Research ArticleBiochemistry

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

See allHide authors and affiliations

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

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

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.

  • This PDF file includes:

    • 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.

    [Download PDF]

Navigate This Article