Role of Tyrosine residues in MRGPRX 2 on G protein and β-arrestin-mediated signaling in response to substance P

1 Role of Tyrosine residues in MRGPRX2 on G protein and β-arrestin-mediated signaling in response to substance P Chalatip Chompunud Na Ayudhya, DDS; Abdulaziz A. Alblaihess, DDS; Aetas Amponnawarat, DDS; Hydar Ali, PhD chalatip@upenn.edu Department of Basic and Translational Sciences, University of Pennsylvania School of Dental Medicine, Philadelphia, PA-19104, USA Recent evidence suggests that substance P (SP) contributes to neurogenic inflammation, chronic idiopathic urticaria and atopic dermatitis through mast cell activation via Mas-related G protein-coupled receptor (GPCR)-X2 (MRGPRX2). We found that SP serves as a balanced agonist for MRGPRX2 as it activated both G protein and b-arrestin-mediated signaling pathways. MRGPRX2 contains six tyrosine residues, five located in transmembrane domains and one in the intracellular loop 2. However, the role of MRGPRX2’s tyrosine residues on G protein and β-arrestin-mediated signaling is unknown. To address this question, we prepared cDNA encoding MRGPRX2 mutants; Y67A, Y89A, Y113A, Y137A, Y222A and Y279A, and generated transient transfectants in RBL-2H3 cells. We found that cells expressing Y67A, Y113A, Y137A or Y279A mutant were unresponsive to SP-induced Ca2+ mobilization and degranulation. Tyrosine Y279 in MRGPRX2’s seventh transmembrane domain within the NPXXY motif is important for G protein coupling. To determine if Y279 also contributes to barrestin-mediated signaling, we generated Y279A mutant in a plasmid used for transcriptional activation following arrestin translocation (TANGO) assay, and transiently transfected in engineered HEK293 cells (HTLA). We found that Y279A mutation resulted in significant loss of β-arrestin-mediated gene expression and receptor internalization in response to SP when compared to the wild-type receptor. These findings provide the first demonstration that a single tyrosine residue of MRGPRX2 may be responsible for both G protein and β-arrestinmediated signaling. Funding: This work was supported by National Institutes of Health grants R01-AI124182, R01-AI149487 and R01-AI143185 to Hydar Ali.