Mechanisms and regulation of MRGPRX2-regulated exocytosis in mast cells

While best known for their role in IgE-mediated allergy, a subset of mast cells (MCs) respond independently of IgE to a range of positively charged substances, commonly known as basic secretagogues (BS). The latter, including physiological ligands, such as substance P (SP) that is involved in neurogenic inflammation, FDA–approved drugs and the synthetic compound 48/80, activate MCs by binding to Mas-related G protein-coupled receptors (Mrgprs). Although conserved through evolution, ligand recognition and affinity of Mrgprs are species specific. RBL-2H3 cells, a rat MC line ectopically expressing MRGPRX2, the human Mrgpr, respond to BS. However, to ascertain that RBL-MRGPRX2 are an adequate model for studies of the human receptor, we have undertaken a broad comparative study of the responses elicited by endogenous MRGPRX2 in human LAD-2 MCs versus the ectopic receptor expressed in RBL cells. Studying the patterns of MC activation in response to three BS representatives, i.e. compound 48/80, SP and the drug vancomycin, we demonstrate that RBL-MRGPRX2 and LAD-2 cells display similar dose responses to the three ligands. Further, similar signaling pathways are initiated downstream of MRGPRX2 in both cell types. Finally, a pharmacological screen demonstrates the involvement of protein kinase C, phospholipases C and D, phosphatidylinositol 3 kinases type I and type III, ERK1/2 and IKB kinase β in stimulus-secretion coupling mechanisms of MRGPRX2 in both LAD-2 and RBL-MRGPRX2 cells. Taken together, our results reveal MRGPRX2 downstream signaling and affirm RBL-MRGPRX2 cells as an appropriate model for functional genomics analyses of MRGPRX2 function.