Summary: Mast cells (MCs) are tissue-resident immune cells that are best known for mediating IgE/FcεRI-mediated hypersensitivity and atopic disorders. However, recent excitement in MC research has been the realization that a diverse group of cationic amphipathic peptides and FDA-approved drugs activate human MCs via a novel G protein coupled receptor (GPCR) known as MRGPRX2 (mouse counterpart, MrgprB2). This receptor is implicated in host defense, immediate drug hypersensitivity reactions (IDHRs), neurogenic inflammation/pain and a variety of cutaneous disorders. Not surprisingly, there is an intense interest in developing MRGPRX2 inhibitors for treating MC-mediated disorders. Although several small molecule inhibitors have been developed, a phase 2 clinical trial with a promising lead was recently paused because of toxicity issues, emphasizing the need for additional approach. This proposal is focused on two sets of proteins that directly interact with GPCRs (G proteins and GPCR kinase 2, GRK2) that are traditionally viewed as displaying opposing effects on receptor function. We will test the novel hypothesis that G proteins and GRK2 interact with MRGPRX2 to promote MC-mediated IDHRs, anaphylaxis and cutaneous disorders. In aim 1, we will knockdown the expression of Gαi and Gαq family of G proteins in human MCs and determine the impact of this deletion on MRGPRX2-mediated responses in vitro. We will use retrovirus to express MRGPRX2 in marrow-derived MCs (BMMCs) in the absence of G proteins. These BMMCs will be engrafted into MC-deficient mice. This approach will be used to determine the role of specific G proteins on MRGPRX2-mediated IDHRs, anaphylaxis, rosacea and psoriasis in vivo. We will express MRGPRX2 variants with defective coupling to different G proteins in BMMCs and these cells will be engrafted into MC-deficient to mice. This strategy will be used to determine the impact of disrupting MRGPRX2/G protein interaction on disease phenotype in vivo. In aim 2,