Factors modulating the assembly of human β₂-adrenergic receptor-β-arrestin complexes

β-arrestins, pivotal regulators of G protein-coupled receptor (GPCR) signaling, assemble with hundreds of GPCRs. How this assembly rises to functionally distinct complexes in which β-arrestin engages the GPCR tail, core or both, remains a central question. Here employing single-molecule force spectroscopy and molecular dynamics simulations, we monitor assembly of β₂-adrenergic receptor (β₂AR)-β-arrestin2 (βarr2) tail, core and tail-core complexes in phospholipid membranes and dissect their mechanical and kinetic stabilities. We show that βarr2 engages the phosphorylated β₂AR carboxy-terminus (C-tail) within milliseconds, much faster than the active receptor core. In addition, the phospholipid membrane contributes substantially to complex stability, with phosphatidylinositol 4,5-bisphosphate (PIP₂) modulating stability and conformation. While PIP₂ stabilizes the β₂AR-βarr2 core, it precludes βarr2 from concomitantly binding the phosphorylated β₂AR C-tail. βarr2 activation and PIP₂ strengthen βarr2-membrane association through insertion of the C-edge and finger loop. These findings establish PIP₂, alongside ligand binding and receptor phosphorylation, as a central determinant of β₂AR-βarr2 complex assembly, offering mechanistic insight into the regulation of GPCR signaling.