MRGPRX2 Signaling on Mast Cell-Mediated Pseudoallergy and Neuroimmune Interaction
In addition to high affinity IgE receptor, human mast cells (MCs) express a newly identified receptor known as Mas-related G protein-coupled receptor-X2 (MRGPRX2; mouse ortholog MrgprB2). This receptor is predominantly expressed on only one subtype of MCs and it can be activated by a diverse group of cationic agonists including host defense peptides, Food and Drug Administration (FDA)-approved drugs associated with pseudoallergy and neuropeptides secreted from sensory nerve endings. Not surprisingly, MRGPRX2 has been implicated in several MC-mediated health and disease, ranging from host defense and wound healing to drug-induced pseudoallergic reactions, neurogenic inflammation and pain. However, there is a controversy regarding its role on rocuronium-induced hypersensitivity. Furthermore, the molecular mechanisms underlying MRGPRX2 regulation remain largely unknown. In this dissertation, we first investigated the role of MRGPRX2 on rocuronium-induced hypersensitivity. The effect of MRGPRX2 mutations (M196I, L226P and L237P) identified in a patient with rocuronium hypersensitivity were also tested. We found that rocuronium induced degranulation in murine and human MCs via MrgprB2 and MRGPRX2, respectively, but with different affinities, indicating important functional differences between these receptors. This indicates that mice expressing MrgprB2 may not be a suitable model to study human MRGPRX2 function and highlights the need to develop better animal models. It is now realized that activation of MCs by substance P (SP) via MRGPRX2 contributes to neurogenic inflammation, pain and itch. We sought to identify the mechanisms underlying MRGPRX2 signaling and regulation on SP-activated MC responses. Using pertussis toxin and YM-254890, we demonstrated that SP induces MRGPRX2-mediated Ca2+ mobilization and degranulation via both Gαi and Gαq. Next, we utilized information obtained from both structural modeling and naturally occurring MRGPRX2 missense variants to identify putative G protein coupling regions. In addition, several gain- and loss-of-function missense single nucleotide polymorphisms (SNPs) in MRGPRX2 have been discovered. Finally, we demonstrated that SP can activate β-arrestin recruitment and receptor internalization. A tyrosine residue in the highly conserved NPxxY motif of MRGPRX2 (Tyr279) is crucial for SP-induced β-arrestin recruitment and receptor internalization. This study reveals the novel findings that activation of MRGPRX2/B2 by SP is regulated by β-arrestins and that a highly conserved tyrosine residue is responsible for MRGPRX2 signaling and regulation.