Mas-related gene X2 (MrgX2) is a novel G protein-coupled receptor for the antimicrobial peptide LL-37 in human mast cells: resistance to receptor phosphorylation, desensitization, and internalization - PubMed (original) (raw)
Mas-related gene X2 (MrgX2) is a novel G protein-coupled receptor for the antimicrobial peptide LL-37 in human mast cells: resistance to receptor phosphorylation, desensitization, and internalization
Hariharan Subramanian et al. J Biol Chem. 2011.
Abstract
Human LL-37 is a multifunctional antimicrobial peptide that promotes inflammation, angiogenesis, wound healing, and tumor metastasis. Most effects of LL-37 are mediated via the activation of the cell surface G protein-coupled receptor FPR2 on leukocytes and endothelial cells. Although LL-37 induces chemotaxis, degranulation, and chemokine production in mast cells, the receptor involved and the mechanism of its regulation remain unknown. MrgX2 is a member of Mas-related genes that is primarily expressed in human dorsal root ganglia and mast cells. We found that a human mast cell line LAD2 and CD34(+) cell-derived primary mast cells, which natively express MrgX2, responded to LL-37 for sustained Ca(2+) mobilization and substantial degranulation. However, an immature human mast cell line, HMC-1, that lacks functional MrgX2 did not respond to LL-37. shRNA-mediated knockdown of MrgX2 in LAD2 mast cell line and primary CD34(+) cell-derived mast cells caused a substantial reduction in LL-37-induced degranulation. Furthermore, mast cell lines stably expressing MrgX2 responded to LL-37 for chemotaxis, degranulation, and CCL4 production. Surprisingly, MrgX2 was resistant to LL-37-induced phosphorylation, desensitization, and internalization. In addition, shRNA-mediated knockdown of the G protein-coupled receptor kinases (GRK2 and GRK3) had no effect on LL-37-induced mast cell degranulation. This study identified MrgX2 as a novel G protein-coupled receptor for the antibacterial peptide LL-37 and demonstrated that unlike most G protein-coupled receptors it is resistant to agonist-induced receptor phosphorylation, desensitization, and internalization.
Figures
FIGURE 1.
LL-37 stimulates degranulation and Ca2+ mobilization in LAD2 mast cells. A, LAD2 mast cells were stimulated with different concentrations of LL-37 (0.1–10 μ
m
), and percent degranulation (β-hexosaminidase release) was determined. B, cells were treated with or without PTx (100 ng/ml, 16 h), loaded with Indo-1AM, and Ca2+ mobilization in response to LL-37 (1 μ
m
) was determined. Indo-1-loaded cells were also exposed to La3+ (1 μ
m
), and LL-37-induced Ca2+ mobilization was determined. C and D, LAD2 cells were pretreated with vehicle or PTx (100 ng/ml) (C) or La3+ (1 μ
m
; 5 min) (D) and stimulated with different concentrations of LL-37 (1–10 μ
m
), and percent degranulation was determined. Data are mean ± S.E. of three experiments. Statistical significance was determined by one-way ANOVA (A) or two-way ANOVA with Bonferroni's post test (C and D). ** indicates p < 0.001.
FIGURE 2.
Cortistatin and LL-37 induce Ca2+ mobilization in LAD2 mast cells but not in HMC-1 cells. LAD2 (A–C) and HMC-1 (D–F) cells were loaded with Indo-1AM and stimulated with C3a (10 n
m
; A and D), CST (1 μ
m
) (B and E), or 10 μ
m
LL-37 (C and F), and intracellular Ca2+ mobilization was determined. E and F, cells were also stimulated at 500 s with 10 n
m
C3a following initial stimulation. Data shown are representative of three similar experiments.
FIGURE 3.
LL-37 induces Ca2+ mobilization, chemotaxis, and chemokine CCL4 generation in HMC-1 cells stably expressing MrgX2. A, representative histogram of expression level of HA-MrgX2 (MrgX2) in receptor-transfected (thick bold line) or mock-transfected control cells (dotted line) as analyzed by flow cytometry is shown. B, mock-transfected HMC-1 cells or cells expressing MrgX2 were loaded with Indo-1, and intracellular Ca2+ mobilization in response to LL-37 (3 μ
m
) was determined. C and D, cells (as in B) were exposed to different concentrations of LL-37, and chemotaxis as well as chemokine CCL4 generation were determined. Data are means ± S.E. of three experiments. Statistical significance was determined by two-way ANOVA with Bonferroni's post test. * indicates p < 0.01, and ** indicates p < 0.001.
FIGURE 4.
LL-37 induces degranulation in RBL-2H3 cells stably expressing MrgX2 but not MrgX1. RBL-2H3 cells stably expressing MrgX2 (A) or MrgX1 (B) were stimulated with buffer (bar C), BAM-22P, cortistatin (CST) (both at 1 μ
m
) or LL-37 (3 μ
m
) for 30 min, and β-hexosaminidase release was measured. Data are mean ± S.E. of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. ** indicates p < 0.001.
FIGURE 5.
Knockdown of MrgX2 inhibits LL-37-induced Ca2+ mobilization and degranulation in LAD2 mast cells. LAD2 mast cells were stably transduced with scrambled shRNA control lentivirus or shRNA lentivirus targeted against MrgX2. A, quantitative PCR was performed to determine mRNA expression in control and MrgX2 knockdown (KD) cells. Results are expressed as a ratio of MrgX2 to GAPDH mRNA levels. B, shRNA control or C, MrgX2 KD cells were loaded with Indo-1 and stimulated with LL-37 (100 n
m
) at 100 s followed by a second stimulation of C3a (10 n
m
) at 300 s, and intracellular Ca2+ mobilization was determined. D, shRNA control and MrgX2 KD cells were stimulated with different concentrations of LL-37 (1–10 μ
m
), CST (10 and 100 n
m
), or C3a (1 n
m
), and percent degranulation (β-hexosaminidase release) was determined. Data are mean ± S.E. of three experiments. Statistical significance was determined by t test (A) or two-way ANOVA with Bonferroni's post test (D). * indicates p < 0.01, and ** indicates p < 0.001.
FIGURE 6.
MrgX2 is resistant to agonist-induced phosphorylation. A, sequence comparisons of the C termini of C3aR and MrgX2 is shown. B and C, HEK-293T cells transiently expressing HA-C3aR or HA-MrgX2 were incubated with anti-HA antibody followed by PE-conjugated anti-mouse IgG. Representative histograms of expression levels of C3aR (B) and MrgX2 (C) (thick bold lines) in receptor-transfected and mock-transfected cells (dotted lines) are shown. D, HEK-293T cells expressing C3aR were labeled with 32P and stimulated with C3a (100 n
m
), and C3aR phosphorylation was determined. Similarly, cells expressing MrgX2 were stimulated with CST (100 n
m
) or LL-37 (10 μ
m
) for 5 min, and receptor phosphorylation was determined. An autoradiograph from a 3-h exposure is shown. Data shown are representative of three independent experiments.
FIGURE 7.
MrgX2 is resistant to desensitization and internalization. A and B, LAD2 cells were incubated with Indo-1AM, washed in a Ca2+-free buffer, and stimulated with C3a (10 n
m
) (A) and LL-37 (3 μ
m
) (B), and Ca2+ mobilization in the absence of extracellular Ca2+ was determined. After 5 min, cells were exposed to Ca2+ at a final concentration of 1 m
m,
and intracellular Ca2+ mobilization was again determined. Traces represent results from three similar experiments. C, MrgX2-expressing HMC-1 cells were exposed to C3a (100 n
m
) for different time periods, and cell surface C3aR expression was determined by flow cytometry. MrgX2-expressing HMC-1 cells were exposed to LL-37 (1 μ
m
) (D) or PMA (50 ng/ml) (E) for different time periods, and MrgX2 expression levels were determined by flow cytometry using 12CA5 antibody. Data are represented as percent receptor surface expression levels of control. Data are mean ± S.E. of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. ** indicates p < 0.001.
FIGURE 8.
Knockdown of GRK2 or GRK3 has no effect on cortistatin and LL-37-induced degranulation in LAD2 mast cells. LAD2 mast cells were stably transduced with scrambled shRNA control lentivirus or shRNA lentivirus targeted against GRK2 or GRK3. A, representative immunoblot of LAD2 cells with GRK2 knockdown is shown. B, quantitative PCR was performed to assess GRK3 mRNA levels in shRNA control and GRK3 knockdown (KD) cells. Results are expressed as a ratio of GRK3 to GAPDH mRNA levels. C and D, control shRNA, GRK2, and GRK3 KD cells were stimulated with different concentrations of C3a (0.1 and 1 n
m
) (C) or LL-37 (1 and 3 μ
m
) and CST (100 n
m
) (D), and percent degranulation was determined. Data are mean ± S.E. of three experiments. Statistical significance was determined by t test (B) and two-way ANOVA with Bonferroni's post test. * indicates p < 0.01 and ** indicates p < 0.001.
FIGURE 9.
LL-37 activates CD34+ cell-derived primary mast cells via MrgX2. A, CD34+ primary mast cells were stimulated with different concentrations of LL-37 (1–10 μ
m
), and percent degranulation was determined. B, cells were incubated with Indo-1AM in Ca2+-free buffer and stimulated with 3 μ
m
LL-37 at 100–200 s, and intracellular Ca2+ mobilization was determined. After 5 min, cells were exposed to 1 m
m
Ca2+, and intracellular Ca2+ mobilization was determined. C, mast cells were stably transduced with scrambled shRNA control lentivirus or shRNA lentivirus targeted against MrgX2. Quantitative PCR was performed to assess MrgX2 mRNA levels in shRNA control and MrgX2 knockdown (KD) cells. Results are expressed as a ratio of MrgX2 to GAPDH mRNA levels. D, control shRNA or MrgX2 KD cells were stimulated with different concentrations of LL-37 (3 and 10 μ
m
), and percent degranulation was determined. Data are mean ± S.E. of three experiments. Statistical significance was determined by one-way ANOVA (A), t test (C), or two-way ANOVA with Bonferroni's post test (D). ** indicates p < 0.001.
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