Mechanistic basis for GPCR phosphorylation-dependent allosteric signaling specificity of β -arrestin 1 and 2

β-arrestins (βarr1 andβarr2) are key transducers of G protein-coupled receptor (GPCR) signaling, orchestrating both shared and isoform-specific intracellular pathways. Phosphorylation of the receptor C-terminal tail by GPCR kinases encodes regulatory “barcodes” that modulateβ-arrestin conformations and interactions with downstream effectors. However, how distinct phosphorylation patterns shapeβ-arrestin structure and function remains poorly understood. Here, we combine all-atom molecular dynamics simulations with machine learning and graph neural networks to systematically map the barcode-dependent conformational landscapes ofβ-arrestins bound to the phosphorylated vasopressin receptor 2 tail (V2Rpp). We find that V2Rpp engagesβarr1 more stably thanβarr2, mediated by isoform-specific residue contacts that trigger distinct allosteric responses. These include differential interdomain rotations and rearrangements in key structural motifs, potentially facilitating selective effector protein engagement. Furthermore, we identify critical residue networks that transmit phosphorylation signals to effector-binding interfaces in a barcode- and isoform-specific manner. Notably,βarr1 exhibits stronger allosteric coupling between V2Rpp and c-edge loop 2 compared toβarr2, which is consistent with its enhanced membrane association. Together, these findings advance our understanding of the molecular mechanisms by whichβ-arrestins interpret GPCR phosphorylation signatures, offering a framework that could aid in designing pathway-selective therapeutics.