Functional Switching of BLT1 via Graded Phosphorylation: Transition From a High‐Affinity Form‐Mediated ERK Pathway to a Low‐Affinity State‐Mediated

ABSTRACT

BLT1, activated by leukotriene B ₄ (LTB ₄ ), triggers diverse neutrophil responses in a ligand concentration‐dependent manner: migration begins at low‐LTB ₄ , whereas degranulation requires high concentrations. How BLT1 discriminates between these two regimes has remained unclear. Here, by analyzing BLT mutants lacking specific phosphorylation sites and examining chemotaxis and β‐hexosaminidase release, we reveal that this regulation is governed by a four‐stage phosphorylation process in the C‐terminus. Stage‐1‐phos (Ser ³¹⁴ and Ser ³²⁰ ) involves a post‐translational modification essential for the recruitment of β‐arrestin1/2 to BLT1. Stage‐2‐phos (Ser ³¹⁰ ) occurs immediately after stimulation with low‐LTB ₄ . This modification triggers β‐arrestin1/2 recruitment (in coordination with Stage‐1‐phos), termination of ERK1/2 signaling, and the subsequent structural conversion of BLT1 to a low‐affinity state. Loss of Stage‐2‐phos left migration at low‐LTB ₄ intact but caused sustained ERK1/2 activation and impaired chemotaxis at high‐LTB ₄ . Stage‐3‐phos (Thr ³⁰⁸ ) occurs upon BLT1 reactivation by high‐LTB ₄ and is essential for the subsequent Stage‐4‐phos. Stage‐4‐phos (Ser ³¹³ , Thr ³¹⁵ , and Thr ³²⁴ ) alters the association state of the BLT1‐β‐arrestin1/2 complex, activates PI3K/AKT pathway, and initiates degranulation. Deficiency in either Stage‐3‐phos or Stage‐4‐phos abolished AKT activation and β‐hexosaminidase release while leaving ERK1/2 activation intact. Thus, stepwise phosphorylation enables BLT1 to sense the ambient LTB ₄ concentration and switch its output from ERK1/2 to PI3K‐AKT.