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.