ABSTRACT/PROJECT SUMMARY Human Mas-related G protein coupled receptor (GPCR)-X2 (MRGPRX2) and its mouse orthologue MrgprB2 are predominantly expressed in connective tissue type mast cells and contribute to drug-induced pseudoallergy, non-histaminergic itch and neurogenic inflammation. Emerging evidence suggests that MRGPRX2/B2 is activated by a wide spectrum of cationic ligands but the molecular mechanisms involved in its regulation are largely unknown. Classical GPCRs are regulated by a process of desensitization via phosphorylation by GPCR kinases (GRKs) to prevent the detrimental effects of sustained signaling. In particular, GRK2, the most widely studied member of this family of kinases, has the ability to phosphorylate both GPCRs and non-receptor substrates and interact with a diverse repertoire of protein partners in addition to its function in receptor desensitization. It was recently demonstrated that GRK2 positively regulates FcεRI and negatively regulates anaphylatoxin C3a receptor (C3aR) signaling in mast cells. However, the possibility that GRK2 modulates MRGPRX2/B2 signaling in mast cells has yet to be determined. My expectation was that GRK2 would serve to desensitize MRGPRX2 responses in mast cells such that its overexpression would attenuate signaling and that silencing its expression would enhance the response. However, my preliminary data demonstrated the opposite suggesting that as for FceRI, GRK2 contributes to MRGPRX2/B2 signaling in mast cells. Based on my unexpected findings, I propose to test the novel hypothesis that GRK2 promotes MrgprB2-mediated mast cell signaling in vitro and contributes to pseudoallergy, non-histaminergic itch, neurogenic inflammation and in vivo. Because global GRK2 knockout mice are embryonic lethal, I have generated mice with mast cell-specific deletion of GRK2. Studies in Aim 1 will determine the effects of GRK2-deletion on mast cell degranulation and cytokine/chemokine generation in response to MrgprB2 ligands implicated in pseudoallergy (Ciprofloxacin, Icatibant), non-histaminergic itch (Proadrenomedullin N- terminal 20 peptide, fragment 9-20 (PAMP9-20) and neurogenic inflammation (Substance P, (SP)).The hypothesis that GRK2 mediates its effect on MrgprB2-mediated responses via the modulation of Syk, phospholipase Cg, protein kinase B (Akt) and NF-kB signaling pathways will be tested. MrgprB2-mediated mast cell degranulation (early response) is responsible for pseudoallergy and non-histaminergic itch whereas neurogenic inflammation depends on the cytokine/chemokine generation (late response). Studies in Aim 2 will test the hypothesis that mast cell-specific deletion of GRK2 modulate MrgprB2-mediated pseudoallergy, non-histaminergic itch and neurogenic inflammation in vivo. A comprehensive understanding of the mechanism via which GRK2 regulates MRGPRX2/B2 function in mast cells may lead to novel approaches for modulating pseudoallergy, non-histaminergic itch and neurogenic inflammation.