Regulation of MRGPRX2-mediated mast cell function by competence-stimulating peptide 1 and pro-adrenomedullin peptide

Mas-related G protein-coupled receptor (GPCR)-X2 (MRGPRX2, mouse ortholog MrgprB2) is predominantly expressed in mast cells (MCs) and is activated by a wide range of cationic ligands such as bacterial quorum sensing molecules (QSMs) and pro-adrenomedullin peptide 9-20 (PAMP-12). Activation of MrgprB2 by competence-stimulating peptide-1 (CSP-1), a QSM produced by Gram-positive bacteria, has recently been shown to promote antibacterial immunity, whereas its activation by PAMP-12 has been implicated in allergic contact dermatitis (ACD) and itch. Mechanisms via which the activation of the same receptor by different agonists contributes to different functional outcomes are unknown. GPCR agonists that activate both the G protein and receptor phosphorylation-mediated β-arrestin pathways are known as balanced agonists but agonists that activate only the G proteins are known as G protein-biased agonists. The goals of this study were to determine if CSP-1 and PAMP-12 serve as balanced or biased MRGPRX2 agonists and to investigate the differences in receptor internalization and desensitization in response to these agonists.

Bioluminescence resonance energy transfer 2 (BRET2) in HEK293T cells expressing MRGPRX2 was utilized to investigate G protein coupling following MRGPRX2 activation. β-arrestin recruitment studies were performed using Transcriptional Activation Following Arrestin Translocation (Tango) assay. Effects of PAMP-12 and CSP-1 on MRGPRX2 phosphorylation, desensitization, receptor internalization, extracellular-signal-regulated kinase (ERK) phosphorylation and degranulation were determined in RBL cells stably expressing MRGPRX2.

PAMP-12 and CSP-1 coupled to Gαq, Gαi1, and Gαi3 and induced ERK phosphorylation and degranulation at similar levels. However, PAMP-12 caused MRGPRX2 phosphorylation and desensitization, but CSP-1 did not. PAMP-12 caused ~50-fold increase in β-arrestin recruitment and this was associated with ~60% internalization of cell surface MRGPRX2. By contrast, CSP-1 caused ~5-fold increase in β-arrestin recruitment and ~20% receptor internalization.

These findings demonstrate that PAMP-12 and CSP-1 utilize shared G proteins to induce ERK phosphorylation and degranulation. However, they display substantial differences in their ability to cause MRGPRX2 phosphorylation, β-arrestin recruitment, receptor internalization and desensitization. These differences in G protein-biased and balanced signaling may dictate the ability of CSP-1 and PAMP-12 to contribute to host defense and ACD, respectively.