Mast cell secretory granule fusion with amphisomes coordinates their homotypic fusion and release of exosomes.

Secretory granule (SG) fusion is an intermediate step in SG biogenesis. However, the precise mechanism of this process is not completely understood. We show that Golgi-derived mast cell (MC) SGs enlarge through a mechanism that is dependent on phosphoinositide (PI) remodeling and fusion with LC3+ late endosomes (amphisomes), which serve as hubs for the fusion of multiple individual SGs. Amphisome formation is regulated by the tyrosine phosphatase PTPN9, while the subsequent SG fusion event is additionally regulated by the tetraspanin protein CD63 and by PI4K. We also demonstrate that fusion with amphisomes imparts to SGs their capacity of regulated release of exosomes. Finally, we show that conversion of PI(3,4,5)P3 to PI(4,5)P2 and the subsequent recruitment of dynamin stimulate SG fission. Our data unveil a key role for lipid-regulated interactions with the endocytic and autophagic systems in controlling the size and number of SGs and their capacity to release exosomes.

The Underlying Rab Network of MRGPRX2-Stimulated Secretion Unveils the Impact of Receptor Trafficking on Secretory Granule Biogenesis and Secretion.

MRGPRX2, the human member of the MAS-related G-protein-coupled receptors (GPCRs), mediates the immunoglobulin E (IgE)-independent responses of a subset of mast cells (MCs) that are associated with itch, pain, neurogenic inflammation, and pseudoallergy to drugs. The mechanisms underlying the responses of MRGPRX2 to its multiple and diverse ligands are still not completely understood. Given the close association between GPCR location and function, and the key role played by Rab GTPases in controlling discrete steps along vesicular trafficking, we aimed to reveal the vesicular pathways that directly impact MRGPRX2-mediated exocytosis by identifying the Rabs that influence this process. For this purpose, we screened 43 Rabs for their functional and phenotypic impacts on MC degranulation in response to the synthetic MRGPRX2 ligand compound 48/80 (c48/80), which is often used as the gold standard of MRGPRX2 ligands, or to substance P (SP), an important trigger of neuroinflammatory MC responses. Results of this study highlight the important roles played by macropinocytosis and autophagy in controlling MRGPRX2-mediated exocytosis, demonstrating a close feedback control between the internalization and post-endocytic trafficking of MRGPRX2 and its triggered exocytosis.

GRK2 differentially regulates FcεRI and MRGPRB2-mediated responses in mast cells.

In addition to high-affinity IgE receptor (FcεRI), a subtype of mouse mast cells (MCs) expresses a G protein-coupled receptor known as Mas-related G protein-coupled receptor (GPCR)-B2 (MRGPRB2; human ortholog MRGPRX2). GPCR kinase 2 (GRK2) is a Serine/Threonine kinase that phosphorylates GPCRs to promote their desensitization and internalization. We previously showed that silencing GRK2 expression in mouse bone marrow-derived MCs (BMMCs) blocks IgE-mediated degranulation. Compound 48/80 (C48/80), substance P (SP) and LL-37 cause degranulation in human and mouse MCs via MRGPRX2 and MRGPRB2, respectively. We also reported that C48/80 and SP cause desensitization and internalization of MRGPRX2, but LL-37 does not. Here, we generated mice with MC-specific deletion of Grk2 (Cpa3Cre+/Grk2fl/fl ) to determine its role on IgE-mediated responses and to assess whether it differentially regulates degranulation in response to LL-37, C48/80 and SP. Absence of GRK2 substantially inhibited IgE-mediated tyrosine phosphorylation of STAT5, calcium mobilization, and degranulation in mouse primary lung-derived MCs (PLMCs). By contrast, peritoneal MCs (PMCs) from Cpa3Cre+/Grk2fl/fl mice demonstrated significant enhancement of degranulation in response to C48/80 and SP, but not LL-37. Deletion of Grk2 in MCs attenuated IgE-mediated passive cutaneous anaphylaxis (PCA) and itch but not passive systemic anaphylaxis (PSA). Surprisingly, PSA was significantly reduced in Mrgprb2-/- mice. These findings suggest that GRK2 contributes to PCA and itch but not PSA. By contrast, GRK2 desensitizes MRGPRX2/B2-mediated responses to C48/80 and SP but not LL-37. However, IgE-mediated PSA likely involves the activation of MRGPRB2 by LL-37 or a similar agonist, whose function is resistant to modulation by GRK2.

Inhibition of MRGPRX2 but not FcεRI or MrgprB2-mediated mast cell degranulation by a small molecule inverse receptor agonist.

Mas-related G protein-coupled receptor-X2 (MRGPRX2) expressed on mast cells (MCs) contributes to hypersensitivity reactions to cationic US-Food and Drug Administration (FDA) approved drugs such as the neuromuscular blocking agent, rocuronium. In addition, activation of MRGPRX2 by the neuropeptide substance P (SP) and the pro-adrenomedullin peptide (PAMP-12) is associated with a variety of cutaneous conditions such as neurogenic inflammation, pain, atopic dermatitis, urticaria, and itch. Thus, small molecules aimed at blocking MRGPRX2 constitute potential options for modulating IgE-independent MC-mediated disorders. Two inverse MRGPRX2 agonists, named C9 and C9-6, have recently been identified, which inhibit basal G protein activation and agonist-induced calcium mobilization in transfected HEK293 cells. Substance P serves as a balanced agonist for MRGPRX2 whereby it activates both G protein-mediated degranulation and ß-arrestin-mediated receptor internalization. The purpose of this study was to determine if C9 blocks MRGPRX2's G protein and ß-arrestin-mediated signaling and to determine its specificity. We found that C9, but not its inactive analog C7, inhibited degranulation in RBL-2H3 cells stably expressing MRGPRX2 in response to SP, PAMP-12 and rocuronium with an IC50 value of ~300 nM. C9 also inhibited degranulation as measured by cell surface expression of CD63, CD107a and ß-hexosaminidase release in LAD2 cells and human skin-derived MCs in response to SP but not the anaphylatoxin, C3a or FcϵRI-aggregation. Furthermore, C9 inhibited ß-arrestin recruitment and MRGPRX2 internalization in response to SP and PAMP-12. We found that a G protein-coupling defective missense MRGPRX2 variant (V282M) displays constitutive activity for ß-arrestin recruitment, and that this response was significantly inhibited by C9. Rocuronium, SP and PAMP-12 caused degranulation in mouse peritoneal MCs and these responses were abolished in the absence of MrgprB2 or cells treated with pertussis toxin but C9 had no effect. These findings suggest that C9 could provide an important framework for developing novel therapeutic approaches for the treatment of IgE-independent MC-mediated drug hypersensitivity and cutaneous disorders.

GRK2 inhibitors, paroxetine and CCG258747, attenuate IgE-mediated anaphylaxis but activate mast cells via MRGPRX2 and MRGPRB2.

G protein-coupled receptor (GPCR) kinase 2 (GRK2), which phosphorylates agonist-occupied GPCRs to promote their desensitization, has been investigated as an attractive therapeutic target for cardiovascular and metabolic diseases. Several GRK2-targeted inhibition strategies have been reported including the use of direct pharmacological inhibitors such as paroxetine (a widely prescribed antidepressant) and its analogs such as compound CCG258747. Cross-linking of high affinity IgE receptor (FcϵRI) on mast cells (MCs) and the resulting degranulation causes anaphylaxis and allergic asthma. Using gene silencing strategy, we recently showed that GRK2 contributes to FcεRI signaling and MC degranulation. The purpose of this study was to determine if the GRK2 inhibitors paroxetine and CCG258747 modulate FcεRI-mediated MC responses in vitro and in vivo. Utilizing rat basophilic leukemia (RBL-2H3) cells and primary mouse lung MCs (LMCs), we found that paroxetine and CCG258747 inhibit FcϵRI-mediated calcium mobilization and degranulation. Furthermore, intravenous administration of paroxetine and CCG258747 in mice resulted in substantial reduction of IgE-mediated passive cutaneous anaphylaxis. Unlike LMCs, human cutaneous MCs abundantly express a novel GPCR known as MRGPRX2 (mouse; MRGPRB2). We found that in contrast to their inhibitory effects on FcεRI-mediated MC responses, both paroxetine and CCG258747 induce calcium mobilization and degranulation in RBL-2H3 cells stably expressing MRGPRX2 but not in untransfected cells. Furthermore, paroxetine and CCG258747 induced degranulation in peritoneal MCs from Wild-type (WT) mice in vitro and caused increased cutaneous vascular permeability in vivo, but these responses were substantially reduced in Mrgprb2-/- mice. Additionally, upon intradermal injection, paroxetine also induced neutrophil recruitment in WT but not Mrgprb2-/- mice. These findings suggest that in addition to their potential therapeutic utility against cardiovascular and metabolic disorders, paroxetine-based GRK2-inhibitors may serve to modulate IgE-mediated anaphylaxis and to enhance cutaneous host defense by harnessing MC's immunomodulatory property through the activation of MRGPRX2/MRGPRB2.

Spatiotemporal Patterns of Substance P-Bound MRGPRX2 Reveal a Novel Connection Between Macropinosome Resolution and Secretory Granule Regeneration in Mast Cells.

MRGPRX2, the human member of the MAS-related G protein coupled receptors (Mrgprs), serves as the cellular target of human mast cells (MCs) for innate ligands, including neuropeptides and antimicrobial peptides. In addition, MRGPRX2 also functions as the receptor for multiple FDA-approved drugs. As such, MRGPRX2 is a mediator of MC responses in neurogenic inflammation, host defense and pseudoallergy. We analyzed the spatiotemporal patterns of MRGPRX2 following its binding of the neuropeptide substance P (SP). Herein, we show that MRGPRX2 internalizes via both endocytosis and macropinocytosis, followed by its distribution between a perinuclear region and the secretory granules (SGs). Further, we show that MRGPRX2-containing macropinosomes undergo resolution by a mechanism that involves dynamin and LC3, giving rise to the incorporation of both LC3 and MRGPRX2 into the SGs. SP then promotes the acidification of the LC3-associated SGs, presumably by stimulating their fusion with lysosomes. Taken together, our results reveal a unique mode of MRGPRX2 trafficking that complements endocytosis and involves macropinocytosis, autophagic machinery-assisted macropinosome resolution and receptor delivery to the SGs.

Role of MrgprB2 in Rosacea-Like Inflammation in Mice: Modulation by ß-Arrestin 2.

Cathelicidin LL-37‒mediated activation of mast cells (MCs) has been implicated in the pathogenesis of rosacea, but the receptor involved and the mechanism of its activation and regulation remain unknown. We found that skin biopsies from patients with rosacea display higher frequencies of MCs expressing MRGPRX2 (mouse counterpart MrgprB2) than normal skin. Intradermal injection of LL-37 in wild-type mice resulted in MC recruitment, expression of inflammatory mediators, and development of rosacea-like inflammation. These responses were substantially reduced in MrgprB2‒/‒ mice and abolished in MC deficient Wsh/Wsh mice. ß-arrestin 2 is an adaptor protein that regulates G protein-coupled receptor function by receptor desensitization and also by activation of downstream signaling. We found that LL-37‒induced rosacea-like inflammation was significantly reduced in mice with MC-specific deletion of ß-arrestin 2 compared with that in control mice. Interestingly, the absence of ß-arrestin 2 resulted in enhanced cofilin phosphorylation and substantial inhibition of LL-37‒induced chemotaxis of mouse peritoneal MCs. Furthermore, LL-37‒induced extracellular signal‒regulated kinase 1/2 phosphorylation, NF-κB activation, and proinflammatory cytokine/chemokine production were reduced in ß-arrestin 2‒/‒ peritoneal MCs compared with those in wild-type cells. These findings suggest that MRGPRX2/B2 participates in rosacea and that ß-arrestin 2 contributes to its pathogenesis by promoting cofilin dephosphorylation, extracellular signal‒regulated kinase 1/2 and NF-κB phosphorylation, MC chemotaxis, and chemokine/cytokine generation.

Mas-Related G Protein-Coupled Receptor-X2 and Its Role in Non-immunoglobulin E-Mediated Drug Hypersensitivity.

A diverse group of Food and Drug Administration-approved cationic drugs including antibiotics, neuromuscular blocking drugs, opioids, antidepressants, and radiocontrast media activate mast cells and cause hypersensitivity reactions by both an immunoglobulin E IgE-dependent and independent manner. The recent discovery that these drugs activate mast cells via the G protein-coupled receptor known as Mas-related GPCR-X2 (MRGPRX2) has represented a paradigm shift of how drug hypersensitivity reactions are viewed. This article provides an overview of the current status of the role of MRGPRX2 on non-IgE-mediated drug hypersensitivity. Potential risk factors and evaluation for suspected MRGPRX2-mediated drug reactions are also discussed.

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 ß-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 ß-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 ß-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.

Substance P Serves as a Balanced Agonist for MRGPRX2 and a Single Tyrosine Residue Is Required for ß-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 ß-arrestins, which contributes to receptor internalization and desensitization (balanced agonists). We found that SP caused ß-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 ß-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 ß-arrestin recruitment and internalization. This study reveals the novel findings that activation of MRGPRX2 by SP is regulated by ß-arrestins and that a highly conserved tyrosine residue within MRGPRX2's NPxxY motif contributes to both G protein- and ß-arrestin-mediated responses.

Multifaceted MRGPRX2: New insight into the role of mast cells in health and disease.

Cutaneous mast cells (MCs) express Mas-related G protein-coupled receptor-X2 (MRGPRX2; mouse ortholog MrgprB2), which is activated by an ever-increasing number of cationic ligands. Antimicrobial host defense peptides (HDPs) generated by keratinocytes contribute to host defense likely by 2 mechanisms, one involving direct killing of microbes and the other via MC activation through MRGPRX2. However, its inappropriate activation may cause pseudoallergy and likely contribute to the pathogenesis of rosacea, atopic dermatitis, allergic contact dermatitis, urticaria, and mastocytosis. Gain- and loss-of-function missense single nucleotide polymorphisms in MRGPRX2 have been identified. The ability of certain ligands to serve as balanced or G protein-biased agonists has been defined. Small-molecule HDP mimetics that display both direct antimicrobial activity and activate MCs via MRGPRX2 have been developed. In addition, antibodies and reagents that modulate MRGPRX2 expression and signaling have been generated. In this article, we provide a comprehensive update on MrgprB2 and MRGPRX2 biology. We propose that harnessing MRGPRX2's host defense function by small-molecule HDP mimetics may provide a novel approach for the treatment of antibiotic-resistant cutaneous infections. In contrast, MRGPRX2-specific antibodies and inhibitors could be used for the modulation of allergic and inflammatory diseases that are mediated via this receptor.

Revisiting the role of MRGPRX2 on hypersensitivity reactions to neuromuscular blocking drugs.

Anaphylaxis is caused by a variety of triggers including Food and Drug Administration (FDA)-approved antibiotics, contrast media and neuromuscular blocking drugs (NMBDs). Traditionally, drug-induced anaphylaxis was thought to result mainly from IgE-mediated histamine release from mast cells. Recently, a G protein-coupled receptor known as MRGPRX2 has been identified and shown to be highly expressed on human skin but not lung mast cells. The demonstration that many NMBDs induce degranulation in human mast cells via MRGPRX2 led to the idea that this receptor contributes to NMBD-induced hypersensitivity reactions. However, other studies have raised doubts regarding its role in drug-induced hypersensitivity. This review discusses the current status and controversy on MRGPRX2's role on NMBD-induced hypersensitivity.

Authentic and Ectopically Expressed MRGPRX2 Elicit Similar Mechanisms to Stimulate Degranulation of Mast Cells.

The identification of the Mas-related G-protein-coupled receptors (Mrgpr) as targets of diverse stimuli of mast cells (MCs), including neuropeptides and pseudo-allergy causing drugs, has placed these receptors at a prime position in MC research. However, the species-dependent diversity of these receptors raises the need for an adequate model for investigating the human MRGPRX2 receptor. RBL-2H3 cells, stably transfected with MRGPRX2 (RBL-MRGPRX2), are increasingly used for this purpose. Therefore, we investigated whether ectopically expressed MRGPRX2, in rat MCs, recapitulates its authentic signaling. To this purpose, we performed a broad comparative study of the responses of human LAD-2 MCs that express MRGPRX2 endogenously, and RBL-MRGPRX2 cells to compound 48/80, substance P and vancomycin, three proto-type ligands of MRGPRX2. We demonstrate that both models share similar dose-response relationships, kinetics and sensitivities to a wide range of signaling targeting drugs. Therefore, our results indicate that ectopically expressed MRGPRX2 preserves the signaling pathways employed to evoke human MC degranulation, which we show to rely on ERK1/2 MAP kinases, phospholipase C (PLC) and autophagy-related signaling. Importantly, we also show that the underlying mechanisms of MRGPRX2-triggered MC degranulation in either LAD-2 or RBL-MRGPRX2 cells are different from those elicited by its rodent orthologs.

Mast Cell-Specific MRGPRX2: a Key Modulator of Neuro-Immune Interaction in Allergic Diseases.

PURPOSE OF REVIEW: Atopic dermatitis (AD) and allergic asthma are complex disorders with significant public health burden. This review provides an overview of the recent developments on Mas-related G protein-coupled receptor-X2 (MRGPRX2; mouse counterpart MrgprB2) as a potential candidate to target neuro-immune interaction in AD and allergic asthma. RECENT FINDINGS: Domestic allergens directly activate sensory neurons to release substance P (SP), which induces mast cell degranulation via MrgprB2 and drives type 2 skin inflammation in AD. MRGPRX2 expression is upregulated in human lung mast cells and serum of asthmatic patients. Both SP and hemokinin-1 (HK-1 generated from macrophages, bronchial cells, and mast cells) cause degranulation of human mast cells via MRGPRX2. MrgprB2 contributes to mast cell-nerve interaction in the pathogenesis of AD. Furthermore, asthma severity is associated with increased MRGPRX2 expression in mast cells. Thus, MRGPRX2 could serve as a novel target for modulating AD and asthma.

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 seminal observation 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 caused degranulation 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 compared to the wild-type receptor. These findings provide the first demonstration that rocuronium induces degranulation in human MCs via MRGPRX2. Furthermore, the important differences between MrgprB2 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.