PROJECT SUMMARY Mast cells are tissue resident innate immune cells that have been best characterized for their role in mediating allergic diseases. A recent development in mast cell research has been the identification and characterization of a novel G protein coupled receptor, Mas-Related G-Protein-coupled Receptor X2 (MRGPRX2). This receptor promotes pseudoallergic reaction to U.S. Food and Drug Administration (FDA) approved drugs and chronic inflammation in diseases such as asthma, rosacea and hives (urticaria). While the role of MRGPRX2 in promoting allergic reactions has been investigated, the molecular mechanisms utilized by this receptor is poorly understood. Our data suggests that a Ca2+ sensor protein, stromal interaction molecule 1 (STIM1), which is an essential component of the store operated Ca2+ entry (SOCE) pathway, regulates MRGPRX2- and MrgprB2- (the mouse ortholog of the human receptor) induced responses in mast cells. Specifically, silencing the expression of STIM1 or pharmacological targeting of SOCE pathway results in reduced intracellular Ca2+ mobilization, degranulation and cytokine release following mast cell activation via MRGPRX2/MrgprB2 in vitro. Consistent with this data, inflammation is also reduced in in vivo mouse models of rosacea and paw edema when the SOCE pathway is pharmacologically inhibited. This proposal builds up on these observations; the central hypothesis is that STIM1 is a critical regulator of MRGPRX2 responses in mast cells. We will determine the role of STIM1 in regulating human MRGPRX2 and mouse MrgprB2 response in vitro and in vivo (Aim 1). STIM1 couples to the Orai1 channel to mediate SOCE in several cell types. In Aim 1, we will also test whether the STIM1-Orai1 interaction regulates mast cells responses following MRGPRX2/MrgprB2 activation. Similar to data obtained with STIM1 knockdown mast cells, silencing the expression of Na+/H+ exchanger regulatory factor (NHERF)1, an adaptor protein reduced MRGPRX2/MrgprB2-dependent Ca2+ mobilization in vitro and passive systemic anaphylaxis in vivo. How NHERF1 regulates STIM1 functions and affect intracellular Ca2+ mobilization is currently unknown. In Aim 2, we will explore the contribution of the NHERF1-STIM1-Orai1 axis in modulating mast cell response via the MRGPRX2/MrgprB2 receptors. Given the critical role of mast cell MRGPRX2 in causing pseudoallergic reactions and chronic inflammation in allergic diseases, elucidation of the mechanisms by which STIM1 and NHERF1 regulates mast cell MRGPRX2-mediated allergic response is of significant scientific and clinical importance. The successful completion of the proposed studies will likely lead to the identification of potential targets of the MRGPRX2 pathway that can provide insights into the future development of drugs for not only pseudoallergic reactions but also other mast cell-mediated inflammatory diseases.