Membrane-domain compartmentalization of active GPCRs by β-arrestins through PtdIns(4,5)P₂ binding

Upon ligand-induced activation, G-protein-coupled receptors (GPCRs) recruit β-arrestins (βarrs) to the plasma membrane, where phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) stabilizes the GPCR-βarr complex. Although PtdIns(4,5)P₂ is reported to form nanoscale clusters, the spatiotemporal dynamics of how the GPCR-βarr-PtdIns(4,5)P₂ complex assembles and organizes in living cells remain unexplored. Here we demonstrate that multiple PtdIns(4,5)P₂-binding sites on βarrs cooperatively promote GPCR-βarr assembly in membrane domains. Using molecular dynamics simulations, we identify a noncanonical (NC) PtdIns(4,5)P₂-binding site, distinct from the known canonical site. Biochemical assays confirm that both sites are essential for βarr binding to PtdIns(4,5)P₂-containing liposomes, while NanoBiT assays reveal synergistic contributions of both sites for βarr recruitment in living cells. Notably, single-molecule imaging demonstrates that the NC site is required for the rapid accumulation of the GPCR-βarr-PtdIns(4,5)P₂ complex into immobile membrane domains upon ligand stimulation. Collectively, our findings highlight how multivalent βarr-PtdIns(4,5)P₂ interactions drive GPCR-βarr compartmentalization, adding complexity to GPCR signaling dynamics.