Role of beta-arrestin-2 on IgE-mediated cofilin dephosphorylation and mast cell activation

 描述(申请人提供)：哮喘是世界上最常见的呼吸道疾病之一；目前有3亿人正在接受治疗，全球经济成本超过结核病和艾滋病毒/艾滋病的总和。因此，哮喘仍然是一个相当未得到满足的医疗需求领域。过敏性哮喘是由Th2细胞对过敏原的过度免疫反应引起的，其中免疫球蛋白E(IgE)和肥大细胞起关键作用。因此，变应原对肥大细胞的高亲和力ε受体(Fc-IgE RI)的聚集导致组胺的释放以及脂质和细胞因子的产生，这与哮喘的表现有关。本实验室的工作重点是研究肥大细胞中G蛋白偶联受体(GPCR)信号的调控。GPCRs的功能是通过一组被称为GPCRKs(GRKs)的蛋白激酶家族的磷酸化和支架分子？arrestin的募集来调节的。受体/？-ARR复合体用于 减弱G蛋白的激活，导致受体脱敏。B-ARR还促进下游的MAPK信号通路，而不依赖于G蛋白的激活。据我们所知，ç-ARR在肥大细胞Fc-εRI信号转导中的作用尚未见报道。基于我们意想不到的初步数据，我们假设ç-arr2作为一种支架蛋白，促进FcεRI介导的肥大细胞中的Cofilin去磷酸化。我们进一步假设，这种？arr2介导的cofilin去磷酸化有助于肥大细胞在肺中的募集。 促进慢性哮喘的发展。在目标1中，将利用逆转录病毒将不同结构域的？arr2转导入？？arr2-/-肥大细胞，并测定它们促进Fc-εRI介导的cofilin去磷酸化和肥大细胞趋化的能力。然后，我们将在肥大细胞中过表达不同的B-ARR2亚域，内源性表达BE-ARR2，并确定它们干扰Fc、ε、RI介导的cofilin去磷酸化和肥大细胞趋化的能力。在目标2中，我们将通过培育ç-arr2-Flosed小鼠和羧基肽酶-3(CPA-3)cre小鼠来删除肥大细胞中的？-arr2。我们还将通过将？arr2转导的BMMCs移植到肥大细胞缺陷的Wsh/Wsh小鼠体内，在肥大细胞中过表达？arr2。这些互补的方法将被用来检验这样的假设，即肥大细胞中表达的ç-arr2有助于它们在肺中的招募，并促进慢性哮喘的发展。最后，我们将在体内肥大细胞中表达B-arr2的cofilin和多肽结构域。这些策略将被用来确定肥大细胞特异性表达的cofilin和ç-arr2结构域是否调节慢性哮喘。这项研究可能会为肥大细胞在体内外趋化中的作用提供新的信息，并可能为慢性哮喘新疗法的开发提供更好的理论基础。

 DESCRIPTION (provided by applicant): Asthma is one of the most common respiratory diseases world-wide; 300 million people are currently receiving treatment and global economic cost exceeds those of tuberculosis and HIV/AIDS combined. Thus, asthma remains an area of considerable unmet medical need. Allergic asthma is caused by an overzealous Th2 immune response to allergens in which immunoglobulin E (IgE) and mast cells play critical roles. Thus, aggregation of high affinity IgE receptor (FcεRI) by allergen on mast cells results in histamine release and the generation of lipids and cytokines, which are responsible for the manifestations of asthma. The focus of our laboratory has been to study the regulation of G protein coupled receptor (GPCR) signaling in mast cells. Functions of GPCRs are regulated via their phosphorylation by a family of protein kinases known as GPCR kinases (GRKs) and the recruitment of the scaffolding molecule ß-arrestin (ß-arr). The receptor/ß-arr complex serves to attenuate G protein activation leading to receptor desensitization. ß-arr also promotes downstream MAPK signaling pathways independent of G protein activation. To the best of our knowledge, the role of ß-arr on FcεRI signaling in mast cells has never been reported. Based on our unexpected preliminary data, we hypothesize that ß-arr2 serves as a scaffolding protein to promote FcεRI- mediated cofilin dephosphorylation in mast cells. We further hypothesize that this ß-arr2-mediated cofilin dephosphorylation contributes to mast cell recruitment in the lung to promote the development chronic asthma. In aim 1, retrovirus will be used to transduce different domains of ß-arr2 into ß-arr2-/- mast cells and their ability to promote FcεRI-mediated cofilin dephosphorylation and mast cell chemotaxis will be determined. We will then overexpress different sub-domains of ß-arr2 into mast cells endogenously expressing ß-arr2 and their ability to interfere with FcεRI-mediated cofilin dephosphorylation and mast cell chemotaxis will be determined. In aim 2, we will delete ß-arr2 in mast cells by breeding ß-arr2-floxed mice and carboxypeptidase-3 (Cpa-3) cre mice. We will also overexpress ß-arr2 in mast cells in vivo by engrafting ß-arr2-transduced BMMCs into mast cell-deficient Wsh/Wsh mice. These complementary approaches will be used to test the hypothesis that ß-arr2 expressed in mast cells contributes to their recruitment in the lung and promotes the development of chronic asthma. Finally, we will express cofilin and peptide domains of ß-arr2 in mast cells in vivo. These strategies will be used to determine if mast cell-specific expression of cofilin and ß-arr2 domains modulate chronic asthma. This study will likely generate new information regarding the roles of ß-arr2 and cofilin in mast cell chemotaxis in vitro and in vivo and may provide a better rationale for the development of novel therapeutics for chronic asthma.