The NHERF1-STIM1-Orai1 axis regulates MRGPRX2 responses in mast cells

项目总结 肥大细胞是一种驻留在组织中的先天免疫细胞，因其在 过敏性疾病。肥大细胞研究的最新进展是鉴定和鉴定 一种新的G蛋白偶联受体，Mas相关的G蛋白偶联受体X2(MRGPRX2)。这种受体 促进对美国食品和药物管理局(FDA)批准的药物和慢性药物的假性过敏反应 哮喘、酒渣鼻和麻疹等疾病的炎症。而MRGPRX2在促进 过敏反应已被研究，但该受体利用的分子机制知之甚少。 我们的数据表明，一种钙离子感受器蛋白，基质相互作用分子1(STIM1)，这是一种必不可少的 商店操作的钙内流(SOCE)途径的组成部分，调节MRGPRX2-和MRGPrB2-(小鼠 人类受体的同源同源物)在肥大细胞中诱导反应。具体地说，抑制STIM1的表达 或药物靶向SOCE途径导致细胞内钙动员减少，脱颗粒 在体外通过MRGPRX2/MRGPrB2激活肥大细胞后释放细胞因子。与这一数据一致， 当SOCE途径被激活时，体内酒渣鼻和足肿胀的小鼠模型中的炎症也被减轻 被药物抑制了。这一提议建立在这些观察的基础上；中心假设是STIM1 是肥大细胞中MRGPRX2反应的关键调节因子。我们将确定STIM1在调节中的作用 人MRGPRX2和小鼠MRGPrB2在体内外的反应(目标1)。STIM1连接到Orai1 在几种细胞类型中调节SOCE的通道。在目标1中，我们还将测试STIM1-Orai1相互作用 调节肥大细胞在MRGPRX2/MRgprB2激活后的反应。与使用STIM1获得的数据相似 敲除肥大细胞，沉默适配器Na+/H+交换调节因子(NHERF)1的表达 蛋白质降低MRGPRX2/MRgprB2依赖的体外钙离子动员和小鼠被动全身过敏反应 活着。NHERF1如何调节STIM1的功能和影响细胞内钙离子的动员目前尚不清楚。在……里面 目的2，我们将探讨NHERF1-STIM1-Orai1轴在调节肥大细胞反应中的作用 MRGPRX2/MRgprB2受体。鉴于肥大细胞MRGPRX2在引起假性变态反应中的关键作用 变态反应性疾病的反应和慢性炎症，阐明STIM1和STIM1的机制 NHERF1调节肥大细胞MRGPRX2介导的变态反应具有重要的科学和临床意义 重要性。拟议研究的成功完成将可能导致确定潜在的 MRGPRX2途径的靶点，不仅可以为药物的未来发展提供见解 假性过敏反应，也包括其他肥大细胞介导的炎症性疾病。

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.