Naturally Occurring Missense MRGPRX2 Variants Display Loss of Function Phenotype for Mast Cell Degranulation in response to substance P and Human β-Defensin-3

Objectives: Mast cells (MCs) play an important role in periodontitis but the mechanisms of their activation and regulation remain unknown. MCs express a novel G protein-coupled receptor (GPCR) known as Mas-related GPCR X2 (MRGPRX2). In periodontitis, the expression of epithelium derived host defense peptides (human β-defensin-3 and neuropeptides (substance P) which activate MCs via Mas-related G protein coupled receptor X2 (MRGPRX2) is increased. For most GPCRs, the extracellular (ECL) domains and their associated transmembrane (TM) domains display the greatest structural diversity and are responsible for binding different ligands. The goal of the current study was to determine if MRGPRX2 is expressed in human gingival mast cells and if naturally occurring missense variants within MRGPRX2's ECL/TM domains contribute to gain or loss of function phenotype for MC degranulation in response to neuropeptides (substance P) and host defense peptide (human β-defensin-3). Methods: Immunofluorescence (IF) staining was performed on samples from human healthy and Chronic Periodontitis (CP) gingiva. Also, we constructed eight naturally occurring missense variants within MRGPRX2's using site directed mutagenesis technique. The constructs were then transfected individually into rat basophilic leukemia-2H3 cells and degranulation and calcium mobilization assays were done. Results: MCs were detected in normal gingiva and their numbers increased in CP. Moreover, MRGPRX2 is also expressed in normal gingival tissue and the number of cells expressing this receptor is increased in periodontitis. We found that all variants express in rat basophilic leukemia cells. However, four natural MRGPRX2 variants, G165E (rs141744602), D184H (rs372988289), W243R (rs150365137), and H259Y (rs140862085) failed to respond to any of the ligands tested. Conclusions: MRGPRX2-expressing mast cells are present in normal gingiva and that their numbers are elevated in patients with chronic periodontitis. Individual harboring loss of function missense mutation may be protected against chronic inflammatory diseases such as periodontitis.