The role of ubiquitin in GPCR signaling

摘要 G蛋白偶联受体、C-X-C趋化因子受体4及其同源配体CXCL12 已经与内皮细胞迁移有关，内皮细胞迁移是健康和疾病中血管生成的必要组成部分。 具体地说，动脉表达CXCR4促进了心肌梗死后心脏新生血管的形成。 但控制CXCR4信号转导的机制仍然存在 人们对此知之甚少。更好地了解CXCR4信号有助于开发改进的治疗方法 心血管疾病。CXCR4的丰度和信号受翻译后修饰的调节 被称为泛素化，但其机制仍鲜为人知。这个项目的目标是更好地 了解泛素化如何管理CXCR4信号和贩运。CXCL12刺激诱导 CXCR4上C端赖氨酸残基的泛素化，作为进入多囊泡的分选信号 转运所需的体内/内体分选复合体(MVB/ESCRT)途径用于后续降解 在溶酶体中导致信号下调。此外，CXCR4激动剂的激活促进 泛素化的STAM1，ESCRT-0复合体的一个组成部分。STAM1的泛素化需要 内吞接头蛋白b-arrestin1参与调控CXCR4的运输和信号转导。尽管 这一关键作用对STAM1泛素化知之甚少。初步数据显示，b-arrestin1 通过E3泛素连接酶AIP4增加STAM1的泛素化，与b-arrestin1作为一种 用于STAM1泛素化的适配器。质谱分析表明，STAM1被泛素修饰在 几个赖氨酸残基和潜在的多种类型的泛素连接。值得注意的是，其中一个 泛素连接对应于线状或MET1连接的泛素链，这是一种以前只知道的连接类型 在NFkB信号传导的背景下，由E3连接酶复合体LUBAC形成。进一步的生化研究表明 在STAM1上特定的赖氨酸残基上形成M1连接的线性泛素链需要b-arrestin1 由AIP4。根据初步数据，我们假设b-arrestin1参与了AIP4对赖氨酸的选择。 介导STAM1的线性泛素化。为了验证这一假设，我们将使用几种生化、分子 和生物物理方法(目标1)通过以下方式识别和表征STAM1的位点特定的线性泛素化 通过AIP4和(Aim 2)确定线性多泛素链在GPCR运输中的作用和 发信号。在项目结束时，我们将确定M1连接泛素的一个意想不到的角色 GPCR信号和贩运中的链条。该项目将在威斯康星州医学院进行 在Adriano Marchese博士的指导下，泛素在GPCR信号和转录因子中的作用专家 贩卖人口。赞助实验室和威斯康星医学院的整个科学界提供 成功完成拟议研究的出色环境。

ABSTRACT The G protein-coupled receptor (GPCR) C-X-C chemokine receptor 4 (CXCR4) and its cognate ligand CXCL12 have been linked to endothelial cell migration, a necessary part of angiogenesis in health and disease. Specifically, arterial expression of CXCR4 promotes neovascularization of the heart following myocardial infarction, reducing damage and leading to recovery, yet the mechanisms that control CXCR4 signaling remain poorly understood. Better understanding of CXCR4 signaling could aid in developing improved therapeutics for cardiovascular disease. CXCR4 abundance and signaling is regulated by the post-translational modification termed ubiquitination, but the mechanisms remain poorly understood. The objective of this project is to better understand how ubiquitination governs CXCR4 signaling and trafficking. Stimulation with CXCL12 induces ubiquitination of C-terminal lysine residues on CXCR4, serving as a sorting signal into the multi-vesicular body/endosomal sorting complexes required for transport (MVB/ESCRT) pathway for subsequent degradation in lysosomes leading to downregulation of signaling. In addition, agonist activation of CXCR4 promotes ubiquitination of STAM1, a component of the ESCRT-0 complex. Ubiquitination of STAM1 requires the endocytic adaptor proteins b-arrestin1 and is implicated in regulating CXCR4 trafficking and signaling. Despite this pivotal role very little is known concerning STAM1 ubiquitination. Preliminary data suggest that b-arrestin1 increases ubiquitination of STAM1 by the E3 ubiquitin ligase AIP4, consistent with b-arrestin1 serving as an adaptor for STAM1 ubiquitination. Mass spectroscopy analysis revealed that STAM1 is modified by ubiquitin at several lysine residues and with potentially multiple types of ubiquitin linkages. Remarkably, one of the ubiquitin linkages corresponded to linear or Met1-linked ubiquitin chains, a linkage type only previously known to form by the E3 ligase complex LUBAC in the context of NFkB signaling. Further biochemical studies suggest that b-arrestin1 is required for M1-linked linear ubiquitin chain formation at a specific lysine residue on STAM1 by AIP4. Based on the preliminary data, we hypothesize that b-arrestin1 coordinates lysine selection by AIP4 to mediate linear ubiquitination of STAM1. To test this hypothesis, we will use several biochemical, molecular and biophysical approaches (Aim 1) to identify and characterize site-specific linear ubiquitination of STAM1 by b-arrestin1 via AIP4 and (Aim 2) to determine the role of linear poly-ubiquitin chains on GPCR trafficking and signaling. At the conclusion of the project, we will have identified an unexpected role for M1-linked ubiquitin chains in GPCR signaling and trafficking. This project will be carried out at the Medical College of Wisconsin under the mentorship of Dr. Adriano Marchese, an expert on the role of ubiquitin on GPCR signaling and trafficking. The sponsor lab and the overall scientific community at the Medical College of Wisconsin provides an outstanding environment to successfully complete the proposed studies.