4 research outputs found
Murepavadin, a Small Molecule Host Defense Peptide Mimetic, Activates Mast Cells via MRGPRX2 and MrgprB2
Pseudomonas aeruginosa is a frequent cause of hospital-acquired wound infection and is difficult to treat because it forms biofilms and displays antibiotic resistance. Previous studies in mice demonstrated that mast cells (MCs) not only contribute to P. aeruginosa eradication but also promote wound healing via an unknown mechanism. We recently reported that host defense peptides (HDPs) induce human MC degranulation via Mas-related G protein-coupled receptor-X2 (MRGPRX2). Small molecule HDP mimetics have distinct advantages over HDPs because they are inexpensive to synthesize and display high stability, bioavailability, and low toxicity. Murepavadin is a lipidated HDP mimetic, (also known as POL7080), which displays antibacterial activity against a broad panel of multi-drug-resistant P. aeruginosa. We found that murepavadin induces Ca2+ mobilization, degranulation, chemokine IL-8 and CCL3 production in a human MC line (LAD2 cells) endogenously expressing MRGPRX2. Murepavadin also caused degranulation in RBL-2H3 cells expressing MRGPRX2 but this response was significantly reduced in cells expressing missense variants within the receptor’s ligand binding (G165E) or G protein coupling (V282M) domains. Compound 48/80 induced β-arrestin recruitment and promoted receptor internalization, which resulted in substantial decrease in the subsequent responsiveness to the MRGPRX2 agonist. By contrast, murepavadin did not cause β-arrestin-mediated MRGPRX2 regulation. Murepavadin induced degranulation in mouse peritoneal MCs via MrgprB2 (ortholog of human MRGPRX2) and caused increased vascular permeability in wild-type mice but not in MrgprB2-/- mice. The data presented herein demonstrate that murepavadin activates human MCs via MRGPRX2 and murine MCs via MrgprB2 and that MRGPRX2 is resistant to β-arrestin-mediated receptor regulation. Thus, besides its direct activity against P. aeruginosa, murepavadin may contribute to bacterial clearance and promote wound healing by harnessing MC’s immunomodulatory property via the activation of MRGPRX2. © Copyright © 2021 Amponnawarat, Chompunud Na Ayudhya and Ali
Substance p Serves as a Balanced Agonist for Mrgprx2 and a Single Tyrosine Residue is Required for β‐Arrestin Recruitment and Receptor Internalization
Mrgprx2 Activation by Rocuronium: Insights From Studies with Human Skin Mast Cells and Missense Variants
Perioperative hypersensitivity (POH) to the neuromuscular blocking drug (NMBD) rocuronium was previously thought to be IgE and mast cell (MC)-mediated. However, the recent seminalobservation that rocuronium induces degranulation in murine peritoneal MCs (PMCs) via Mas-related G protein-coupled receptor B2 (MrgprB2) led to the idea that POH to this drug involves the activation of MRGPRX2 (human ortholog of MrgprB2). Furthermore, based on the demonstration that a patient with POH to rocuronium displayed three missense mutations (M196I, L226P and L237P) in MRGPRX2’s transmembrane domains, it was proposed that this hypersensitivity reaction resulted from aberrant activation of this receptor. We found that rocuronium at 20 μg/mL causeddegranulation in mouse PMCs via MrgprB2 but required at least 500 μg/mL to induce degranulation in human MCs via MRGPRX2. Furthermore, RBL-2H3 cells transiently expressing M196I, L226P and L237P variants did not display enhanced degranulation in response to rocuronium when comparedto the wild-type receptor. These findings provide the first demonstration that rocuronium induces degranulation in human MCs via MRGPRX2. Furthermore, the important differences between Mrg-prB2 and MRGPRX2 and the inability of rocuronium to induce enhanced response in cells expressing MRGPRX2 variants suggest that the mechanism of its POH is more complex than previously thought
Inhibition of Orai Channel Function Regulates Mas-Related G Protein-Coupled Receptor-Mediated Responses in Mast Cells
Mast cells (MCs) are tissue resident immune cells that play important roles in the pathogenesis of allergic disorders. These responses are mediated via the cross-linking of cell surface high affinity IgE receptor (FcϵRI) by antigen resulting in calcium (Ca2+) mobilization, followed by degranulation and release of proinflammatory mediators. In addition to FcϵRI, cutaneous MCs express Mas-related G protein-coupled receptor X2 (MRGPRX2; mouse ortholog MrgprB2). Activation of MRGPRX2/B2 by the neuropeptide substance P (SP) is implicated in neurogenic inflammation, chronic urticaria, mastocytosis and atopic dermatitis. Although Ca2+ entry is required for MRGPRX2/B2-mediated MC responses, the possibility that calcium release-activated calcium (CRAC/Orai) channels participate in these responses has not been tested. Lentiviral shRNA-mediated silencing of Orai1, Orai2 or Orai3 in a human MC line (LAD2 cells) resulted in partial inhibition of SP-induced Ca2+ mobilization, degranulation and cytokine/chemokine generation (TNF-α, IL-8, and CCL-3). Synta66, which blocks homo and hetero-dimerization of Orai channels, caused a more robust inhibition of SP-induced responses than knockdown of individual Orai channels. Synta66 also blocked SP-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation and abrogated cytokine/chemokine production. It also inhibited SP-induced Ca2+ mobilization and degranulation in primary human skin MCs and mouse peritoneal MCs. Furthermore, Synta66 attenuated both SP-induced cutaneous vascular permeability and leukocyte recruitment in mouse peritoneum. These findings demonstrate that Orai channels contribute to MRGPRX2/B2-mediated MC activation and suggest that their inhibition could provide a novel approach for the modulation of SP-induced MC/MRGPRX2-mediated disorders. Copyright © 2022 Chaki, Alkanfari, Roy, Amponnawarat, Hui, Oskeritzian and Ali