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.

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.

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.

The actin cytoskeleton and mast cell function.

The application of high and super-resolution microscopy techniques has extended the possibilities of studying actin dynamics in mast cells (MCs). These studies demonstrated the close correlation between actin-driven changes in cell morphology and the functions that MC perform during their life cycle. Dynamic conversions between actin polymerization and depolymerization support MC degranulation and leading to the release of the preformed, secretory granule (SG)-contained, inflammatory mediators. Cell flattening inflicting an actin porous geometry and clearing of cortical actin, characterize the secretory actin phenotype. In contrast, pericentral actin clusters, that entrap the SGs, characterize the migratory actin phenotype, which supports MC migration, but restricts MC degranulation. Multiple actin binding and actin interacting proteins regulate these actin rearrangements, in compliance with the signals elicited by the respective activating receptors. Here, we review recent findings on the interplay between the actin cytoskeleton and MC migration and degranulation.

Mammalian diaphanous-related formin 1 (mDia1) coordinates mast cell migration and secretion through its actin-nucleating activity.

BACKGROUND: Actin remodeling is a key regulator of mast cell (MC) migration and secretion. However, the precise mechanism underlying the coordination of these processes has remained obscure. OBJECTIVE: We sought to characterize the actin rearrangements that occur during MC secretion or chemotactic migration and identify the underlying mechanism of their coordination. METHODS: Using high-resolution microscopy, we analyzed the dynamics of actin rearrangements in MCs triggered to migration by IL-8 or prostaglandin E2 or to FcεRI-stimulated secretion. RESULTS: We show that a major feature of the actin skeleton in MCs stimulated to migration is the buildup of pericentral actin clusters that prevent cell flattening and converge the secretory granules (SGs) in the cell center. This migratory phenotype is replaced on encounter of an IgE cross-linking antigen that stimulates secretion through a secretory phenotype characterized by cell flattening, reduction of actin mesh density, ruffling of cortical actin, and mobilization of SGs. Furthermore, we show that knockdown of mammalian diaphanous-related formin 1 (mDia1) inhibits chemotactic migration and its typical actin rearrangements, whereas expression of an active mDia1 mutant recapitulates the migratory actin phenotype and enhances cell migration while inhibiting FcεRI-triggered secretion. However, mice deficient in mDia1 appear to have normal numbers of MCs in various organs at baseline. CONCLUSION: Our results demonstrate a unique role of actin rearrangements in clustering the SGs and inhibiting their secretion during MC migration. We identify mDia1 as a novel regulator of MC response that coordinates MC chemotaxis and secretion through its actin-nucleating activity.