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

Abstract

AbstractG 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.