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.

Zhao Wang,Magda Babina,Kristin Franke,Torsten Zuberbier,Zhuoran Li,Gürkan Bal

doi:10.3390/cells11060953

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.

Highlights

With the discovery of Mas-related G protein-coupled receptor member X2 (MRGPRX2)as a key receptor of IgE-independent mast cell (MC) degranulation, two effective routes of degranulation are established in skin MCs [1,2]
We analyzed the effects of the G protein inhibitors PTX and YM-254890, as well as the Ca++ channel blockers 2-APB and La3+ on degranulation of skin-derived MCs
In skin-derived precultured MCs, we found that ERK1/2 as well as PI3K, significantly contribute to degranulation elicited by the MRGPRX2 agonists 48/80 and SP (Figure 2A,B)

Summary

Introduction

With the discovery of Mas-related G protein-coupled receptor member X2 (MRGPRX2). As a key receptor of IgE-independent mast cell (MC) degranulation, two effective routes of degranulation are established in skin MCs [1,2]. Since MRGPRX2 was discovered fairly recently, signaling cascades elicited by its ligands are still ill-defined. As a G protein-coupled receptor (GPCR), MRGPRX2 initiates signaling by activation of G proteins. Gαq specific [12], have been variably shown to interfere with MRGPRX2 signaling [13–15]; it is assumed that the cell type expressing MRGPRX2 plays a decisive role, but no data are currently available for skin MCs. cutaneous MCs are the most abundant MC population in the body [16], but they express the highest levels of MRGPRX2 and respond most vigorously to its ligands [17–20]. Skin MCs critically contribute to acute hypersensitivity reactions and anaphylaxis [21–24]

Methods

Results

Discussion

Conclusion