The Signal Transduction Mechanism of Interleukin-5

描述（由申请人提供）：哮喘的特征是嗜酸性粒细胞增多， 呼吸道的炎症。白细胞介素-5（IL-5）是一种重要的 嗜酸性粒细胞分化和功能的调节剂。IL-5激活 嗜酸性粒细胞通过刺激一些酪氨酸激酶。然而， 酪氨酸激酶在转导IL-5特异性信号中起重要作用， 未知基于初步结果，我们假设林恩激酶在细胞内起作用。 在IL-5刺激的嗜酸性粒细胞分化中的重要和非冗余作用 以及气道嗜酸性粒细胞炎症的发展。上 具体的目的，我们将研究林恩与IL-5受体的物理联系 α亚基（IL-5Ra），并研究其磷酸化受体的能力。的 林恩与干细胞向嗜酸性粒细胞分化的生物学相关性将 使用林恩敲除小鼠进行检查。此外，我们将研究林恩的影响， 空突变对气道变应性炎症发展的影响 卵清蛋白致敏的哮喘小鼠模型。第二个具体目标是 阐明IL-5 Ra激活林恩激酶的分子机制。 为了鉴定可能激活林恩的IL-5 Ra相关分子， 激酶，我们已经进行了酵母双杂交筛选的人cDNA文库 并克隆了一个新的SH2/SH3配体--IRIP（ImmunoReceptor-Interactive Protein）。 我们假设IRIP通过其SH 3相互作用激活林恩激酶 域为了检验上述假设，我们将绘制IRJP的结合位点， 和使用重组林恩结构域和定点诱变的林恩激酶。的 IRIP SH2/SH3基序对林恩活化的重要性将用 基序肽和定点诱变。最后，IRIP的相关性 将通过过度表达野生型和 IRIP在小鼠干细胞和HL-60细胞中的显性负突变体， 检查它们分化成嗜酸性粒细胞。拟议的研究是 旨在识别关键的IL-5特异性信号分子， 用于嗜酸性粒细胞分化和嗜酸性粒细胞炎症。克隆和 新型Src型酪氨酸激活剂IRIP的功能表征 激酶将推进基础知识，并将有助于了解分子 受体相关酪氨酸激酶的激活机制。Lyn 和IRIP可能是哮喘干预的良好治疗靶点 和其他过敏性疾病。

DESCRIPTION (provided by applicant): Asthma is characterized by eosinophilic inflammation of the airways. Interleukin-5 (IL-5) is one of the most important regulators of eosinophil differentiation and function. IL-5 activates eosinophils by stimulating a number of tyrosine kinases. However, which tyrosine kinase plays an essential role in transducing IL-5-specific signals is unknown. Based upon preliminary results we hypothesize that Lyn kinase plays an essential and non-redundant role in IL-5 -stimulated eosinophil differentiation and in the development of airway eosinophilic inflammation. In the first specific aim we will examine the physical association of Lyn with IL-5 receptor alpha subunit (IL-5Ra) and study its ability to phosphorylate the receptor. The biological relevance of Lyn for eosinophil differentiation from stem cells will be examined using Lyn knockout mice. Further, we will examine the effect of Lyn null mutation on the development of airway allergic inflammation using an ovalbumin-sensitized mouse model of asthma. The second specific aim is devoted to delineating the molecular mechanism of activation of Lyn kinase by IL-5Ra. In order to identify IL-5Ra-associated molecules that might activate Lyn kinase, we have performed yeast two-hybrid screening of a human cDNA library and cloned a novel SH2/SH3 ligand--IRIP (ImmunoReceptor-Interactive Protein). We hypothesize that IRIP activates Lyn kinase by interacting through its SH3 domain. To test the foregoing hypothesis we will map the binding sites of IRJP and Lyn kinase using recombinant Lyn domains and site directed mutagenesis. The importance of IRIP SH2/SH3 motifs for Lyn activation will be examined using motif peptides and site directed mutagenesis. Finally, the relevance of IRIP for eosinophil differentiation will be studied by overexpressing wild type and dominant negative mutants of IRIP in murine stem cells and HL-60 cells and by examining their differentiation into eosinophils. The proposed studies are designed to identify key IL-5-specific signaling molecules that are important for eosinophil differentiation and eosinophilic inflammation. The cloning and functional characterization of IRIP as a novel activator of Src-type tyrosine kinases will advance basic knowledge and will help understand the molecular mechanism of activation receptor-associated tyrosine kinases in general. Lyn and IRIP may represent excellent therapeutic targets for intervention in asthma and other allergic diseases.