Quality control of the primary cilium proteome

项目概要 初级纤毛组织信号传导途径，例如视觉、嗅觉和刺猬信号传导。的 信号受体进出纤毛的运动对于正确调节这些信号至关重要 途径，但我们对通过纤毛控制信号受体运输的基本机制的理解 仍然是碎片化的。该实验室过去的工作鉴定并表征了 BBSome，这是一种蛋白质复合物， 将信号受体运送出纤毛。 BBSome 与人类健康和疾病的相关性是证据 事实上，BBSome 功能障碍会导致 Bardet-Biedl 综合征 (BBS)，这是一种遗传性疾病，其特征是 肥胖、视网膜变性、多指畸形和肾脏畸形。 该提案的主要目标是确定 BBSome 如何选择信号受体进行去除 来自纤毛。本资助期的重点将是研究泛素在标记膜中的作用 用于从纤毛中去除的蛋白质。初步数据表明存在一种能够识别 激活 GPCR，对其进行泛素化，并将泛素化的 GPCR 从纤毛中分选出来。我们将表征 使用信号依赖性 GPCR 退出的定量分析来作用于每个步骤的分子。的 将泛素固定并读取需要退出的蛋白质的分子齿轮和杠杆的分层排序 纤毛承诺揭示一个多步骤途径，让人想起负责 降级分选。最后，退出纤毛的 GPCR 的命运将通过单分子成像进行追踪 确定内吞作用是否与纤毛出口相关，或者 GPCR 是否扩散到血浆中 离开纤毛后形成膜。 拟议的研究将为如何调节纤毛丰度提供新的线索，并打开 纤毛质量控制的机械研究之门。

PROJECT SUMMARY Primary cilia organize signaling pathways such as vision, olfaction and Hedgehog signaling. The movements of signaling receptors into, inside and out of cilium are critical for the correct regulation of these pathways, yet our understanding of the basic mechanisms governing signaling receptor trafficking through cilia remains fragmentary. Past work from the lab identified and characterized the BBSome, a protein complex that ferries signaling receptors out of cilia. The relevance of the BBSome to human health and disease is evidence by the fact that BBSome dysfunction causes Bardet-Biedl Syndrome (BBS), a hereditary disease characterized by obesity, retinal degeneration, polydactyly and kidney malformations. The major goal of this proposal is to determine how the BBSome selects signaling receptors for removal from cilia. The emphasis in this funding period will be on investigating the role of ubiquitin in tagging membrane proteins for removal from cilia. Preliminary data indicate the existence of a ciliary machinery that recognizes activated GPCRs, ubiquitinates them and sorts ubiquitinated GPCRs out of cilia. We will characterize the molecules acting at each of these steps using quantitative assays for signal-dependent GPCR exit. The hierarchical ordering of the molecular cogs and levers that affix and read ubiquitin on proteins that need to exit cilia promises to uncover a multi-step pathway reminiscent of the ESCRT machinery responsible for degradative sorting. Finally, the fate of GPCRs that exit cilia will be tracked by single-molecule imaging to determine whether endocytosis is coupled to ciliary exit or whether GPCRs instead diffuse into the plasma membrane after exiting cilia. The proposed studies will cast new light on how the ciliary abundance of proteins is regulated and open the door to a mechanistic investigation of ciliary quality control.