Research ArticleBiochemistry

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

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Science Signaling  21 Aug 2018:
Vol. 11, Issue 544, eaao5390
DOI: 10.1126/scisignal.aao5390

Phosphorylation order matters

The 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.

Abstract

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.

INTRODUCTION

With more than 900 members, G protein–coupled receptors (GPCRs) are the largest family of cellular receptors (1). Binding of an agonist to a GPCR induces a conformational change in the receptor, which stimulates dissociation of the heterotrimeric G proteins associated with the receptor. The dissociated Gα and Gβγ subunits stimulate diverse cellular responses through effector proteins and intracellular second messengers (2). When the activated receptors are phosphorylated on Ser and Thr residues within the intracellular loops and the carboxyl tail, cellular responses to subsequent stimuli are usually attenuated, a phenomenon known as desensitization. The functional significance of GPCR phosphorylation has also been characterized in contexts other than desensitization. For example, the distinct GPCR phosphorylation profiles mediated by different kinases (the phospho-barcodes) explain the multidimensional features of GPCR signaling networks (36). Thus, precise analysis of the phosphorylation of specific GPCRs is useful for clarifying the biological functions of each receptor.

Leukotriene B4 (LTB4) is a lipid mediator that is derived from arachidonic acid in the plasma membrane and released into the extracellular milieu (79), where it acts as a potent chemoattractant for neutrophils and lymphocytes (10). Experiments using microscale arrays have demonstrated that the LTB4 chemotactic gradient is important for triggering neutrophil swarming (11, 12). The LTB4 receptor type 1 (BLT1) is a high-affinity LTB4 receptor that is abundant in and contributes to the activation and migration of phagocytic and immune cells, such as neutrophils, eosinophils, dendritic cells, macrophages, B cells, and T cells (13). BLT1 is a GPCR and activates at least two different Gα family members, including the pertussis toxin (PTX)–sensitive Gi/o and PTX-insensitive Gq alpha proteins (14, 15). The activation of these Gα proteins by BLT1 results in phosphoinositide hydrolysis, intracellular Ca2+ mobilization, and a decrease in cellular cyclic adenosine monophosphate, followed by chemotaxis and degranulation (15, 16). Like other GPCRs, BLT1 is phosphorylated in its C-terminal tail after activation. Several studies have reported the phosphorylation of BLT1 (1719), yet the precise phosphorylation sites and their biological importance remain to be determined.

Here, we determined the amino acid residues responsible for basal (constitutive) and ligand-induced phosphorylation of human BLT1 using Phos-tag SDS–polyacrylamide gel electrophoresis (PAGE), in which a small molecule that is incorporated into the polyacrylamide gel matrix slows the mobility of phosphorylated proteins by specifically binding to phosphate groups. Subsequent mutational analyses established the phosphorylation hierarchies of these residues. To better understand the biological roles of these phosphorylation events, we examined chemotaxis and β-hexosaminidase release elicited by different concentrations of LTB4 using BLT1 mutants lacking the identified phosphorylation sites. We found that an LTB4 gradient boosted BLT1 phosphorylation in a stepwise manner to facilitate the precise migration of immune cells and the initiation of local responses, including degranulation.

RESULTS

BLT1 is phosphorylated basally and LTB4 inducibly

Because BLT1 on the cell surface is covalently modified with N-glycan moieties of various molecular masses, wild-type human BLT1 that is N-terminally tagged with hemagglutinin (HA-BLT1/WT) and expressed in HeLa cells appears as diffuse bands by SDS-PAGE (fig. S1A), making it difficult to detect the phosphorylation of BLT1 as a band shift in Western blot analysis. To eliminate this problem, we generated a BLT1 mutant in which the two putative N-glycosylation sites in the extracellular domain, Asn2 and Asn164, were mutated to Ala. The resulting protein, HA-BLT1/0N, is a prospective N-glycosylation–deficient receptor. When expressed in HeLa cells, HA-BLT1/0N appeared as a sharp band on the SDS-PAGE (fig. S1A), although the band migrated slightly more slowly than that of endoglycosidase-treated HA-BLT1/WT (fig. S1A). This difference could be due to posttranslational modifications other than N-glycosylation. The trafficking of HA-BLT1/0N to the cell surface was identical to that of HA-BLT1/WT (fig. S1B). Furthermore, HA-BLT1/0N showed LTB4-elicited intracellular Ca2+ mobilization, although this activity was slightly lower than that of HA-BLT1/WT (fig. S1C). These results indicate that HA-BLT1/0N is a useful tool for the analysis of BLT1 phosphorylation sites.

We performed Western blot analysis of HA-BLT1/0N expressed in HeLa cells with or without LTB4 stimulation. Although LTB4 stimulation led to the appearance of a shifted band on the polyacrylamide gel, the separation of phosphorylated and nonphosphorylated HA-BLT1/0N was not sufficient for precise analysis (Fig. 1A). Therefore, we used Phos-tag technology with SDS-PAGE, which allows for the specific separation of various phosphorylated forms of proteins (20). HA-BLT1/0N migrated as multiple bands on the Phos-tag SDS-PAGE without LTB4 stimulation (Fig. 1A), suggesting that various LTB4-independent phosphorylations occur at multiple sites. Moreover, additional slower migrating bands appeared after LTB4 stimulation. These shifted bands disappeared with alkaline phosphatase (AP) treatment (Fig. 1B), demonstrating that these shifted bands were due to the phosphorylation of HA-BLT1/0N. We also examined the phosphorylation states of HA-BLT1/WT from LTB4-treated cells after treatment with endoglycosidase by Phos-tag SDS-PAGE. There was no substantial difference in the band patterns between endoglycosidase-treated HA-BLT1/WT and HA-BLT1/0N (Fig. 1C), suggesting that N-glycosylation has no effect on these LTB4-induced modifications. Moreover, BLT1 endogenously expressed in retinoic acid–stimulated human promyelocytic leukemia (HL-60) cells shared a phosphorylation pattern similar to that of exogenously expressed HA-BLT1/WT (Fig. 1D). Together, these data demonstrate that BLT1 was phosphorylated to various extents even in the resting state (henceforth referred to as “basal phosphorylation”) and that LTB4 stimulation induced additional phosphorylation events (henceforth referred to as “LTB4-induced phosphorylation”).

Fig. 1 Phosphorylation of human BLT1.

(A) HeLa cells expressing HA-tagged BLT1/0N (HA-BLT1/0N) were stimulated with LTB4, and membrane fractions were subjected to SDS-PAGE and Phos-tag SDS-PAGE. Receptors were detected by immunoblotting (IB) for the HA tag. β-Actin is an experimental and loading control. Mock, empty vector; WCL, whole-cell lysates. (B) HeLa cells expressing HA-BLT1/0N were stimulated with LTB4, and membrane fractions were treated with calf intestine alkaline phosphatase (CIAP) before Phos-tag SDS-PAGE and immunoblotting for HA. (C) HeLa cells expressing wild-type BLT1 (BLT1/WT) or a form of BLT1 lacking the extracellular N-glycosylation sites (BLT1/0N) were stimulated with LTB4, and membrane fractions were treated with peptide-N-glycosidase F (PNGase-F) before Phos-tag SDS-PAGE and immunoblotting for HA. (D) HL-60 cells cultivated in the presence of all-trans retinoic acid (ATRA) were stimulated with LTB4, and membrane fractions were treated with PNGase-F before Phos-tag SDS-PAGE and immunoblotting for BLT1. All blots are representative of at least three independent experiments.

BLT1 has seven phosphorylated residues in its C terminus

GPCR phosphorylation occurs on Ser and Thr residues in the cytoplasmic domains (21). There are 21 Ser and Thr residues that are conserved among the cytoplasmic domains of human, mouse, rat, guinea pig, and zebrafish BLT1s (fig. S2). Because kinases require specific motifs around the target residue, we excluded Ser343 and Ser344 from our analysis because of the low homology surrounding these residues among different species. To identify the amino acid residues critical for basal phosphorylation of BLT1, we divided the remaining 19 residues into 10 subgroups based on the proximity of the residues and generated 10 mutant HA-BLT1/0N receptors in which these residues were replaced with Ala (Fig. 2A). The Phos-tag SDS-PAGE analyses revealed that Ser and Thr residues in the intracellular loops were not part of the basal phosphorylation profile; however, the iL-1 mutant (S51A and T53A) was not produced in HeLa cells, as previously reported (Fig. 2B) (22). The bands for mutants iL-2B (S125A and T130A) and iL-3B (S216A and T219A) showed a slight difference in mobility compared to HA-BLT1/0N (Fig. 2B), possibly because we used a twofold greater volume of the mutant samples relative to HA-BLT1/0N to generate comparable signal intensity. On the other hand, the mutant forms of HA-BLT/0N bearing substitution mutations in the C terminus, CT-3 (S313A, S314A, and T315A) and CT-4 (S320A and T324A), displayed aberrant basally phosphorylated bands after Phos-tag SDS-PAGE (Fig. 2B). Next, we examined the amino acid residues involved in LTB4-induced phosphorylation. Previous reports demonstrated that the substitution of all Ser and Thr residues in the C-terminal domain with Ala led to a notable disappearance of LTB4-induced phosphorylation, suggesting that the phosphorylation of these sites depends on LTB4 (17). Thus, we focused our mutational analyses on Ser and Thr residues in the C-terminal domain. Mutant receptor CT-2 (T308A and S310A) did not show any band shifts upon LTB4 treatment (Fig. 2C). These experiments identified Ser313, Ser 314, Thr315, Ser320, and Thr324 as candidates for basal phosphorylation and Ser310 and Thr308 as candidates for LTB4-induced phosphorylation.

Fig. 2 Residues essential for phosphorylation of BLT1 cytoplasmic domains.

(A) Conserved Ser (S) and Thr (T) residues in the cytoplasmic domains of human BLT1. The indicated 10 human HA-BLT1/0N mutants were generated. (B) HA-BLT1 mutants were expressed in HeLa cells, and membrane fractions were examined by Phos-tag SDS-PAGE followed by immunoblotting for HA. β-Actin is an experimental and loading control. (C) HeLa cells expressing the indicated HA-tagged receptors were treated with 100 nM LTB4 for 40 min before separation of membrane fractions by Phos-tag SDS-PAGE and immunoblotting for HA. (D) The indicated HA-BLT1/0N mutants were constructed to determine the phosphorylation sites in the BLT1 C-terminal domain. Thr308, Ser310, Ser313, Ser314, Thr315, Ser320, and Thr324 were substituted with Ala (A) as indicated. 7TM, transmembrane domain 7; H8, helix 8. (E) HA-BLT1/0N and the indicated mutants were expressed in HeLa cells, and membrane fractions were examined by Phos-tag SDS-PAGE followed by immunoblotting for HA. (F) Membrane fractions from HeLa cells expressing the indicated mutant forms of BLT1 treated with 10 or 100 nM LTB4 for 40 min were separated by Phos-tag SDS-PAGE and immunoblotted for HA. All blots are representative of at least three independent experiments.

To further characterize these seven potential basal and LTB4-induced phosphorylation sites, we generated 12 additional mutants in which these residues were mutated individually and in various combinations (Fig. 2D). Mutants CT-3 and CT-4 have a combined five Ser and Thr residues, Ser313, Ser314, Thr315, Ser320, and Thr324, mutated to Ala. To determine whether all or only a subset of these residues was basally phosphorylated, we constructed five mutants in which only one of these five residues was not substituted with Ala (BLT1/0N/S313, BLT1/0N/S314, BLT1/0N/T315, BLT1/0N/S320, and BLT1/0N/T324). Each of these five mutants migrated more slowly, and substitution of all five residues (BLT1/0NΔb-phos) led to the complete disappearance of shifted bands in the absence of LTB4 (Fig. 2, C, E, and F). These results indicate that all five Ser and Thr residues in this C-terminal region (Ser313, Ser314, Thr315, Ser320, and Thr324) contribute to the basal phosphorylation of HA-BLT1/0N. To address the residues mutated in the CT-2 mutant that showed aberrant LTB4-induced phosphorylation (Ser310 and Thr308), we generated mutants in which only one of these two residues was mutated (BLT1/0N/T308 and BLT1/0N/S310, respectively) and mutants in which one of these two residues was mutated in addition to the five basally phosphorylated residues (Δb/T308 and Δb/S310, respectively). Each of these mutants still exhibited the LTB4-induced shifts, indicating that both residues were involved in LTB4-induced phosphorylation (Fig. 2F). Moreover, the BLT1/0NΔb-phos mutant, completely devoid of basal phosphorylation, displayed LTB4-stimulated band shifts (Fig. 2F), demonstrating that basal phosphorylation was not required for LTB4-induced phosphorylation.

We next compared three mutant receptors in which all five of the residues that were basally phosphorylated were mutated (HA-BLT1/0NΔb-phos), both of the residues that exhibited LTB4-induced phosphorylation were mutated (HA-BLT1/0NΔi-phos), or all seven residues were mutated (HA-BLT1/0NΔphos) (Fig. 2D). HA-BLT1/0NΔb-phos and HA-BLT1/0NΔi-phos exhibited greatly reduced basal phosphorylation and LTB4-induced phosphorylation, respectively, when expressed in HeLa cells (fig. S3A). Furthermore, bands corresponding to both basal phosphorylation and LTB4-induced phosphorylation disappeared in cells expressing HA-BLT1/0NΔphos (fig. S3A). These phenomena were reproduced in Chinese hamster ovary (CHO-K1) cells, murine interleukin-3–dependent pro-B (Ba/F3) cells, and rat basophilic leukemia (RBL-2H3) cells, implying that the observed modifications occur in many different cell types (fig. S3B). The seven BLT1 residues that we identified as basally phosphorylated and phosphorylated in an LTB4-dependent manner are conserved among humans, mice, rats, guinea pigs, and zebrafish (fig. S2). We observed similar phosphorylation of the corresponding residues in mouse BLT1 by generating and analyzing mBLT1/0N, mBLT1/0NΔb-phos, mBLT1/0NΔi-phos, mBLT1/0NΔphos, mBLT1/0N/S313, mBLT1/0N/S314, mBLT1/0N/T315, mBLT1/0N/T320, mBLT1/0N/T324, mBLT1/0N/T308, and mBLT1/0N/S310 mutants (fig. S4A). In mouse, Ser313 and Ser314 contributed to basal phosphorylation, whereas phosphorylation of Thr308 and Ser310 occurred upon stimulation with LTB4 (fig. S4B). Thus, these data further support that the observed modifications are not species-specific. Collectively, our data suggest that human BLT1 has five basal phosphorylation sites (Ser313, Ser314, Thr315, Ser320, and Thr324) and two LTB4-induced phosphorylation sites (Thr308 and Ser310) in its C-terminal region. The presence of many distinctly migrating bands for the wild-type protein and each mutant form indicates that phosphorylation of these residues may occur in many different combinations.

Phosphorylation of BLT1 is facilitated by dose-dependent LTB4 stimulation

To evaluate whether different concentrations of LTB4 affected the extent of human BLT1 phosphorylation, we stimulated HeLa cells expressing HA-BLT1/0N or HA-BLT1/0NΔb-phos with various concentrations of LTB4. The LTB4-induced phosphorylation of both HA-BLT1/0N and HA-BLT1/0NΔb-phos increased in a dose-dependent manner (Fig. 3, A and B). For the HA-BLT1/0NΔb-phos mutant, one of the LTB4-induced phosphorylated species initially appeared at 1 nM LTB4, but the other LTB4-induced phosphorylated species (phosphorylated on both Ser310 and Thr308) only appeared in LTB4 concentrations at or above 30 nM (Fig. 3A). On the basis of the position of the band, the faster-migrating phosphorylated species appearing at 1 nM LTB4 likely corresponded to phosphorylation on Ser310 (Fig. 2F), and the slower migrating band likely corresponded to phosphorylation on both Ser310 and Thr308. These results raise the possibility that each residue that is phosphorylated in an LTB4-dependent manner can act as a sensor for different LTB4 concentrations. Moreover, the total phosphorylation of BLT1/0N was initially enhanced by 1 nM LTB4, followed by a gradual increase, with the maximal phosphorylation observed at concentrations more than 30 nM (Fig. 3B). These data further suggest that the phosphorylation of each LTB4-induced phosphorylation site could trigger additional phosphorylation events at the sites initially identified as basal phosphorylation sites.

Fig. 3 Enhancement of BLT1 phosphorylation by ligand in a dose-dependent manner.

(A and B) HeLa cells expressing HA-BLT1/0NΔb-phos, which lacks the five basal phosphorylation sites (A) or HA-BLT1/0N (B), were stimulated with the indicated LTB4 concentrations for 40 min. Membrane fractions were subjected to Phos-tag SDS-PAGE followed by immunoblotting for HA. The positions of BLT1 phosphorylated on Ser310 (p-Ser310) and Thr308 (p-Thr308) are noted. β-Actin is an experimental and loading control. Blots are representative of at least three independent experiments. p, phosphorylated.

Stepwise addition of LTB4 enhances BLT1 phosphorylation

The cell responses to activation of BLT1, such as an increase in intracellular [Ca2+], are immediately shut down after the initial stimulation of LTB4 (23). The rise in intracellular [Ca2+] in HeLa cells expressing HA-BLT1/0N in response to 100 nM LTB4 decreased when the cells were prestimulated with 10 nM LTB4 (Fig. 4A). Exposing cells to 100 nM LTB4 after stimulation with 10 nM LTB4 increased the extent to which BLT1/0N was phosphorylated compared to stimulation with 10 nM LTB4 alone (Fig. 4B) and did not stimulate additional activation of extracellular signal–regulated kinases 1 and 2 (ERK1/2) after the cells were desensitized by the primary stimulation (Fig. 4B). Similar results were obtained in CHO-K1, Ba/F3, and RBL-2H3 cells (Fig. 4C). In HeLa cells expressing BLT1/0N, the additional phosphorylation in response to 100 nM LTB4 treatment after prestimulation was greatly diminished by cotreatment with an equal amount of BLT1 antagonist, CP105696 or ZK158252 (Fig. 4, D and E) (24). Previously, Okuno et al. found that the conformation of human BLT1 switches from a high-affinity state to a low-affinity state after activation of the downstream G proteins (25). These results raise the possibility that a low-affinity form of BLT1 remains on the cell surface after ligand stimulation and could be modified by additional phosphorylation events upon exposure to higher LTB4 concentrations. To investigate the existence of BLT1 on the cell surface after stimulation with 10 nM LTB4, we fluorescently labeled BLT1 using sortase A (SrtA)–mediated transpeptidation, known as sortagging (26, 27), and examined the trafficking of the labeled BLT1 after stimulation with LTB4. It has been reported that only a portion (~30%) of BLT1 undergoes internalizations after stimulation with LTB4 (17, 19, 28, 29). In keeping with these findings, a substantial amount of both the labeled BLT1/0N and BLT1/0NΔphos remained on the surface of Ba/F3 cells at least 20 min after stimulation with 10 nM LTB4, with only ~5% detected in the intracellular vesicles. These data suggest that BLT1 remains on the cell surface after ligand stimulation and thus could be modified by additional phosphorylation events upon exposure to higher LTB4 concentrations (Fig. 4, F and G).

Fig. 4 Enhanced phosphorylation of BLT1 by sequential exposure to increasing concentrations of LTB4.

(A) Intracellular [Ca2+] in HeLa cells expressing HA-BLT1/0N that were pretreated with 10 nM LTB4 or vehicle before being stimulated with 100 nM LTB4. The response to adenosine triphosphate (ATP) was included as a positive control. (B) HeLa cells expressing HA-BLT1/0N were stimulated with LTB4 as indicated. Membrane fractions were subjected to Phos-tag SDS-PAGE and immunoblotting for HA. WCLs were subjected to SDS-PAGE and immunoblotting for ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2). β-Actin is an experimental and loading control. (C) RBL-2H3, CHO-K1, and Ba/F3 cells expressing HA-BLT1/0N were stimulated with LTB4 as indicated. Membrane fractions were separated by Phos-tag SDS-PAGE and immunoblotted for HA. (D) HeLa cells expressing HA-BLT1/0N were stimulated with LTB4 in the presence or absence of 100 nM CP105696 (CP) or ZK158252 (ZK). Membrane fractions were separated by Phos-tag SDS-PAGE and immunoblotted for HA. (E) HeLa cells expressing HA-BLT1/0N were incubated with 10 nM LTB4 for 20 min followed by the addition of 100 nM LTB4 alone or 100 nM LTB4 plus the presence or absence of 100 nM CP105696 or ZK158252. Membrane fractions were separated by Phos-tag SDS-PAGE and immunoblotted for HA. (F) Confocal microscopic images of surface and intracellular BLT1 in Ba/F3 cells expressing SrtA-HA-BLT1/0N or SrtA-HA-BLT1/0NΔphos. The cytosol of the cells expressing SrtA-HA-BLT1/0N was stained with CytoRed (red), and the cytosol of cells expressing SrtA-HA-BLT1/0NΔphos was stained with ViVidFluor Cell Blue CMAC (blue). These cells were mixed and immobilized on a collagen-coated dish. The BLT1 proteins were expressed with a SrtA tag, which allowed them to be labeled with Alexa Fluor 488 (AF488) on the cell surface (green). Cells were imaged immediately after the addition of 10 nM LTB4 (left) or 20 min later (right). Clockwise from upper left: AF488-labeled BLT1/0N and BLT1/0NΔphos (green), differential interference contrast image of the cells, BLT1/0N in the cytoplasm (red), and BLT1/0NΔphos in the cytoplasm (blue). Scale bars, 20 μm. (G) Changes in the distribution of AF488-labeled BLT1/0N and BLT1/0NΔphos between the plasma membrane and cytoplasm after 20 min of LTB4 stimulation. The mean relative brightness values of the plasma membrane and the intracellular compartment in the cells expressing BLT1/0N (red) and BLT1/0NΔphos (blue) were calculated by averaging the 10 cell values in (F). Data are means ± SEM, n = 10 cells. *P < 0.05 by Student’s t test. All data are representative of at least three independent experiments.

LTB4-induced phosphorylation facilitates the degree of BLT1 basal phosphorylation

We investigated whether LTB4-induced phosphorylation affected the degree to which basal phosphorylation sites were modified during the stepwise addition of LTB4 in HeLa cells. After primary (10 nM) and stepwise (100 nM) additions of LTB4, a larger proportion of BLT1/0N was highly phosphorylated compared to nonphosphorylated BLT1/0N (Fig. 5, A and B). In contrast, the highly phosphorylated forms of BLT1/Δi-phos were substantially reduced, and the nonphosphorylated forms were increased, compared to BLT1/0N, even after stimulation with 100 nM LTB4 (Fig. 5, A and B). Because the total phosphorylation of BLT1 increased in parallel with the modification of each LTB4-induced phoshorylation site (Fig. 3), the reduction in highly phosphorylated forms observed with BLT1/Δi-phos could be due to a decrease in modification of basal phosphorylation sites that depended on LTB4-induced phosphorylation events. Eliminating one of the two LTB4-induced phosphorylation sites (mutants BLT1/0N/S310 and BLT1/0N/T308) resulted in an increase in partial phosphorylations upon stepwise stimulation (Fig. 5C), supporting the requirement of LTB4-induced phosphorylation for further phosphorylation at basal sites. Because the phosphorylation of Ser310 occurred at a lower LTB4 concentration than did the phosphorylation of Thr308 (Figs. 2F and 5C), we propose a model for the sequential phosphorylation of human BLT1 (Fig. 5D). Because BLT1 is exposed to a gradual increase in LTB4 concentration during the migration of neutrophils, the extent of phosphorylation may increase in parallel with these physiological changes in LTB4.

Fig. 5 Facilitation of basal phosphorylation site modification by LTB4-induced phoshorylation.

(A) HeLa cells expressing HA-BLT1/0N or HA-BLT1/0NΔi-phos, which lacks both LTB4-induced phosphorylation sites, were stimulated with LTB4 as indicated. Membrane fractions were subjected to Phos-tag SDS-PAGE and immunoblotted for HA. The two areas surrounded by dashed lines are defined as Y and X, the nonphosphorylated and highly phosphorylated bands, respectively. β-Actin is an experimental and loading control. (B) The signal intensities of areas X and Y were determined by densitometric analysis, and the ratios were expressed as the percentage of the total intensity. Data are means ± SEM, n = 3 experiments. NS, not significant; *P < 0.05 and **P < 0.01 by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. (C) HeLa cells expressing the indicated forms of HA-BLT1 were stimulated with LTB4 as indicated. Membrane fractions were subjected to Phos-tag SDS-PAGE and immunoblotted for HA. All data are representative of at least three independent experiments. (D) Schematic model of the sequential phosphorylations of BLT1. In the absence of LTB4, some basal sites are phosphorylated. Low concentrations of LTB4 elicit phosphorylation of Ser310, which stimulates additional phosphorylation of basal phosphorylation sites. Higher concentrations of LTB4 stimulate phosphorylation ofThr308, which, in turn, promotes further phosphorylation events at basal sites in the C-terminal region.

Activation of Gi is required for LTB4-induced phosphorylation

We examined the functional significance of LTB4-induced stepwise phosphorylation of human BLT1 in HeLa cells. First, surface trafficking similar to that of BLT1/0N was confirmed for each of three mutants, BLT1/0NΔb-phos, BLT1/0NΔi-phos, and BLT1/0NΔphos, indicating that these mutations did not affect the surface trafficking of BLT1 (fig. S5A). Previously, several reports demonstrated that GPCR-dependent chemotaxis by polymorphonuclear (PMN) leukocytes requires both ERK1/2 and the phosphoinositide 3-kinase (PI3K)–protein kinase B (also called AKT) pathways to facilitate proper cell polarity and motility (16, 3036). On the other hand, an intracellular [Ca2+] increase and the PI3K-AKT pathway are required for PMN leukocyte degranulation (16, 3036), but signaling through mitogen-activated protein kinases (MAPKs) such as ERK1/2 is not (fig. S5B) (37). Because the activation of Gi by BLT1 is a prerequisite for the initiation of both motility and degranulation, we assessed the importance of Gi activation with respect to BLT1 phosphorylation. Pretreatment with PTX, which prevents Gi proteins from being activated by GPCRs, before stepwise LTB4 stimulation substantially reduced ligand-induced phosphorylation of BLT1, suggesting that the activation of Gi is required for these phosphorylations (fig. S5C).

[Ca2+] increase is a prerequisite for LTB4-induced phosphorylation

With respect to the relationship between the intracellular Ca2+ mobilization and BLT1 phosphorylation, there is no difference in the LTB4 dependency of the Ca2+ influx between BLT1/0N and BLT1/0NΔphos (fig. S6), implying that the basal phosphorylation is not essential for triggering a [Ca2+] response. The BLT1/0N/DE mutant, in which all phosphorylation sites are replaced with Asp or Glu, was also used to simulate the fully phosphorylated form (38). The finding that BLT1/0N/DE requires higher doses of ligand to obtain a response similar to that of BLT1/0N may be due to the reduced affinity for LTB4 (17). LTB4-induced phosphorylation of BLT1/0N was impaired in the presence of 1 mM EGTA, demonstrating the necessity of the [Ca2+] increase for this modification (fig. S7). Similar results were obtained by the treatment with 100 μM SFK96365, a Ca2+ channel blocker (fig. S7), indicating that the [Ca2+] increase is a prerequisite for LTB4-induced phosphorylation.

Lack of phosphorylation impairs chemotaxis induced by high LTB4 concentration

To gain further insight into the biological importance of BLT1 phosphorylation, we investigated chemotactic activity in CHO-K1 cells expressing human BLT1/0N or BLT1/0NΔphos. Cells expressing BLT1/0NΔphos exhibited impaired chemotaxis at 100 nM LTB4 (Fig. 6, A and B) but exhibited no impairment of chemokinesis at this concentration of LTB4 compared to cells expressing BLT1/0N (Fig. 6B). Real-time monitoring of cell migration using TAXIScan-FL revealed that 100 nM LTB4 significantly reduced the migration directionality, but increased the migration velocity, of cells expressing hBLT1/0NΔphos compared to cells expressing hBLT1/0N (Fig. 6C). Because BLT1/0N/DE-expressing cells showed a chemotactic activity similar to that of cells expressing BLT1/0N (Fig. 6, A and C), the aberrant chemotaxis of BLT1/0NΔphos at 100 nM LTB4 could be due to the lack of phosphorylation. Previous reports demonstrated that chemotaxis requires an LTB4-elicited intracellular Ca2+ influx followed by the activation of MAPKs (30, 31). At 100 nM LTB4, cells expressing BLT1/0NΔphos exhibited a sustained [Ca2+] increase compared to the transient response of cells expressing BLT1/0N, although the peak Ca2+ influx was similar (Fig. 6D). Moreover, sustained activation of ERK1/2 was observed after stimulation with 100 nM LTB4 in cells expressing BLT1/0NΔphos, whereas activation was transient in cells expressing BLT1/0N (Fig. 6E). Accordingly, we observed increased expression of a MAPK signaling reporter in cells expressing BLT1/0NΔphos compared to cells expressing BLT1/0N (Fig. 6F). In contrast, these cells showed substantially reduced AKT activation after the stepwise addition of 100 nM LTB4 (Fig. 6G). This abnormal signaling in cells expressing BLT1/0NΔphos at high concentrations of LTB4 might be related to the aberrant chemotaxis displayed by these cells at high concentrations of LTB4.

Fig. 6 Effect of phosphorylation deficiency on chemotaxis.

(A) Chemotaxis of CHO-K1 cells expressing the indicated forms of BLT1 in the presence of the indicated concentrations of LTB4. Chemotaxis was quantified by the number of cells that migrated across a porous barrier as measured by an optical density at 595 nm (OD595). Data are means ± SEM, n = 3 experiments. **P < 0.01 by two-way ANOVA followed by Tukey’s post hoc test. (B) LTB4-induced chemotaxis and chemokinesis of CHO-K1 cells expressing the indicated forms of BLT1. Data are means ± SEM, n = 5 experiments. ***P < 0.001 by two-way ANOVA followed by Tukey’s post hoc test. (C) Directionality and velocity of CHO-K1 cells expressing the indicated forms of BLT1 as they migrated along an LTB4 gradient (0 to 100 nM). Each data point represents an individual cell. Data are means ± SEM, n > 20 cells. *P < 0.05; ***P < 0.001 by Kruskal-Wallis test with Dunn’s post hoc multiple comparisons tests. (D) Intracellular Ca2+ mobilization in response to the indicated doses of LTB4 in CHO-K1 cells expressing the indicated forms of BLT1. (E) Western blot showing ERK1/2 and p-ERK1/2 at the indicated time points after LTB4 stimulation in WCLs from CHO-K1 cells expressing the indicated forms of BLT1. β-Actin is an experimental and loading control. (F) MAPK reporter activity in LTB4-stimulated HeLa cells expressing the indicated forms of BLT1, serum response element (SRE)–firefly luciferase (MAPK-responsive), and cytomegalovirus (CMV)–Renilla luciferase (constitutively expressed). Reporter activity was quantified as the ratio of firefly/Renilla luciferase activity. Data are means ± SEM, n = 3 experiments. *P < 0.05 by two-way ANOVA followed by Tukey-Kramer test. (G) Western blot showing AKT and phosphorylated AKT (p-AKT) in lysates from CHO-K1 cells expressing BLT1/0N or BLT1/0NΔphos at the indicated points after stepwise LTB4 stimulation. All data are representative of at least three independent experiments.

Phosphorylation defect reduces the release of LTB4-induced β-hexosaminidase

Previous research has demonstrated that the LTB4-BLT1 axis is involved in degranulation of both human PMN leukocytes (39) and RBL-2H3 cells stably expressing BLT1 (16, 40). The PI3K-AKT pathway (36), but not the MAPK pathway (37), plays a key role in degranulation of PMN leukocytes. Because signaling through BLT1/0NΔphos produced an aberrant AKT response to LTB4 (Fig. 6G), we next investigated whether the phosphorylation deficiency in this mutant form of BLT1 affected the LTB4-induced release of β-hexosaminidase, a mast cell lysosomal enzyme that is released upon degranulation, in RBL-2H3 cells. First, we assessed the effect of the AKT inhibitor MK-2206 on the release of β-hexosaminidase by these cells. The activation of AKT observed during the stepwise addition of LTB4 was markedly reduced by pretreatment with 5 μM MK-2206, although ERK1/2 activation was not affected (Fig. 7A). The release of β-hexosaminidase elicited by 100 nM LTB4 was significantly decreased, indicating that AKT activation was required for LTB4-induced degranulation in RBL-2H3 cells (Fig. 7B). In RBL-2H3 cells expressing BLT1/0N, the release of β-hexosaminidase was significantly increased by the stepwise addition of LTB4, from 10 to 100 nM. However, the stepwise addition of LTB4 did not enhance the release of β-hexosaminidase in the cells expressing BLT1/0NΔphos (Fig. 7C). Consistent with this, in the BLT1/0NΔphos-expressing cells, the activation of AKT induced by LTB4 was impaired (Fig. 7, D and E), as was also found for CHO-K1 cells overexpressing human BLT1/0NΔphos (Fig. 6G). Together, these data demonstrate that the phosphorylation of BLT1 is pivotal for the promotion of PI3K-AKT signaling, leading to β-hexosaminidase release in RBL-2H3 cells.

Fig. 7 Requirement of phosphorylation for degranulation.

(A) Western blot showing ERK1/2, p-ERK1/2, AKT, and p-AKT in lysates from RBL-2H3 cells expressing HA-BLT1/0N and treated with LTB4 as indicated after pretreatment with vehicle or the AKT inhibitor MK-2206. (B) LTB4-stimulated release of β-hexosaminidase from RBL-2H3 cells expressing HA-BLT1/0N in the presence of MK-2206 or the calcium ionophore A23187. Values for β-hexosaminidase release into the medium are expressed as the percentage of the total β-hexosaminidase in the culture. Column 1, vehicle; column 2, 10 nM LTB4 for 40 min; column 3, 10 nM LTB4 for 20 min and then addition of 100 nM LTB4 for 20 min; column 4, 10 nM LTB4 for 20 min and then addition of 100 nM LTB4 for 20 min (pretreated with MK-2206); column 5, 5 μM A23187 for 20 min. Data are means ± SEM, n = 3 experiments. ***P < 0.001 by one-way ANOVA followed by Tukey’s post hoc test. (C) Release of β-hexosaminidase in RBL-2H3 cells expressing the indicated forms of BLT1 under the indicated treatment conditions. Columns 1 and 5, vehicle; columns 2 and 6, 10 nM LTB4 for 40 min; columns 3, 7, and 9, 10 nM LTB4 for 20 min and then addition of 100 nM LTB4 for 20 min; columns 4 and 8, A23187 for 20 min. Data are means ± SEM, n = 3 experiments. ***P < 0.001 by one-way ANOVA followed by Tukey’s post hoc test. (D) Western blot showing Akt and p-Akt in lysates from RBL-2H3 cells expressing BLT1/0N or BLT1/0NΔphos and treated with LTB4 as indicated. (E) Quantification of AKT activation in RBL-2H3 cells expressing the indicated receptors with or without stepwise LTB4 stimulation (10 nM LTB4 for 20 min followed by 100 nM LTB4 for 20 min). Data are means ± SEM, n = 3 experiments. ***P < 0.001 by two-way ANOVA followed by Tukey’s post hoc test. All data are representative of at least three independent experiments.

Lack of phosphorylation impairs the function of wild-type BLT1

We generated various mutant forms of the prospective N-glycosylation–deficient receptor BLT1/0N (fig. S1A) and performed functional studies using these mutants, demonstrating aberrant migration and impaired enzyme release resulting from the lack of BLT1 phosphorylation. To eliminate issues regarding surface trafficking, stability, and ligand affinity when using the glycosylation-deficient BLT1s, we further carried out similar functional experiments using human wild-type BLT1 (BLT1/WT and BLT1/WTΔphos), in which the glycosylation sites were intact. There was no difference in the surface trafficking or stability between BLT1/WT and BLT1/WTΔphos (fig. S8, A and B). We observed similar intracellular [Ca2+] increases upon LTB4 stimulation in cells expressing BLT1/WT or BLT1/WTΔphos, although the [Ca2+] response in cells expressing BLT1/WTΔphos was slightly sustained at 100 nM, similar to the nonglycosylated mutant (fig. S8C). In contrast, we found significant reductions in chemotaxis and β-hexosaminidase release in BLT1/WTΔphos elicited by 100 nM LTB4 (fig. S8, D and E). All these data are consistent with those from experiments using glycosylation-deficient receptors, indicating that the results are not specific to glycosylation-deficient receptors and supporting the biological importance of phosphorylation in native glycosylated forms of the protein.

Phosphorylation is necessary for the low-affinity conformation

Previously, Okuno et al. indicated that human BLT1 changes conformation from a high-affinity to low-affinity form after the activation of coupled G proteins. Because BLT1s harboring a mutated helix 8, BLT1/ΔH8 and BLT1/LLAA (fig. S9A), show increased [3H]LTB4 binding compared to wild-type BLT1, this region is likely to be important for the conformational change to the low-affinity state (25, 41). Here, we obtained evidence supporting the notion that the disruption of helix 8 leads to defective BLT1 phosphorylation (fig. S9B); thus, we propose the involvement of phosphorylation in the conformational change. Moreover, phosphorylation was necessary for the expression of a reporter gene that is activated by the low-affinity conformation (fig. S10A) and for the shedding of transforming growth factor–α (TGFα), which requires high concentrations of [LTB4] (fig. S10B) (42). In agreement with the prediction that phosphorylation is required for BLT1 to undergo the conformational change from the high- to low-affinity state, we found that BLT1/WT/DE required a high dose of LTB4 for its activation, compared to wild-type, indicating that it was in a low-affinity conformation. In contrast, BLT1/WTΔphos showed hyperactivity, indicating the high-affinity form, in both the reporter assay and the TGFα shedding assay. Collectively, these data support our hypothesis that primary BLT1 phosphorylation is essential for the high-to-low affinity conformational change (Fig. 8).

Fig. 8 Sequential BLT1 phosphorylation.

(i) In the absence of LTB4, the basal phosphorylation sites in the BLT1 C-terminal domain are variably phosphorylated, yielding a mixture of phosphorylated species. The receptor is not active, and the downstream Gi protein is bound to guanosine diphosphate (GDP). (ii) Low concentrations of LTB4 stimulate BLT1 to adopt a high-affinity conformation, which stimulates GDP-GTP (guanosine triphosphate) exchange on Gi and triggers primary neutrophil responses such as the initiation of chemotaxis. (iii) After primary signaling, Gi exchanges GTP for GDP, and Ser310 is phosphorylated. Additional phosphorylation of basal sites in BLT1 then occurs, leading to a return to a low-affinity state. (iv) As neutrophils migrate up the LTB4 gradient, higher LTB4 concentrations stimulate phosphorylation of The308, which, in turn, promotes additional phosphorylation events at the basal sites, leading to secondary responses, such as degranulation.

We could not rule out the possibility that a distinct signaling pathway through the phosphorylated form is triggered by a high dose of LTB4. The phosphorylation may play a role in triggering the recruitment of effectors, such as β-arrestins. Here, we found the reduced binding of β-arrestins, β-arrestin–1 and β-arrestin–2, to LTB4-stimulated BLT1/WTΔphos, although substantial amounts of β-arrestins remained bound (fig. S11). These results are consistent with previous findings regarding the phosphorylation-independent recruitment of β-arrestin being essential for the internalization of BLT1 (17).

DISCUSSION

It has been demonstrated that the distinct GPCR phosphorylation profiles that result from the actions of different kinases are responsible for various signaling outcomes. This concept is known as the “phosphorylation barcode” and is critical for the multidimensional features of GPCR signaling (36, 4345). Thus, the precise analysis of BLT1 phosphorylation is important to clarify the biological functions of this receptor. Many studies have attempted to understand the potential roles of BLT1 phosphorylation (1719); however, a comprehensive analysis of these phosphorylations is still lacking. Here, we aimed to more extensively characterize the mapping, dynamics, and biological importance of BLT1 phosphorylation events by using Phos-tag technology in combination with site-directed mutagenesis. Although this approach does not provide direct evidence for the phosphorylation of specific sites, as would be revealed by mass spectrometry, it identifies the precise phosphorylation sites involved (4648). Here, we determined the precise sites of basal and LTB4-induced phosphorylations in human BLT1. Furthermore, we demonstrated hierarchical and sequential phosphorylation of BLT1 in response to increasing concentrations of LTB4. This work demonstrates that Phos-tag SDS-PAGE analysis can be used to assess GPCR phosphorylation and may be useful for research on other GPCRs.

Here, we demonstrate that the lack of BLT1 phosphorylation leads to aberrant neutrophil migration and impaired enzyme release (Fig. 9A). It was reported that RBL-2H3 cells overexpressing a human BLT1-Ala mutant, in which all the Ser and Thr residues in the C terminus were replaced with Ala, responded to various concentrations of LTB4 similarly to cells overexpressing the wild-type receptor in a chemotaxis assay (17). There are no clear explanations for this discrepancy; however, the specific cell lines, mutated residues, and the experimental conditions (96-well versus 48-well chambers) could account for these differences. Previous reports demonstrated the importance of the ligand-elicited intracellular Ca2+ influx followed by the activation of ERK1/2 in BLT1-stimulated chemotaxis (30, 31). For example, Ichiki et al. found that lack of the receptor for advanced glycation end products (RAGE) caused attenuation of the BLT1-dependent activation of ERK1/2, leading to reduced velocity of neutrophil motility, although the directionality of migration was slightly enhanced (49). In contrast, we observed a sustained increase in ERK1/2 activity in cells expressing BLT1/0NΔphos after stimulation with 100 nM LTB4, resulting in the opposite behavior—a decline in directionality and increased velocity. These findings highlight the importance of ERK1/2 signaling for proper cell motility. There is evidence suggesting that cell polarity in neutrophil chemotaxis is controlled by phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] (3235, 50). Nishio et al. demonstrated that during the initiation of chemotaxis, neutrophils become partially polarized without PtdIns(3,4,5)P3 and are able to migrate with correct directionality. Thereafter, PI3Kγ, phosphatase and tensin homolog (PTEN), and Src homology 2 domain–containing inositol-5-phosphatase 1 (SHIP1) cooperatively localize PtdIns(3,4,5)P3 to the prospective leading edge. This process leads to the assembly of the leading edge and full polarization, which are critical for proper directionality (32). Here, the impaired directionality of the cells expressing BLT1/0NΔphos in chemotaxis could be due to the reduced production of PtdIns(3,4,5)P3, resulting in loss of PtdIns(3,4,5)P3 accumulation at the leading edge (Fig. 9B). The aberrant chemotaxis could also be explained by the reduced desensitization of the [Ca2+] increase and ERK1/2 activation. These impairments may directly or indirectly affect the PI3K-AKT pathway. Further studies with phosphorylation-deficient BLT1, which is hyperactive, as well as the phosphomimic form, are likely to unravel the molecular mechanism(s) underlying these events. Together, our results indicate that the abnormal signaling in cells expressing BLT1/0NΔphos might be involved in deficient migration at high LTB4 concentrations.

Fig. 9 Essential roles of BLT1 phosphorylation.

(A) Highly phosphorylated BLT1 is important for the precise directionality of chemotaxis and degranulation at higher LTB4 concentrations. The absence of these phosphorylations results in aberrant migration and impaired enzyme release. (B) Schematic representation of the proposed model for PtdIns(3,4,5)P3-dependent polarization in the chemotaxis of neutrophils. During the initiation of chemotaxis, neutrophils become partially polarized without PtdIns(3,4,5)P3 and are able to migrate with correct directionality. Thereafter, PI3Kγ, PTEN, and SHIP1 cooperatively localize PtdIns(3,4,5)P3 to the prospective leading edge. This process leads to the assembly of the leading edge and full polarization, both of which are critical for proper directionality. The impaired directionality of cells expressing phosphorylation-deficient BLT1 could be due to the reduced production of PtdIns(3,4,5)P3, resulting in the loss of PtdIns(3,4,5)P3 accumulation at the leading edge where LTB4 concentrations are greatest.

Furthermore, we found a deficiency of β-hexosaminidase release in cells expressing BLT1/0NΔphos. Because β-hexosaminidase release requires a high dose of LTB4 (ca. >100 nM) (16, 37, 40), the phenomenon is likely triggered through the low-affinity form of BLT1, harboring increased phosphorylation. In keeping with this idea, a substantial reduction in β-hexosaminidase release was induced by 100 nM LTB4 in RBL-2H3 cells expressing BLT1/0NΔphos. Because the PI3K-AKT pathway, but not the ERK1/2 pathway, plays a key role in the degranulation of PMN leukocytes (36, 37), we suggest that the reduced β-hexosaminidase release could be, at least in part, due to impairment of the PI3K-AKT pathway.

Human BLT1 is highly abundant in phagocytic and immune cells, especially neutrophils (13), but it is difficult to use human neutrophils for the analysis of BLT1 mutants at present because of the low transfection efficiency. Here, we identified phosphorylation sites in mouse BLT1 and performed functional studies in cultured cells. To provide further support for our conclusions, we hope to generate knock-in mice expressing phosphorylation-deficient forms of BLT1 in our future work to elucidate the in vivo role of BLT1 phosphorylation, similarly to what has been done for other GPCRs (51). Extensive studies using these mice, such as assessing the effect of the in vivo LTB4 gradient as found in many disease models in mice, will be required to clarify the regulatory mechanisms of BLT1 involved in chemotaxis and degranulation. Recently, Majumdar et al. reported that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils to the primary chemoattractant and in a paracrine fashion to mediate the recruitment of neighboring neutrophils (52). To further clarify the relationship between the exosome-mediated LTB4 relay and phosphorylation-dependent regulation of BLT1 in chemotaxis, these mice will be useful tools in our future works. In addition, to further clarify the involvement of BLT1 phosphorylation in the degranulation in vivo, it is important to discriminate between pathophysiogical degranulation to the extracellular milieu and granule mobilization into the phagocytic lysosomal vacuole. In neutrophils, there are several types of intracellular granules that can be mobilized for releasing their contents into the primary phagocytic vacuole. For example, innate immune phagocytes do not degranulate in response to LTB4 under physiologic conditions. Because the release of enzymes to the extracellular milieu occurs only under pathophysiologic conditions, it is crucial to distinguish between specific and azurophilic granules in neutrophils and to clarify their mobilization to the primary phagocytic vacuole in response to LTB4. Although it is challenging to introduce the various BLT1 mutants into human phagocytic cells, the BLT1 phosphorylation–deficient knock-in mice will be ideal tools to address these issues.

Antibodies that are able to recognize specific phosphorylated forms of BLT1 would be useful tools for elucidating the spatial and temporal dynamics of individual phosphorylation events as well as to identify the kinases involved. The generation of a panel of phosphorylation site–specific antibodies would enable the study of this modification in situ, allowing insights into dynamic protein phosphorylations in the spatially complex structure of cells (53). Such antibodies would also be useful for identifying the kinases responsible for these modifications, which is also critical for understanding the biological importance of BLT1 phosphorylation.

In 1984, Goldman and Goetzl revealed the existence of high-affinity (Kd = 0.36 nM) and low-affinity (Kd = 76 nM) forms of a putative LTB4 receptor in human PMN leukocytes (39). They further demonstrated that PMN leukocytes, which were deactivated by 10 nM LTB4, selectively lost the high-affinity binding sites although low-affinity binding sites remained. Hence, the PMN leukocytes pretreated with 10 nM LTB4 exhibited a substantially diminished chemotactic response to subsequent LTB4 stimulation, whereas β-hexosaminidase release was still observed when elicited by higher LTB4 (300 nM). The findings of the current study show that the loss of high LTB4 affinity after pretreatment with LTB4 is likely due to the phosphorylation of BLT1, followed by a conformational change to a low-affinity state. The remaining low-affinity form acts as a LTB4 sensor and serves to release β-hexosaminidase once the receptor is exposed to a higher dose of LTB4.

During the migration of neutrophils, the concentration of LTB4 to which BLT1 is exposed gradually increases as the cells migrate toward the source. In other words, the degree of BLT1 phosphorylation can be enhanced in parallel with the concentration of LTB4 such that the graded increase in the phosphorylation of BLT1 acts as a LTB4 sensor. BLT1 is not phosphorylated and in the high-affinity conformation when the cell encounters low concentrations of LTB4, which trigger cell migration. As neutrophils migrate toward the source, BLT1 becomes progressively more phosphorylated and switches to the low-affinity conformation. When BLT1 is exposed to a concentration of LTB4 high enough to activate the low-affinity form, it triggers other cell responses, such as degranulation. Although more extensive studies are needed to better understand the functional significance of BLT1 modifications, we suggest that basal and LTB4-induced phosphorylations are indispensable for BLT1 maturation during cell migration to potentiate degranulation at inflammatory loci.

MATERIALS AND METHODS

Materials

LTB4 and the antibody recognizing human BLT1 (#120114) were purchased from Cayman Chemical Company. The antibody recognizing the HA tag (clone 3F10), PNGase-F, and the cOmplete EDTA-free protease inhibitor cocktail were purchased from Roche Applied Science. Phycoerythrin (PE)–conjugated antibody recognizing rat immunoglobulin G (IgG) was from Beckman Coulter. Phos-tag was purchased from NARD Institute Ltd. Antibodies recognizing phosphorylated p44/42 MAPK (ERK1/2) (Thr202/Tyr204), p44/42 MAPK (ERK1/2), phospho-AKT (Ser473), and AKT were from Cell Signaling Technology. The β-actin antibody, ATRA, and calcium ionophore A23187 were from Sigma-Aldrich Corporation. PTX and MK-2206 were from Funakoshi. SKF96365 was from Wako. BLT1 antagonists CP 105696 and ZK 158252 were gifts from Pfizer Inc. and Schering AG, respectively.

Construction of mutant human BLT1 expression plasmids

N-terminally HA-BLT1 was prepared as described previously (25). The BLT1 mutants were constructed by overlap extension polymerase chain reaction (PCR) using HA-BLT1 as a template (54). The amplified PCR products digested with Bam HI and Eco RI were subcloned into the pcDNA3 vector, and the sequences were confirmed. The primer sets used are listed in table S1.

Cell culture and transfection

HeLa cells and RBL-2H3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS). CHO-K1 cells were cultured in Ham’s F-12 medium (Nacalai Tesque) supplemented with 10% FBS. HL-60 cells and Ba/F3 cells were cultured in RPMI 1640 (Nacalai Tesque) supplemented with 10% FBS. These cells were transfected with a plasmid harboring the wild-type or mutant receptors using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. For differentiation of HL-60 cells, these cells were cultivated for 4 days in the presence of 1 μM ATRA and then stimulated with or without 100 nM LTB4.

Stable expression of BLT1

HeLa cells, CHO-K1 cells, and RBL-2H3 cells stably expressing wild-type or mutant receptors were established as follows. These cells were transfected with the HA-tagged receptor-expression plasmid. Stable transfectants were selected with geneticin (1 mg/ml) for 20 days. The existence of the HA epitope on the cell surface was confirmed by flow cytometry. After labeling the drug-resistant cells with PE using antibody recognizing HA tag (clone 3F10) and PE-conjugated antibody recognizing rat IgG, a group of HA-positive cells was sorted using MACS Cell Separation Columns and Anti-PE MicroBeads (Miltenyi Biotec).

Flow cytometry

For staining, cells were incubated with the antibody recognizing the HA tag (clone 3F10) in phosphate-buffered saline (PBS) containing 2% goat serum at room temperature for 30 min, followed by staining with PE-conjugated antibody recognizing rat IgG at room temperature for 30 min. The EPICS XL (Beckman Coulter) was used for flow cytometry.

Calcium mobilization assay

Transiently transfected HeLa cells were plated in a 96-well plate (4 × 104 cells per well) and incubated for 16 hours. The cells were then incubated with loading buffer A [1× Hanks’ balanced salt solution (HBSS), 1.25 mM probenecid (Sigma-Aldrich Corporation), 1 mM CaCl2, 0.5 mM MgCl2, 0.1% (w/v) bovine serum albumin (BSA), 20 mM Hepes-NaOH (pH 7.4)] containing 4 μM Fluo-3 AM (Dojindo) and 0.04% pluronic acid (Molecular Probes) at 37°C for 1 hour. The cells were then washed once with buffer A. Intracellular Ca2+ mobilization was monitored with the FlexStation scanning fluorometer (Molecular Devices) by measuring the emission fluorescence at 525 nm in response to excitation at 485 nm. Relative fluorescence units (maximum-minimum) are indicated.

To investigate the desensitization and shutdown of intracellular Ca2+ responses, cells were loaded with 3 μM Fura-2 AM (Dojindo) in a modified Hepes-Tyrode’s BSA buffer [25 mM Hepes-NaOH (pH 7.4), 140 mM NaCl, 2.7 mM KCl, 1.0 mM CaCl2, 12 mM NaHCO3, 5.6 mM d-glucose, 0.37 mM NaH2PO4, 0.49 mM MgCl2, 0.1% (w/v) BSA] containing 0.01% pluronic acid (Molecular Probes) at 37°C for 1 hour. The cells were washed and resuspended in Hepes-Tyrode’s BSA buffer at a density of 1 × 106 cells/ml. The cell suspension (0.5 ml) was applied to a CAF-100 system (JASCO Corp.), and 5 μl of ligand solution was added. The ligands were dissolved in Hepes-Tyrode’s BSA buffer after evaporation of the stock solution in ethanol under a nitrogen stream and used immediately. The intracellular Ca2+ concentration was measured by determining the ratio of emission fluorescence of 500 nm by excitation at 340 and 380 nm. The free Ca2+ concentration was calculated from the equation Ca2+i = Kd[(F − Fmin)/(Fmax − F)], where Kd represents the Ca2+ binding dissociation constant (224 nM for Fura-2), F is the 500-nm fluorescence ratio, Fmax is the maximal fluorescence ratio determined after the addition of 0.1% Triton X-100 to permeabilize the cells in the presence of 1 mM Ca2+, and Fmin is the minimal fluorescence ratio determined after permeabilization and the addition of 5 mM EGTA.

Preparation of cell membrane fractions

After stimulation of the cells expressing mutant or wild-type BLT1 with 100 mM LTB4, the cells were washed with ice-cold tris buffer [20 mM tris-HCl (pH 7.4), 0.3 M sucrose, 1 mM sodium orthovanadate] and collected in Hepes buffer [25 mM Hepes-NaOH (pH 7.4), 10 mM MgCl2, 0.3 M sucrose, 1 mM sodium orthovanadate, supplemented with cOmplete protease inhibitor cocktail]. Subsequently, the cells were sonicated for 20 min using a Bioruptor (Cosmo Bio) and centrifuged at 9000g for 10 min at 4°C to remove cellular debris. The resulting supernatant was ultracentrifuged at 100,000g for 1 hour at 4°C. The microsome pellet was resuspended in a buffer containing the abovementioned Hepes buffer. The samples were then subjected to SDS-PAGE or Phos-tag SDS-PAGE as cell membrane fractions.

Phos-tag SDS-PAGE and immunoblotting

For Western blot analyses, protein samples were separated on SDS-PAGE gels and transferred to a nitrocellulose membrane. To detect the band shift that represents phosphorylated proteins, an SDS-PAGE gel containing 50 μM Phos-tag acrylamide with 200 μM MnCl2 was used. After a blocking step using 1% skimmed milk in TBS-T [20 mM tris-buffered saline (pH 7.4) and 0.1% Tween 20], blots were probed with the primary antibody [antibody against HA tag (3F10) or antibody recognizing human BLT1] for 1 hour. The membrane was washed with TBS-T and incubated with horseradish peroxidase–conjugated antibody against rat (for 3F10) or mouse (for human BLT1 antibody) IgG for 30 min. The signal was visualized using an enhanced chemiluminescence Western blotting detection system (Nacalai Tesque).

Endoglycosidase treatment

Protein samples containing BLT1 were obtained from the cell membrane fractions described above. Samples were treated with 1 U of PNGase-F in 50 μl of buffer [139.2 mM Na2HPO4, 40.8 mM NaH2PO4, 0.4% SDS, 20 mM EDTA, 2% 2-mercaptoethanol] for 16 hours at 4°C. The resultant samples were suspended in a sampling buffer [25 mM tris-HCl (pH 6.5), 5% glycerol, 1% SDS, 0.05% bromophenol blue] and subjected to Western blot analyses.

AP treatment

Protein samples containing wild-type and mutant BLT1 were obtained from the microsome pellet described above without sodium orthovanadate. Samples were suspended in buffer [25 mM Hepes-NaOH (pH 7.4), 10 mM MgCl2, 0.3 M sucrose, and cOmplete protease inhibitor cocktail]. The samples were then treated with 60 U of CIAP (Takara Bio) in 50 μl of AP buffer [50 mM tris-HCl (pH 9.0) and 1 mM MgCl2] for 1 hour at 37°C.

Construction of SrtA-HA–tagged BLT1 expression plasmids

The linearized vector was prepared by PCR using two primers (pMK-HA, forward and reverse) derived from pMK-SrtA-HA-G2A-IRES-puro that encodes the LPETGGGGG(SrtA)-HA–tagged G2 accumulation (G2A) receptor and an internal ribosomal entry site (IRES)–puromycin cassette (27). BLT1/0N and BLT1/0NΔphos were amplified by PCR using two primers (BLT1/0N, forward and reverse), respectively. Then, the linearized vector and the insert were fused using the In-Fusion HD enzyme (Clontech), generating pMK-SrtA-HA-BLT1/0N-IRES-puro and pMK-SrtA-HA-BLT1/0NΔphos. The constructs were confirmed by sequencing.

Analysis of AF488-labeled BLT1 trafficking

The cytosol of the Ba/F3 cells expressing SrtA-HA-BLT1/0N and SrtA-HA-BLT1/0NΔphos were stained with CytoRed and ViVidFluor Cell Blue CMAC, respectively. These cells were mixed the same population, immobilized on a biocompatible anchor for membrane–collagen–coated glass base dish (Matsunami) (55) and preincubated in RPMI 1640 (0.1% BSA) for 1 hour at 37°C, 5% CO2. SrtA-mediated labeling of BLT1 was carried out as follows (27). The medium was replaced with RPMI 1640 (0.1% BSA, 30 μM SrtA, 1 mM Gly3) followed by incubation for 1 hour at 37°C, 5% CO2. After washing the cells with RPMI 1640 (0.1% BSA) to remove excess Gly3, the cells were incubated for labeling BLT1 with AF488 in RPMI 1640 (0.1% BSA, 15 μM AF488-DLPETGG, 30 μM SrtA) for 1 hour at 37°C, 5% CO2. The cells were washed twice with RPMI 1640 (0.1% BSA), and then RPMI 1640 (0.1% BSA) was added at 1 ml per dish for confocal microscopy observation. The single-cell analysis of AF488-labeled BLT1/0N and BLT1/0NΔphos was simultaneously performed in RPMI 1640 (0.1% BSA). Time-lapse images of AF488-labeled BLT1/0N and BLT1/0NΔphos were acquired using an LSM 510 confocal microscope equipped with an incubation system to maintain 37°C. Immediately after the addition of LTB4 (10 nM final concentration), the time-lapse image was acquired for 20 min at 1-min intervals. The acquired images were automatically analyzed using OpenCV.

Chemotaxis assay

Polycarbonate filters with an 8-μm pore size (Neuro Probe) were coated with fibronectin (13.3 μg/ml) (Sigma-Aldrich Corporation) in PBS for 60 min. A dry coated filter was placed on a 96-well blind chamber (Neuro Probe) containing LTB4 (10−10 to 10−6 M). CHO-K1 cells expressing wild-type and mutant BLT1/0N (200 μl, 8 × 104 cells per well) were added to the top wells. The ligand solution and cell suspension were prepared in the same buffer [Ham’s F-12 medium containing 0.1% (w/v) BSA]. After incubation at 37°C in 5% CO2 for 3 hours, the filter was disassembled. The cells on the filter were fixed with methanol and stained with a Diff-Quick staining kit (International Reagent). The upper side of the filter was then scraped free of cells. The number of cells that migrated to the lower side was determined by measuring the OD595 using a 96-well microplate reader.

To evaluate motility during chemotaxis, CHO-K1 cells expressing wild-type and mutant BLT1/0N were allowed to migrate in the chamber of a TAXIScan-FL optical assay device (Effector Cell Institute), along with the LTB4 gradient (0 to 100 nM). Phase-contrast images of migrating cells were acquired at 10-s intervals over 15 min. Images were imported as stacks to ImageJ (National Institutes of Health). Velocity and directionality were calculated with manual tracking using the chemotaxis and migration tools in the ImageJ program.

Reporter gene assay

HeLa cells were plated in a 24-well plate (1 × 105 cells per well) and transfected with 0.8 μg per well of plasmid DNA using Lipofectamine 2000 (Invitrogen). The ratio of transfected plasmid DNA was kept constant at 17:17:1 for SRE–firefly luciferase–pGL4.33 (Promega):BLT1/0N-pcDNA3 or BLT1/0NΔphos-pcDNA3:CMV promoter-driven Renilla luciferase (Promega) (56). The cells were starved for 15 hours and stimulated with 100 nM LTB4 for 6 hours. The luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega) and the MiniLumat LB 9506 luminometer (Berthold). SRE-luc activity was normalized to the Renilla luciferase activity.

For the analyses of BLT1/WT, BLT1/WTΔphos, and BLT1/WT/DE, human embryonic kidney (HEK) 293T cells were plated in a 24-well plate (1 × 105 cells per well) and transfected with 0.8 μg per well of plasmid DNA using Lipofectamine 2000 (Invitrogen). The ratio of transfected plasmid DNA was kept constant at 17:17:1 for SRE-NanoLuc luciferase-pNL(NLucP/SRF/Hygro; Promega):BLT1-pcDNA3:phosphoglycerate kinase (PGK)–firefly luciferase–pGL4.53(luc2/PGK; Promega). The cells were starved for 15 hours and stimulated with 10 or 100 nM LTB4 for 6 hours. The luciferase assay was performed using the Nano-Glo Dual-Luciferase Reporter Assay System (Promega) and the GloMax 20/20 luminometer (Promega). SRE-NanoLuc activity was normalized to the levels of firefly luciferase activity.

β-Hexosaminidase release assay

Degranulation was determined by measuring the release of a granule marker, β-hexosaminidase, as described previously (16). The cells were seeded in 24-well collagen-coated plates at 5 × 105 cells in 1 ml of medium and were serum-starved for 12 hours. Adherent cells were washed twice in prewarmed PBS. Cells were then treated with LTB4 (10 nM for 40 min, or 10 nM for 20 min followed by 100 nM for 20 min), and the activity of β-hexosaminidase was quantified in the supernatant and the cell lysates by spectrophotometric analysis using p-nitrophenyl-N-acetyl-β-d-glucosaminide as a substrate (57). Values for β-hexosaminidase release in the medium were expressed as the percentage of the total β-hexosaminidase, determined from cells lysed in 0.1% Triton X-100. In these experiments, calcium ionophore A23187 (5 μM) was used as a positive-control stimulus to observe the release of β-hexosaminidase.

Pulldown of biotin-labeled HA-BLT1

HeLa cells were plated in a 60-mm dish (1 × 106 cells per dish) and transfected with 6 μg of HA-BLT1/WT or HA-BLT1/WTΔphos using Lipofectamine 2000 (Invitrogen). After 24 hours, the cells were washed once with PBS and then incubated in 3 ml of PBS containing 0.5 M EZ-Link Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific) for 30 min at 4°C. After washing with PBS, the cells were incubated in a culture medium and stimulated with 10 nM LTB4 for 30 min at 37°C. Then, the cells were lysed in 500 μl of lysis buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and supplemented with cOmplete protease inhibitor cocktail] and incubated for 30 min at 4°C with gentle mixing, and the lysates were centrifuged for 10 min at 10,000g. The supernatant was transferred into another tube. Streptavidin Agarose Resin suspension (100 μl) (2× diluted with lysis buffer; Thermo Fisher Scientific) was added to the supernatant and gently mixed for 2 hours at room temperature. The Streptavidin Agarose Resin was collected by centrifugation (10,000g, 1 min), washed twice with Wash Buffer 1 [50 mM tris-HCl (pH 7.5), 500 mM NaCl, 0.1% NP-40, 0.05% sodium deoxycholate], followed by Wash Buffer 2 [50 mM tris-HCl (pH 7.5), 0.1% NP-40, 0.05% sodium deoxycholate]. Next, the biotin-labeled proteins were eluted with 100 μl of 2× SDS sample buffer. The samples were subjected to SDS-PAGE followed by Western blot analyses using an antibody against HA tag (clone 3F10).

TGFα shedding assay

The TGFα shedding assay was carried out as previously described (42). In brief, HEK293T cells were seeded in 12-well plates (2 × 105 cells per well), 24 hours before transfection. The cells were transfected with plasmids encoding AP-TGFα (a gift from S. Higashiyama, Ehime University Proteo-Science Center) (0.25 μg per well), HA-BLT1/WT, HA-BLT1/WTΔphos or HA-BLT1/WT/DE (0.1 μg per well), and Gαq/i1 (0.05 μg per well). After 24 hours, the cells were collected and washed with PBS and then suspended in HBSS containing 5 mM Hepes (pH 7.4). The cell suspension was plated in a 96-well plate (80 μl per well) and stimulated with 20 μl of HBSS containing LTB4 (10 to 150 nM) for 1 hour at 37°C. The 96-well plates were centrifuged for 2 min at 200g, and 80 μl of the supernatant was transferred into another 96-well plate. An 80-μl portion of 2× p-nitrophenyl phosphate disodium salt hexahydrate (pNPP) buffer [10 mM pNPP in 40 mM tris-HCl (pH 9.5), 40 mM NaCl, and 10 mM MgCl2] was added to both the supernatant and the cells. The absorbance at 405 nm (A405) of both plates was read before and after a 1-hour incubation at 37°C using a microplate reader (Thermo Fisher Scientific). AP-TGFα release was calculated by the following formulae:AP-TGFα in supernatant (%)=(ΔOD405Sup/[ΔOD405Sup+ΔOD405Cell])×100AP-TGFα release (%)=AP-TGFα in supernatant under a stimulated condition (%)AP-TGFα in supernatant under a vehicle-treated condition (%)

NanoBiT–β-arrestin recruitment assay

For the NanoBiT–β-arrestin recruitment assay (58), a receptor construct was designed to fuse the small fragment (SmBiT) of the NanoBiT complementation luciferase to the C terminus of HA-BLT1 with a 15–amino acid flexible linker (GGSGGGGSGGSSSGG), whose sequences were recommended by the manufacturer (Promega). The construct (HA-BLT1-SmBiT) was assembled and inserted into a pCAGGS mammalian expression plasmid (a gift from J.-i. Miyazaki, Osaka University) using an NEBuilder HiFi DNA Assembly system (New England Biolabs). A β-arrestin construct was generated by fusing the large fragment (LgBiT), whose nucleotide sequences were gene-synthesized with mammalian codon optimization (GenScript), to the N terminus of human β-arrestin1 or β-arrestin2 (βarr1 or βarr2, respectively) with the 15–amino acid linker. The LgBiT-βarr1 and LgBiT-βarr2 were inserted into the pCAGGS plasmid. HEK293A cells (Thermo Fisher Scientific) were seeded in a 10-cm dish at 2 × 106 cells in 10 ml of DMEM, supplemented with 10% FBS and penicillin (100 U/ml) and streptomycin (100 μg/ml), and cultured for 1 day. The cells were transfected with a mixture of HA-BLT1-SmBiT plasmid (1 μg) and the LgBiT-βarr plasmid (LgBiT-βarr1 or LgBiT-βarr2; 500 ng) by diluting in 500 μl of Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific) and combining 25 μl of polyethylenimine reagent (1 mg/ml) [Polyethylenimine “Max” (molecular weight 40,000), Polysciences] diluted in 500 μl of the Opti-MEM. As a negative control, the pCAGGS plasmid was used. Twenty-four hours after the addition of the transfection solution, the cells were harvested with 5 ml of a 0.53 mM EDTA-containing Dulbecco’s phosphate-buffered saline (D-PBS), followed by rinsing with 5 ml of HBSS containing 5 mM Hepes (pH 7.4). The cells were centrifuged at 190g for 5 min and suspended in 10 ml of 0.01% BSA (fatty acid–free grade; SERVA)–containing HBSS. The cell suspension was seeded in a 96-well white plate at a volume of 80 μl per well and loaded with 20 μl of 50 μM coelenterazine (Carbosynth) diluted in the BSA-HBSS. After incubation at room temperature for 2 hours, background luminescent signals were measured using the luminescent microplate reader SpectraMax L equipped with two detectors (Molecular Devices). Prediluted LTB4 solution (20 μl at final concentrations of 100 pM to 100 nM, 3.2-fold dilution) or vehicle was manually added to the cells. After ligand addition (5 min), luminescent signals were measured for 5 min with 20-s intervals. For each well, the luminescent signal was normalized to the initial count and fold change values over 5 to 10 min after ligand stimulation was averaged. The fold change βarr recruitment signals were fitted to a four-parameter sigmoidal concentration-response curve, and EC50 (median effective concentration) values were obtained, using the Prism 7 software (GraphPad Prism).

Statistical analysis

For each experiment, at least three independent experiments were performed. The images obtained from one representative experiment were presented. To determine statistical significance, values were compared by Student’s t test, one-way or two-way ANOVA with Tukey post hoc test, Bonferroni post hoc test, or Kruskal-Wallis test with Dunn’s post hoc test using Prism 7 software. The differences were considered significant if P values were less than 0.05.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/11/544/eaao5390/DC1

Fig. S1. Properties of N-glycosylation–deficient HA-BLT1/0N.

Fig. S2. Conserved Ser and Thr residues in the cytoplasmic domains of human, mouse, rat, guinea pig, and zebrafish BLT1.

Fig. S3. Confirmation of residues essential for HA-BLT1/0N phosphorylation.

Fig. S4. Phosphorylation of mouse BLT1.

Fig. S5. Phosphorylation at LTB4-induced and basal sites through Gi.

Fig. S6. LTB4 dose dependency of intracellular [Ca2+] increase.

Fig. S7. Effect of blockage of the [Ca2+] increase on BLT1 phosphorylation.

Fig. S8. Effect of phosphorylation deficiency on the functions of wild-type BLT1.

Fig. S9. Effect of helix 8 disruption on BLT1 phosphorylation.

Fig. S10. Importance of phosphorylation for the ligand sensitivity of BLT1.

Fig. S11. Effect of phosphorylation on β-arrestin binding to BLT1.

Table S1. Primer sequences used to generate mutant BLT1s.

REFERENCES AND NOTES

Acknowledgments: We thank S. Higashiyama (Ehime University Proteo-Science Center) for providing the pRc-CMV/AP-TGFα plasmid to construct the TGFα shedding assay system and J.-i. Miyazaki (Osaka University) for providing the pCAGGS vector to develop the NanoBiT–β-arrestin recruitment assay. We also thank A. Kikuchi, M. Segawa (Okayama University of Science), and Y. Aruga (The University of Tokyo) for their research project works. We are grateful to all laboratory members for their constructive comments. Funding: This work was supported by MEXT (Ministry of Education, Culture, Sports, Science and Technology)/Japan Society for the Promotion of Science KAKENHI grant numbers 26460061 (to M.N.), 15H05897 (to J.A.), 15H02319 (to T.N.), 15H05901, 15H05904, and 15H04708 (to T.Y.) and by the Ryobi Teien Memory Foundation (to M.N.), the Naito Foundation (to M.N.), the Mitsubishi Foundation (to M.N.), the Takeda Science Foundation (to M.N. and T.S.), the Science Research Promotion fund by The Promotion and Mutual Aid Corporation for Private Schools of Japan (to M.N.), JST (Japan Science and Technology Agency) grant number JPMJPR1331 (to A.I.), and AMED (Japan Agency for Medical Research and Development) grant numbers JP17gm5910013 (to A.I.) and JP17gm0710001 (to J.A.). Department of Lipid Signaling at the National Center for Global Health and Medicine is financially supported by ONO Pharmaceutical Co. Ltd. (T.S.). Author contributions: Y.N., K.Y., I.T., J.-i.Y., and N.M. performed the experiments. M.T., S. Yamahira, S. Yamaguchi, and T.N. performed SrtA-mediated labeling of BLT1 and analyzed the trafficking of the receptor. T.I. and T.Y. performed a chemotaxis assay using TAXIScan-FL. A.I. and J.A. carried out the β-arrestin–binding assay using the NanoBiT system. Y.N., M.T., T.I., A.I., and M.N. performed statistical analyses. T.S. and M.N. conceived and designed the study and acquired, analyzed, and interpreted the data. Y.N. and M.N. prepared the manuscript. Competing interests: The authors declare that they have no competing financial interests. Data and materials availability: All data needed to evaluate the conclusions in this paper are present in the paper or in the Supplementary Materials. Plasmids require a material transfer agreement from Okayama University of Science.
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