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.
Highlights
Mast cells (MCs) are tissue-resident granulocytes of hematopoietic origin that play a pivotal role in the inflammatory processes due to their ability to release a wide array of proinflammatory mediators and recruit various immune cells upon stimulation [1,2,3]
These findings suggest that Mas-related G protein-coupled receptor-X2 (MRGPRX2) may couple to both pertussis toxin (PTx)-sensitive (Gαi) and insensitive (Gαq) G proteins
It is possible that low-dose substance P (SP) induces MRGPRX2 to preferentially couple to either Gαi or Gαq, whereas a high concentration of SP mediates MRGPRX2 conformational change to couple to both G proteins
Summary
Mast cells (MCs) are tissue-resident granulocytes of hematopoietic origin that play a pivotal role in the inflammatory processes due to their ability to release a wide array of proinflammatory mediators and recruit various immune cells upon stimulation [1,2,3]. MCs are widely distributed throughout the body and are found in close proximity to peripheral nerve endings in various tissues including skin, gastrointestinal mucosa, and respiratory tract [4]. Activation of MCs by SP leads to their degranulation, resulting in vasodilation, plasma extravasation, and the recruitment of immune cells including lymphocytes, neutrophils, and macrophages [5,9,10]. Immune cell recruitment further amplifies local inflammatory responses and facilitates peripheral nerve sensitization, which are critical characteristics of neurogenic inflammation [10]. SP-induced MC activation has been implicated in the pathogenesis of pain and many chronic inflammatory diseases such as sickle cell disease [11], atopic dermatitis [12], and chronic idiopathic urticaria [13]
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