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

总结 肥大细胞（MC）是造血谱系的颗粒状组织驻留细胞，其有助于 先天免疫，但最为人所知的是它们在过敏性疾病如过敏反应、鼻炎、哮喘 慢性荨麻疹和特应性皮炎。除了高亲和力IgE受体（FcεRI）外，MCs还表达 许多G蛋白偶联受体（GPCR），其是最大的膜受体蛋白组 已知并且是药物治疗的最常见靶点。MC研究中最近令人兴奋的发展是 已经认识到，包括抗微生物宿主在内的多种阳离子两亲性肽/蛋白质 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 肽能药物都通过一种称为Mas相关基因-X2（MrgX 2）的新型GPCR激活人类MC。 然而，参与MrgX 2激活/调节的分子机制仍然未知。的 在该提议中待检验的假设是HDPs、NP和嗜酸性粒细胞颗粒蛋白激活MrgX 2 位于三个不同的MC隔室，并有助于红斑痤疮和慢性哮喘。 我们在NHLBI Grand Opportunity中鉴定了42种天然存在的MrgX 2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的严重 哮喘研究计划（SARP） .在目标1中，将转染选定的变体（基于等位基因频率）， 在MC系中，RBL-2 H3细胞和配体（HDPs、NP和嗜酸性粒细胞颗粒蛋白）诱导 将测定信号传导和脱粒。与特定G蛋白相互作用的MrgX 2结构域 （Gαq和Gαi3）的相互作用将被确定，这种相互作用对信号传导和介质释放的影响将被确定。 测定荧光寿命成像（FLIM）技术将用于确定的机制， 真实的时间MrgX 2/G蛋白相互作用。在目的2中，我们将检验MrgX 2介导的MC 脱粒需要来自受体的信号输入整合到三个隔室（细胞表面， 颗粒和脂筏）。我们将确定奥赖通道的作用，颗粒相关的自噬蛋白 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MrgX 2- 将测定介导的MC脱粒。在目标3中，三种体内模型：（a）MrgprB 2-/-小鼠（小鼠 人MrgX 2的直系同源物），（B）用MrgX 2替换MrgprB 2和（c）表达MrgX 2的人源化小鼠 MCs中的MrgX 2将用于确定MgX 2对实验性红斑痤疮和慢性哮喘的作用。的 MrgX 2的结构修饰及其信号转导对酒渣鼻和慢性哮喘的影响将 被确定。这项研究的完成将有助于更好地了解MrgX 2的结构/功能调控 在体外和调节慢性皮肤和肺部疾病（红斑痤疮和哮喘）在体内。

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.