Substance P Serves as a Balanced Agonist for MRGPRX2 and a Single Tyrosine Residue Is Required for β-Arrestin Recruitment and Receptor Internalization

The neuropeptide substance P (SP) mediates neurogenic inflammation and pain and contributes to atopic dermatitis in mice through the activation of mast cells (MCs) via Mas-related G protein-coupled receptor (GPCR)-B2 (MrgprB2, human ortholog MRGPRX2). In addition to G proteins, certain MRGPRX2 agonists activate an additional signaling pathway that involves the recruitment of β-arrestins, which contributes to receptor internalization and desensitization (balanced agonists). We found that SP caused β-arrestin recruitment, MRGPRX2 internalization, and desensitization. These responses were independent of G proteins, indicating that SP serves as a balanced agonist for MRGPRX2. A tyrosine residue in the highly conserved NPxxY motif contributes to the activation and internalization of many GPCRs. We have previously shown that Tyr²⁷⁹ of MRGPRX2 is essential for G protein-mediated signaling and degranulation. To assess its role in β-arrestin-mediated MRGPRX2 regulation, we replaced Tyr²⁷⁹ in the NPxxY motif of MRGPRX2 with Ala (Y279A). Surprisingly, we found that, unlike the wild-type receptor, Y279A mutant of MRGPRX2 was resistant to SP-induced β-arrestin recruitment and internalization. This study reveals the novel findings that activation of MRGPRX2 by SP is regulated by β-arrestins and that a highly conserved tyrosine residue within MRGPRX2’s NPxxY motif contributes to both G protein- and β-arrestin-mediated responses.