Distinct Phosphorylation Patterns of AT1R by Biased Ligands and GRK Subtypes.

G protein-coupled receptors (GPCRs) transmit through G proteins upon agonist activation, followed by phosphorylation by GPCR kinases (GRKs) to initiate β-arrestin signaling. However, the molecular mechanisms underlying GPCR signaling regulation by distinct agonists, GRK subtypes, and phosphorylation patterns remain poorly understood. The angiotensin II (AngII) type 1 receptor (AT1R), a prototypical GPCR, serves as an ideal model for studying biased ligands and signaling. Here, we investigated the wild-type (WT) AT1R and mutants of three potential phosphorylation motifs at its C-terminus (Motif I: S326/S328/S331, Motif II: T332/S335/T336/S338, and Motif III: S346/S347/S348/T349) using unbiased agonist AngII, β-arrestin-biased agonist TRV026, and G protein-biased agonist TRV056, along with GRK2/3/5/6 subtypes. We employed phosphorylation assays, β-arrestin pull-down experiments, molecular dynamics simulations, and AlphaFold3 predictions to dissect these mechanisms. Our results reveal that GRK2-mediated AT1R phosphorylation is abolished by mutations in Motifs I and II, with Motif II exhibiting a more pronounced effect. This phosphorylation was enhanced by Gβγ subunits. In contrast, GRK3-mediated phosphorylation remained unaffected by any mutations. GRK5 specifically phosphorylated Motif II, while GRK6 phosphorylated Motif II with the unbiased agonist AngII and both Motifs I and II with biased agonists TRV026 and TRV056. Notably, Motif II mutations reduced β-arrestin1/2 recruitment by GRK5/6 but not GRK2/3. Molecular dynamics simulations demonstrated that Motif II phosphorylation minimized steric hindrance, facilitating stable β-arrestin interactions, whereas Motif I phosphorylation increased intramolecular contacts that potentially impede recruitment. AlphaFold3 models provided detailed insights into the interactions between Motif II and β-arrestin1/2. Collectively, our findings elucidate diverse AT1R phosphorylation patterns driven by different agonists and GRK subtypes, offering a framework for developing signaling-biased AT1R therapeutics by decoding GRK-specific phosphorylation barcodes.