Role of a novel human mast cell G protein coupled receptor in Allergy and Inflammation

摘要 肥大细胞(MC)是造血系中的颗粒状组织驻留细胞，它有助于 先天免疫，但最为人所知的是它们在过敏性疾病中的作用，如过敏反应，鼻炎，哮喘， 慢性荨麻疹和特应性皮炎。除高亲和力Ig E受体(FcεRI)外，MCs还表达 大量的G蛋白偶联受体(GPCRs)，是膜受体蛋白中最大的一组 已知并且是药物治疗最常见的靶点。最近MC研究中一项令人兴奋的发展 已经认识到一组不同的阳离子两亲性多肽/蛋白质包括抗菌宿主 防御肽(HDPs)、神经肽(NPs)、嗜酸性粒细胞颗粒蛋白和许多FDA批准的 所有的肽能药物都通过一种新的被称为Mas相关基因-X2(MRGX2)的GPCR来激活人的MC。 然而，MRGX2激活/调控的分子机制仍不清楚。这个 在这项提议中要检验的假设是HDPs、NPs和嗜酸性粒细胞颗粒蛋白激活MRgX2 位于三个不同的MC隔间，导致酒渣鼻和慢性哮喘。 我们已经在NHLBI的大好机会中确定了42个自然发生的MRgX2错义变体 外显子组测序项目(NHLBI GO ESP)数据库，其中包含来自NHLBI Severe的测序数据 哮喘研究计划(SARP) 。在目标1中，将对选定的变种(基于等位基因频率)进行转基因 在MC系中，RBL-2H3细胞及其配体(HDPs、NPs和嗜酸性粒细胞颗粒蛋白)诱导 信号和脱颗粒将被确定。MRGX2与特定G蛋白相互作用的结构域 (GαQ和Gαi3)将被确定，这种交互对信令和介体释放的影响将是 下定决心。将利用荧光寿命成像(FLIM)技术来确定 MRGX2/G蛋白的实时相互作用。在目标2中，我们将检验这一假设，即MRGX2介导的MC 脱颗粒需要在三个隔室中整合来自受体的信号输入(细胞表面， 颗粒和脂筏)。我们将确定Orai通道、颗粒相关自噬蛋白的作用 和脂筏对MC脱颗粒的影响。调节这些信号通路中的每一个对MRGX2的影响 将确定介导的MC脱颗粒。在目标3中，三个活体模型：(A)mrgprB2-/-小鼠(小鼠 人类mrgX2的同源基因)，(B)用mrgX2替换mrgprB2和(C)人源化小鼠，表达 MCs中的mrgX2将用于确定mgx2在实验性酒渣鼻和慢性哮喘中的作用。这个 MRgX2的结构修饰及其信号调节对酒渣鼻和慢性哮喘的影响 要下定决心。这项研究的完成将有助于更好地理解MRGX2的结构/功能调控 在体外和体内调节慢性皮肤和肺部疾病(酒渣鼻和哮喘)。

Summary Mast cells (MCs) are granulated tissue-resident cells of hematopoietic lineage which contribute to innate immunity but are best known for their roles in allergic diseases such as anaphylaxis, rhinitis, asthma, chronic urticaria and atopic dermatitis. In addition to the high affinity IgE receptor (FcεRI), MCs express numerous G protein coupled receptors (GPCRs), which are the largest group of membrane receptor proteins known and are the most common targets of drug therapy. A recent exciting development in MC research has been the realization that a diverse group of cationic amphipathic peptides/proteins including antimicrobial host defense peptides (HDPs), neuropeptides (NPs), eosinophil granule proteins and many FDA approved peptidergic drugs all activate human MCs via a novel GPCR known as Mas-related gene-X2 (MrgX2). However, the molecular mechanisms involved in the activation/regulation of MrgX2 remain unknown. The hypothesis to be tested in this proposal is that HDPs, NPs and eosinophil granule proteins activate MrgX2 located in three different MC compartments and contribute to rosacea and chronic asthma. We have identified 42 naturally occurring missense variants of MrgX2 in the NHLBI Grand Opportunity Exome Sequencing Project (NHLBI GO ESP) database, which contains sequencing data from NHLBI's Severe Asthma Research Program (SARP) . In aim 1, selected variants (based on allele frequency) will be transfected in a MC line, RBL-2H3 cells and the ability of ligands (HDPs, NPs and eosinophil granule proteins) to induce signaling and degranulation will be determined. The domains of MrgX2 that interact with specific G proteins (Gαq and Gαi3) will be identified and the impact of this interaction on signaling and mediator release will be determined. Fluorescence lifetime imaging (FLIM) technology will be utilized to determine the mechanisms of MrgX2/G protein interaction in real time. In aim 2, we will test the hypothesis that MrgX2-mediated MC degranulation requires the integration of signaling input from the receptor in three compartments (cell surface, granules and lipid rafts). We will determine the roles of Orai channels, granule-associated autophagy proteins and lipid rafts on MC degranulation. The impact of modulating each of these signaling pathways on MrgX2- mediated MC degranulation will be determined. In aim 3, three in vivo models; (a) MrgprB2-/- mice (mouse ortholog of human MrgX2), (b) replacement of MrgprB2 with MrgX2 and (c) humanized mice that express MrgX2 in MCs will be used to determine the role MgX2 on experimental rosacea and chronic asthma. The effects of structural modification of MrgX2 and modulation of its signaling on rosacea and chronic asthma will be determined. Completion of this study will lead to a better understanding MrgX2 structure/function regulation in vitro and the modulation of chronic skin and lung diseases (rosacea and asthma) in vivo.