Proteasome Regulation of IL-5 Receptor Endocytosis

描述(申请人提供)：白介素5(IL-5)是一种造血细胞因子，在特应性和哮喘的嗜酸性炎症的病理生理学中起核心作用。了解抑制IL-5受体(IL-5R)诱导的炎症信号的机制对于开发新的途径来调节IL-5介导的炎症具有重要意义。本应用的目的是确定通过泛素和蛋白酶体降解途径控制IL-5R内化的分子机制。我们的总体假设是，IL-5诱导的IL-5R内化受两种不同的机制调节：泛素/蛋白酶体降解途径和网状蛋白介导的内吞作用。目的1.验证蛋白酶体通过首先降解(C)胞浆结构域来介导连接的IL-5R内化的假说。为了确定(C)胞浆结构域的一部分的降解是否是IL-5R内化的起始信号，将检测各种胞质截短的(C)突变体是否能够介导连接的IL-5R的蛋白酶体非依赖性内化。此外，还将研究参与细胞因子IL-3和GM-CSF的另一种细胞因子(C)对蛋白酶体活性的要求。目的2.验证一种假设，即IL-5R是通过网状蛋白介导的内吞作用内化的，并且受体的内化发生在(C)细胞质结构域的蛋白酶体降解之前。将使用两种不同的方法来抑制网状蛋白介导的内吞作用，以确定IL-5R是否被这一机制内化，以及在IL-5R内化后是否发生IL-5刺激的信号转导和蛋白酶体降解。目的3.探讨(C泛素化)在IL-5R内化过程中的功能作用和性质。温度敏感的TS20细胞系在泛素结合方面有温度敏感缺陷，a(c突变体在所有形式的泛素化方面都有缺陷，以及显性负泛素异构体，将被用来研究(c泛素化在IL-5R内吞作用中的作用和性质。

DESCRIPTION (provided by applicant): Interleukin-5 (IL-5) is a hematopoietic cytokine that is central to the pathophysiology of eosinophilic inflammation in atopy and asthma. Understanding mechanisms that extinguish IL-5 receptor (IL-5R)-induced inflammatory signals has importance for the development of novel approaches to modulate IL-5-mediated inflammation. The goal of this application is to define the molecular mechanisms controlling IL-5R internalization by the ubiquitin and proteasome degradation pathway. Our overall hypothesis is that IL-5-induced IL-5R internalization is regulated by two different mechanisms: the ubiquitin/proteasome degradation pathway and clathrin-mediated endocytosis. Aim 1. Test the hypothesis that proteasomes mediate internalization of the ligated IL-5R by first degrading the (c cytoplasmic domain. To determine if degradation of a portion of the (c cytoplasmic domain is the initiating signal for IL-5R internalization, various cytoplasmically truncated (c mutants will be examined for their ability to mediate proteasome-independent internalization of the ligated IL-5R. In addition, requirement for proteasome activity in (c internalization by the other (c engaging cytokines, IL-3 and GM-CSF, will be investigated. Aim 2. Test the hypothesis that the IL-5R is internalized by clathrin-mediated endocytosis and that internalization of the receptor occurs before proteasome degradation of the (c cytoplasmic domain. Two different approaches that inhibit clathrin-mediated endocytosis will be used to determine if the IL-5R is internalized by this mechanism and if IL-5-stimulated (c signaling and proteasome degradation occur after IL-5R internalization. Aim 3. Investigate the functional role and nature of (c ubiquitination in IL-5R internalization. The temperature sensitive ts20 cell line which has a temperature sensitive defect in ubiquitin conjugation, a (c mutant that is defective in all forms of ubiquitination, and dominant negative ubiquitin isoforms will be used to examine the role and nature of (c ubiquitination in IL-5R endocytosis.