MRGPRX2-Mediated Degranulation of Human Skin Mast Cells Requires the Operation of Gαi, Gαq, Ca++ Channels, ERK1/2 and PI3K—Interconnection between Early and Late Signaling

Abstract

The recent discovery of MRGPRX2 explains mast cell (MC)-dependent symptoms independently of FcεRI-activation. Because of its novelty, signaling cascades triggered by MRGPRX2 are rudimentarily understood, especially in cutaneous MCs, by which MRGPRX2 is chiefly expressed. Here, MCs purified from human skin were used following preculture or ex vivo and stimulated by FcεRI-aggregation or MRGPRX2 agonists (compound 48/80, Substance P) in the presence/absence of inhibitors. Degranulation was assessed by β-hexosaminidase or histamine release. Phosphorylation events were studied by immunoblotting. As a G protein-coupled receptor, MRGPRX2 signals by activating G proteins; however, their nature has remained controversial. In skin MCs, Gαi and Gαq were required for degranulation, but Gαi was clearly more relevant. Ca++ channels were likewise crucial. Downstream, PI3K was essential for granule discharge initiated by MRGPRX2 or FcεRI. ERK1/2 and JNK were additional participants, especially in the allergic route. Addressing possible points of intersection between early and later events, pERK1/2 and pAKT were found to depend on Gαi, further highlighting its significance. Gαq and Ca++ channels made some contributions to the phosphorylation of ERK. Ca++ differentially affected PI3K activation in FcεRI- vis-à-vis MRGPRX2-signaling, as channel inhibition increased pAKT only when triggered via FcεRI. Collectively, our study significantly extends our understanding of the molecular framework behind granule secretion from skin MCs.

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

“…Both β-arrestin-1, and More Strongly β-arrestin-2, Counter MRGPRX2-Mediated Phosphorylation of ERK1/2 Figure 1 showed that β-arrestin-1 and β-arrestin-2 restrict MRGPRX2's secretory competence to a similar degree, whereas Figure 2 demonstrated that only β-arrestin-1 reduction supports MRGPRX2 expression, calling for an alternative modus operandi in the case of β-arrestin-2. In skin MCs, both ERK1/2 and AKT are crucial to granule discharge activated via MRGPRX2 or FcεRI, as demonstrated recently by us (26). We therefore postulated that β-arrestin-2 deficiency may promote phosphorylation events downstream of MRGPRX2.…”

“…Foreskins from circumcisions were obtained with written informed consent of the patients or their legal guardians (26,42,46). The study was approved by the Ethics Committee of the Charité Universitätsmedizin Berlin (protocol code EA1/204/10, 9 March 2018) and experiments were conducted according to the Declaration of Helsinki Principles.…”

“…Detection of ERK1/2 and AKT phosphorylation was as described (26,30,48). In brief, MCs were stimulated at 5 × 10 5 /ml with c48/80 (10 µg/ml), SP (30 µM), or codeine (100 µg/ml) for 1 min, or FcεRI aggregation by AER-37 (0.1 µg/ml) for 30 min; cells kept without stimulus served as control.…”

“…Signaling via MRGPRX2 is still rudimentarily understood, but involves G αi and/or G αq to initiate cascades that culminate in degranulation (26,27). Recent structure-function studies suggested a unique ligand-transduction mechanism in MRGPRX family receptors, as they lack many of the canonical motifs associated with signaling and ligand recognition in family A G protein-coupled receptors (GPCRs) (28,29).…”

“…Recent structure-function studies suggested a unique ligand-transduction mechanism in MRGPRX family receptors, as they lack many of the canonical motifs associated with signaling and ligand recognition in family A G protein-coupled receptors (GPCRs) (28,29). Interestingly, allergic FcεRI-and pseudo-allergic pathways share the same signaling components further downstream, including extracellular signal-regulated kinase1/2 (ERK1/2), P38 and AKT, though kinetics differ between routes (26,(30)(31)(32). Both pathways also activate the guanosine triphosphatase (GTPase) Cdc42 upstream of actin cytoskeleton rearrangements, processes that involve the unconventional type I myosin 1f (MYO1F) (33).…”