiometry to uncover molecular diversity in β-arr coupling to the neuromodulatory metabotropic glutamate receptors (mGluRs), prototypical, dimeric family C GPCRs. We find that mGluRs couple to β-arrs with variable apparent strength and a 2:1 or 2:2 stoichiometry. Focusing on the mGluR8/β-arr1 interaction, we map the contributions of "tail" and "core" interactions with residues in the receptor C-terminal domain and intracellular loops, respectively. Combinatorial mutagenesis enables the identification of a landscape of β-arr complexes with homo- and hetero-dimeric mGluR8, including both "cis" and "trans" interactions. Finally, we use our SiMPull assay in conjunction with live cell imaging and transcriptomic analysis to reveal that mGluRs can form megacomplexes either by simultaneously binding β-arr1 and β-arr2 or β-arr and G proteins. Together, this study provides a comprehensive framework for mGluR/β-arr complex diversity, expanding the known range of GPCR/transducer coupling modes.