Stepwise phosphorylation of BLT1 defines complex assemblies with -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 B-4 (LTB4 ), as a model, our previous work elucidated that this system functions through the modulation of phosphorylation status on two specific residues: Thr(308) and Ser(310) . Ser(310) phosphorylation occurs at a lower LTB4 concentration than Thr(308) , 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 beta-arrestin and subsequent alterations in signal transduction. Stimulation of the high-affinity BLT1 with LTB4 induces phosphorylation at Ser(310) via the ERK1/2-GRK pathway, resulting in a beta-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 beta-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 The(308) -phosphorylation-dependent basal phosphorylation by PKCs. Within the high-LTB4 -induced complex, beta-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 beta-arrestins perform distinct functions.