Substance P Serves as a Balanced Agonist for MRGPRX2 and a Single Tyrosine Residue Is Required for β-Arrestin Recruitment and Receptor Internalization

Abstract

The neuropeptide substance P (SP) mediates neurogenic inflammation and pain and contributes to atopic dermatitis in mice through the activation of mast cells (MCs) via Mas-related G protein-coupled receptor (GPCR)-B2 (MrgprB2, human ortholog MRGPRX2). In addition to G proteins, certain MRGPRX2 agonists activate an additional signaling pathway that involves the recruitment of β-arrestins, which contributes to receptor internalization and desensitization (balanced agonists). We found that SP caused β-arrestin recruitment, MRGPRX2 internalization, and desensitization. These responses were independent of G proteins, indicating that SP serves as a balanced agonist for MRGPRX2. A tyrosine residue in the highly conserved NPxxY motif contributes to the activation and internalization of many GPCRs. We have previously shown that Tyr²⁷⁹ of MRGPRX2 is essential for G protein-mediated signaling and degranulation. To assess its role in β-arrestin-mediated MRGPRX2 regulation, we replaced Tyr²⁷⁹ in the NPxxY motif of MRGPRX2 with Ala (Y279A). Surprisingly, we found that, unlike the wild-type receptor, Y279A mutant of MRGPRX2 was resistant to SP-induced β-arrestin recruitment and internalization. This study reveals the novel findings that activation of MRGPRX2 by SP is regulated by β-arrestins and that a highly conserved tyrosine residue within MRGPRX2's NPxxY motif contributes to both G protein- and β-arrestin-mediated responses.

Keywords: MRGPRX2; MrgprB2; internalization; mast cells; signaling; substance P; tyrosine.

Figure 1

Substance P (SP) is a balanced agonist for MRGPRX2. (A) HTLA cells stably expressing MRGPRX2 (HTLA-MRGPRX2) were exposed to different concentrations of SP for 16 h, and β-arrestin–mediated gene expression was determined. (B) Rat basophilic leukemia (RBL) cells stably expressing MRGPRX2 (RBL-MRGPRX2) were stimulated with different concentrations of SP for the times indicated, and level of receptor internalization was determined by flow cytometry. (C) RBL-MRGPRX2 cells were cultured in the absence (untreated) or presence of SP (30 μM) for 16 h. Cells were subsequently stimulated with different concentrations of SP for 30 min, and percent degranulation was determined by β-hexosaminidase release assay. All data points are the mean ± SEM of at least three experiments. For comparisons of two samples, two-tailed unpaired t-test was used. For comparisons of multiple samples to a control group, one–way analysis of variance (ANOVA) with Dunnett’s post-hoc test was used. *** p < 0.001 and **** p < 0.0001.

Figure 2

Pertussis toxin (PTx) inhibits SP-induced mast cell (MC) degranulation, but has no effect on β-arrestin recruitment and MRGPRX2 internalization. RBL-MRGPRX2 cells were cultured in the absence or presence of PTx (100 ng/mL, 16 h), and the effects of SP on (A) degranulation, (B) β-arrestin-mediated gene expression, and (C) MRGPRX2 internalization were determined. All data points are the mean ± SEM of at least three experiments. Statistical significance was determined by two-tailed unpaired t-test. **** p < 0.0001.

Figure 3

β-arrestin2 regulates MrgprB2-mediated MC degranulation in response to SP. (A) Peritoneal MCs (PMCs) obtained from wild type (WT) and βArr2^−/− mice displayed similar levels of surface c-Kit and FcεRI expression as determined by flow cytometry. (B) Cells were exposed to either buffer (control) or SP (50 μM) for 30 min, and β-hexosaminidase release was determined. All data points are the mean ± SEM of at least three experiments performed in triplicate. Statistical significance was determined by two-tailed unpaired t-test. * p < 0.05.

Figure 4

Y279A mutation of MRGPRX2 abolishes β-arrestin recruitment in response to SP. (A) HTLA cells were transiently transfected with cDNA encoding either WT-MRGPRX2 or its Y279A mutant, and cell surface receptor expression was determined by flow cytometry using PE-anti-MRGPRX2 antibody. (B) Cells were stimulated with SP (30 μM) for 16 h, and β-arrestin–mediated gene expression was measured. All data points are the mean ± SEM of at least three experiments. Two-tailed unpaired t-test was used. ** p < 0.01. (C) RBL cells co-expressing WT MRGPRX2 or Y279A and βArr2-GFP were stimulated with SP (30 μM) for 1 min and β-arrestin translocation was investigated by confocal microscopy. Scale bar = 10 μm.

Figure 5

Y279A mutation of MRGPRX2 impairs SP-induced receptor internalization. (A) HTLA transiently expressing WT-MRGPRX2 or its Y279A mutant were stimulated with SP (30 μM) for 30 min, and the receptor internalization was determined by flow cytometry. Representative histogram for cell surface receptor expression before (black line) and after SP stimulation (blue line) are shown. (B) The percentage of receptor internalization after SP stimulation was calculated. All data points are the mean ± SEM of at least three experiments. Two-tailed unpaired t-test was used. ** p < 0.001.

Figure 6

Y279A mutation of MRGPRX2 displays resistance to SP-induced receptor trafficking. (A) Schematic showing the dual-color labeling of cell surface and internalized receptors (modified from Carrodus et al., 2014 [42]). (B) Change in receptor trafficking was observed in RBL-2H3 cells expressing WT MRGPRX2 and Y279A mutant after SP stimulation (30 μM; 30 min). Scale bar = 10 μm.