NHERF1 regulates MRGPRX2/MrgprB2 responses in mast cells

项目总结

PROJECT SUMMARY Mast cells are tissue resident innate immune cells that have been best characterized for their role in mediating allergic diseases. A new 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 thoroughly investigated, the molecular mechanisms utilized by this receptor is poorly understood. Our data suggests that Na+/H+ exchanger regulatory factor (NHERF)1, an adaptor protein, regulates MRGPRX2- and MrgprB2- (the mouse ortholog of the human receptor) induced responses in mast cells. Specifically, reducing the expression of NHERF1 in mast cells results in decreased intracellular Ca2+ mobilization and degranulation via MRGPRX2/MrgprB2 in vitro. Consistent with this data, NHERF1 also promoted anaphylaxis in MrgprB2-dependent mouse models of paw edema and passive systemic anaphylaxis in vivo. This proposal builds up on these observations; the central hypothesis is that NHERF1 is a critical regulator of MRGPRX2 responses in mast cells. Using a novel mouse strain that we have recently generated, Cpa3- Cre+:NHERF1fl/fl, primary human CD34+ stem cell-derived mast cells and humanized mice, we will determine the role of mast cell-specific expression of NHERF1 in regulating mouse MrgprB2 and human MRGPRX2 receptors in vitro and in vivo (Aim 1). In Aim 2, we will explore the mechanisms through which NHERF1 modulates mast cell response. Specifically, we will determine whether phosphorylation of NHERF1 is important for NHERF1 regulation of MRGPRX2/MrgprB2. We will identify the components of the NHERF1-interactome and signaling proteins and pathways through which NHERF1 functions in mast cells by using transcriptomic and proteomic approaches. Additionally, we will delineate the role of NHERF1-microRNA (miR)155-suppressor of cytokine signaling (SOCS1)-Src homology 2 (SH2) domain containing inositol polyphosphate 5-phosphatase 1 (SHIP-1) axis in promoting mast cell activation. Given the critical role of mast cell-MRGPRX2 in causing pseudoallergic reactions and chronic inflammation in allergic diseases, elucidation of the mechanisms by which NHERF1 contributes to MRGPRX2-mediated allergic response is of significant scientific and clinical importance. We firmly believe that 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.