β-arrestin-1 and β-arrestin-2 Restrain MRGPRX2-Triggered Degranulation and ERK1/2 Activation in Human Skin Mast Cells

Abstract

As a novel receptor that efficiently elicits degranulation upon binding to one of its numerous ligands, MRGPRX2 has moved to the center of attention in mast cell (MC) research. Indeed, MRGPRX2 is believed to be a major component of pseudo-allergic reactions to drugs and of neuropeptide-elicited MC activation in skin diseases alike. MRGPRX2 signals via G proteins which organize downstream events ultimately leading to granule discharge. Skin MCs require both PI3K and ERK1/2 cascades for efficient exocytosis. β-arrestins act as opponents of G proteins and lead to signal termination with or without subsequent internalization. We recently demonstrated that ligand-induced internalization of MRGPRX2 requires the action of β-arrestin-1, but not of β-arrestin-2. Here, by using RNA interference, we find that both isoforms counter skin MC degranulation elicited by three MRGPRX2 agonists but not by FcεRI-aggregation. Analyzing whether this occurs through MRGPRX2 stabilization under β-arrestin attenuation, we find that reduction of β-arrestin-1 indeed leads to increased MRGPRX2 abundance, while this is not observed for β-arrestin-2. This led us speculate that β-arrestin-2 is involved in signal termination without cellular uptake of MRGPRX2. This was indeed found to be the case, whereby interference with β-arrestin-2 has an even stronger positive effect on ERK1/2 phosphorylation compared to β-arrestin-1 perturbation. Neither β-arrestin-1 nor β-arrestin-2 had an impact on AKT phosphorylation nor affected signaling via the canonical FcεRI-dependent route. We conclude that in skin MCs, β-arrestin-2 is chiefly involved in signal termination, whereas β-arrestin-1 exerts its effects by controlling MRGPRX2 abundance.

Keywords: ERK1/2; MRGPRX2; degranulation; mast cells; signal transduction; skin; β-arrestin.

Figure 1

MRGPRX2 but not FcεRI-driven degranulation is regulated by β-arrestin-1 and β-arrestin-2. Human skin derived MCs were treated with ARRB1-selective, ARRB2-selective or non-target siRNA for 48 h. The cells were then stimulated with (A) c48/80 (10 μg/ml), (B) SP (30 μM), (C) codeine (100 μg/ml), or (D) FcεRI aggregation (AER-37, 0.1 μg/ml). Degranulation was determined by β-hexosaminidase release. Each dot represents an independent culture, the data shown are mean ± SEM of n=5-9. *p < 0.05, **p < 0.01, and ns, not significant.

Figure 2

Knockdown of β-arrestin-1 promotes MRGPRX2 expression. Skin MCs were incubated with non-target siRNA, ARRB1-selective or ARRB2-selective siRNA for 48 h, MRGPRX2 cell surface expression was quantified by flow cytometry. (A) Net median fluorescence intensity (MFI) = (MFI MRGPRX2–MFI isotype) upon knockdown normalized to the respective non-target control (set as 1). The data are the mean ± SEM of n = 10, **p < 0.01, and ns, not significant. (B) Representative histograms of A. blue: MRGPRX2, red: isotype control.

Figure 3

β-arrestin-1 and β-arrestin-2 interfere with MRGPRX2-mediated ERK1/2 phosphorylation. MCs were treated with ARRB1-selective, ARRB2-selective, or non-target siRNA for 48 h. The cells were then activated by (A) c48/80 (10 μg/ml), (B) SP (30 μM), (C) codeine (100 μg/ml) for 1 min, or (D) FcεRI aggregation (AER-37, 0.1 μg/ml) for 30 min, respectively. Cells with no stimulus are given for comparison. Phosphorylated (p) and total (t) ERK as well as β actin and cyclophilin B were detected and quantified consecutively on the same membranes. (A–D) upper panel: relative pERK normalized against tERK expression; lower panel: representative blots showing pERK and all loading controls. The data are shown as mean ± SEM of 4–12 independent experiments (individual cultures). *p < 0.05, **p < 0.01, and ns, not significant.

Figure 4

AKT phosphorylation is unaffected by β-arrestin-1 or β-arrestin-2 knockdown. MCs were treated with ARRB1-selective, ARRB2-selective, or non-target siRNA for 48 h and stimulated with (A) c48/80, (B) SP, (C) codeine for 1 min, or (D) FcεRI aggregation for 30 min exactly as indicated in Figure 3. Phosphorylated (p) and total (t) AKT and loading controls (β-actin and cyclophilin B) detected serially on the same membranes. (A–D) upper panel: relative pAKT normalized against tAKT expression; lower panel: representative blots showing pAKT and all loading controls. Mean ± SEM, n = 4–13 independent experiments, ns, not significant.