Proteasome Regulation of Interleukin-5 Receptor Endocytosis

描述（由申请人提供）：白细胞介素-5（IL-5）是一种造血细胞因子，在特应性和哮喘中嗜酸性粒细胞炎症的病理生理学中起着重要作用。了解消除IL-5受体（IL-5 R）诱导的炎症信号的机制对于开发调节IL-5介导的炎症的新方法具有重要意义。本申请的目的是确定通过泛素和蛋白酶体降解途径控制IL-5 R内化的分子机制。我们的总体假设是，IL-5诱导的IL-5 R内化是由两种不同的机制：泛素/蛋白酶体降解途径和网格蛋白介导的内吞作用。目标1.检验蛋白酶体通过首先降解胞质结构域介导连接的IL-5 R内化的假设。为了确定β胞质结构域的一部分的降解是否是IL-5 R内化的起始信号，将检查各种胞质截短的β突变体介导连接的IL-5 R的蛋白酶体非依赖性内化的能力。此外，还将研究其他参与细胞因子IL-3和GM-CSF内化对蛋白酶体活性的要求。目标2.检验以下假设：IL-5 R通过网格蛋白介导的内吞作用内化，并且受体的内化发生在β胞质结构域的蛋白酶体降解之前。将使用抑制网格蛋白介导的内吞作用的两种不同方法来确定IL-5 R是否通过该机制内化，以及IL-5刺激的β信号传导和蛋白酶体降解是否在IL-5 R内化后发生。目标3：研究β-泛素化在IL-5 R内化中的功能作用和性质。温度敏感性ts 20细胞系在泛素缀合中具有温度敏感性缺陷，是在所有形式的泛素化中都有缺陷的β突变体，并且显性负性泛素同种型将用于检查β泛素化在IL-5 R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.