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Published on: November 6, 2021
Identification of Gain and Loss of Function Missense Variants in MRGPRX2's Transmembrane and Intracellular Domains
Chalatip Chompunud Na Ayudhya1, Saptarshi Roy2, Ibrahim Alkanfari3
1Department of Basic and Translational Sciences, University of Pennsylvania, School of Dental Medicine, Philadelphia PA-19104, USA. chalatip@upenn.edu.
Abstract:
The neuropeptide substance P (SP) contributes to neurogenic inflammation through the activation of human mast cells via Mas-related G protein-coupled receptor-X2 (MRGPRX2). Using pertussis toxins and YM-254890, we demonstrated that SP induces Ca2+ mobilization and degranulation via both the Gαi and Gαq family of G proteins in rat basophilic leukemia (RBL-2H3) cells stably expressing MRGPRX2. To determine the roles of MRGPRX2's transmembrane (TM) and intracellular domains on SP-induced responses, we utilized information obtained from both structural modeling and naturally occurring MRGPRX2 missense variants. We found that highly conserved residues in TM6 (I225) and TM7 (Y279) of MRGPRX2 are essential for SP-induced Ca2+ mobilization and degranulation in transiently transfected RBL-2H3 cells. Cells expressing missense variants in the receptor's conserved residues (V123F and V282M) as well as intracellular loops (R138C and R141C) failed to respond to SP. By contrast, replacement of all five Ser/Thr residues with Ala and missense variants (S325L and L329Q) in MRGPRX2's carboxyl-terminus resulted in enhanced mast cell activation by SP when compared to the wild-type receptor. These findings suggest that MRGPRX2 utilizes conserved residues in its TM domains and intracellular loops for coupling to G proteins and likely undergoes desensitization via phosphorylation at Ser/Thr residues in its carboxyl-terminus. Furthermore, identification of gain and loss of function MRGPRX2 variants has important clinical implications for SP-mediated neurogenic inflammation and other chronic inflammatory diseases.
Insights
Substance P (SP) activates mast cells via MRGPRX2, a receptor crucial for neurogenic inflammation. Specific receptor mutations reveal key domains for SP signaling and potential therapeutic targets for inflammatory diseases.
Area of Science:
- Immunology
- Pharmacology
- Cell Biology
Background:
- Substance P (SP) is a neuropeptide implicated in neurogenic inflammation.
- Mast cell activation by SP is mediated through Mas-related G protein-coupled receptor-X2 (MRGPRX2).
- Understanding MRGPRX2 signaling is crucial for developing treatments for inflammatory conditions.
Purpose of the Study:
- To elucidate the roles of MRGPRX2's transmembrane and intracellular domains in SP-induced mast cell activation.
- To identify specific amino acid residues critical for SP signaling.
- To explore the clinical implications of MRGPRX2 variants in inflammatory diseases.
Main Methods:
- Utilized pertussis toxins and YM-254890 to investigate G protein coupling (Gαi and Gαq).
- Employed structural modeling and naturally occurring MRGPRX2 missense variants in RBL-2H3 cells.
- Assessed Ca2+ mobilization and degranulation responses to SP.
Main Results:
- Conserved residues in TM6 (I225) and TM7 (Y279) are essential for SP-induced Ca2+ mobilization and degranulation.
- Variants in conserved residues and intracellular loops abolished SP response.
- Carboxyl-terminal variants (S325L, L329Q) and Ser/Thr to Ala replacements enhanced mast cell activation by SP.
Conclusions:
- MRGPRX2 utilizes conserved TM and intracellular loop residues for G protein coupling.
- The receptor's carboxyl-terminus, particularly Ser/Thr residues, is involved in desensitization.
- Gain and loss of function MRGPRX2 variants offer potential therapeutic targets for SP-mediated inflammation.
