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.

An in vitro compartmental system underlines the contribution of mitochondrial immobility to the ATP supply in the NMJ.

The neuromuscular junction (NMJ) is the largest, most-complex synapse in the human body. Motor neuron (MN) diseases, such as amyotrophic lateral sclerosis (ALS), specifically target MNs and the NMJs. However, little is known about the reasons for MN-selective neuronal and synaptic vulnerability in MN diseases. Here, utilizing a compartmental microfluidic in vitro co-culture system, we provide a possible explanation for why the NMJ, other than its unusual dimensions, differs from other synapses. By using live-imaging techniques, we discovered that cultured MNs display higher axonal and synaptic mitochondrial immobility compared with sympathetic neurons (SNs), leading to a profound enrichment of mitochondria only in the MN NMJ. Furthermore, by employing a synaptic ATP sensor, we show that mitochondrial respiration is the key contributor to ATP production in MN NMJs but not in SN synapses. Taken together, our data suggest that mitochondrial localization underlies the unique and specific qualities of MN NMJs. Our findings shed light on the role of mitochondria in MN and NMJ maintenance, and possibly indicate how mitochondria may serve as a source for selective MN vulnerability in neurodegenerative diseases.This article has an associated First Person interview with the first author of the paper.