Stepwise phosphorylation of leukotriene B4 receptor 1 defines cellular responses to leukotriene B4

Phosphorylation order mattersThe inflammatory lipid LTB4 stimulates immune cells to migrate toward sites of inflammation and then degranulate. Like other G protein–coupled receptors, the LTB4 receptor BLT1 becomes phosphorylated after ligand-induced activation. Nakanishi et al. identified five residues in the cytoplasmic C-terminal domain of BLT1 that were variably phosphorylated in the absence of LTB4 and two that were phosphorylated upon stimulation with LTB4. Phosphorylation at the LTB4-dependent sites stimulated additional phosphorylation events at the basal phosphorylation sites. The sequence of these phosphorylation events depended on the concentration of LTB4 and was required for chemotaxis and degranulation in response to high concentrations of LTB4. These findings suggest a mechanism whereby increasing concentrations of LTB4 induce distinct responses as cells migrate up an LTB4 gradient.Leukotriene B4 (LTB4) receptor type 1 (BLT1) is abundant in phagocytic and immune cells and plays crucial roles in various inflammatory diseases. BLT1 is phosphorylated at several serine and threonine residues upon stimulation with the inflammatory lipid LTB4. Using Phos-tag gel electrophoresis to separate differentially phosphorylated forms of BLT1, we identified two distinct types of phosphorylation, basal and ligand-induced, in the carboxyl terminus of human BLT1. In the absence of LTB4, the basal phosphorylation sites were modified to various degrees, giving rise to many different phosphorylated forms of BLT1. Different concentrations of LTB4 induced distinct phosphorylation events, and these ligand-induced modifications facilitated additional phosphorylation events at the basal phosphorylation sites. Because neutrophils migrate toward inflammatory sites along a gradient of LTB4, the degree of BLT1 phosphorylation likely increases in parallel with the increase in LTB4 concentration as the cells migrate. At high concentrations of LTB4, deficiencies in these two types of phosphorylation events impaired chemotaxis and β-hexosaminidase release, a proxy for degranulation, in Chinese hamster ovary (CHO-K1) and rat basophilic leukemia (RBL-2H3) cells, respectively. These results suggest that an LTB4 gradient around inflammatory sites enhances BLT1 phosphorylation in a stepwise manner to facilitate the precise migration of phagocytic and immune cells and the initiation of local responses, including degranulation.