Stepwise phosphorylation of BLT1 defines complex assemblies with β-arrestin serving distinct functions

G protein-coupled receptors (GPCRs) utilize complex cellular systems to respond to diverse ligand concentrations. By taking BLT1, a GPCR for leukotriene B₄ (LTB₄ ), as a model, our previous work elucidated that this system functions through the modulation of phosphorylation status on two specific residues: Thr³⁰⁸ and Ser³¹⁰ . Ser³¹⁰ phosphorylation occurs at a lower LTB₄ concentration than Thr³⁰⁸ , leading to a shift in ligand affinity from a high-to-low state. However, the implications of BLT1 phosphorylation in signal transduction processes or the underlying mechanisms have remained unclear. Here, we identify the sequential BLT1-engaged conformations of β-arrestin and subsequent alterations in signal transduction. Stimulation of the high-affinity BLT1 with LTB₄ induces phosphorylation at Ser³¹⁰ via the ERK1/2-GRK pathway, resulting in a β-arrestin-bound low-affinity state. This configuration, referred to as the "low-LTB₄ -induced complex," necessitates the finger loop region and the phosphoinositide-binding motif of β-arrestins to interact with BLT1 and deactivates the ERK1/2 signaling. Under high LTB₄ concentrations, the low-affinity BLT1 again binds to the ligand and triggers the generation of the low-LTB₄ -induced complex into a different form termed "high-LTB₄ -induced complex." This change is propelled by The³⁰⁸ -phosphorylation-dependent basal phosphorylation by PKCs. Within the high-LTB₄ -induced complex, β-arrestin adapts a unique configuration that involves additional N domain interaction to the low-affinity BLT1 and stimulates the PI3K/AKT pathway. We propose that the stepwise phosphorylation of BLT1 defines the formation of complex assemblies, wherein β-arrestins perform distinct functions.