The role of ubiquitin in GPCR signaling

泛素在 GPCR 信号传导中的作用

• 批准号: 10388600
• 负责人: Chandler J McElrath
• 金额: $ 4.68万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10388600
• 关键词: ARRB1 gene; Adaptor Signaling Protein; Address; Agonist; Arginine; Automobile Driving; Biochemical; Biological Assay; Biology; Biophysics; C-terminal; CXC Chemokines; CXCL12 gene; Cardiovascular Diseases; Cardiovascular Physiology; Cardiovascular system; Cells; Communities; Complex; Data; Degradation Pathway; Disease; Down-Regulation; Endosomes; Endothelial Cells; Environment; Functional disorder; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Genetic Transcription; Glycine; Health; Heart; In Vitro; Innate Immune Response; Ligands; Link; Lysine; Lysosomes; Mass Spectrum Analysis; Mediating; Medicine; Mentorship; Methionine; Molecular; Multivesicular Body; Mutate; Myocardial Infarction; Pathway interactions; Peptides; Play; Polyubiquitin; Polyubiquitination; Post-Translational Protein Processing; Proteins; Recovery; Role; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Sorting - Cell Movement; System; Testing; Therapeutic; Translations; Ubiquitin; Ubiquitination; Wisconsin; amino group; angiogenesis; base; biophysical techniques; cardiac repair; cell motility; chemokine receptor; improved; insight; medical schools; neovascularization; novel; protein B; reconstitution; trafficking; ubiquitin-protein ligase

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.

抽象的 G 蛋白偶联受体 (GPCR) C-X-C 趋化因子受体 4 (CXCR4) 及其同源配体 CXCL12 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部分。 具体而言，CXCR4 的动脉表达促进心肌梗死后心脏的新血管形成。 梗塞、减少损伤并导致恢复，但控制 CXCR4 信号传导的机制仍然存在 不太了解。更好地了解 CXCR4 信号传导可能有助于开发改进的治疗方法 心血管疾病。 CXCR4 丰度和信号传导受翻译后修饰调节 称为泛素化，但其机制仍知之甚少。该项目的目标是更好地 了解泛素化如何控制 CXCR4 信号传导和运输。 CXCL12 刺激可诱导 CXCR4 上 C 端赖氨酸残基的泛素化，作为多囊泡的分选信号 运输（MVB/ESCRT）途径所需的身体/内体分选复合物以进行后续降解 在溶酶体中导致信号传导下调。此外，CXCR4 的激动剂激活促进 STAM1（ESCRT-0 复合物的一个组成部分）的泛素化。 STAM1 的泛素化需要 内吞接头蛋白 b-arrestin1 参与调节 CXCR4 运输和信号传导。尽管 关于 STAM1 泛素化的这一关键作用，人们知之甚少。初步数据表明 b-arrestin1 增加 E3 泛素连接酶 AIP4 对 STAM1 的泛素化，与 b-arrestin1 充当 STAM1 泛素化的适配器。质谱分析表明STAM1在以下位置被泛素修饰 几个赖氨酸残基并具有潜在的多种类型的泛素连接。值得注意的是，其中之一 泛素连接对应于线性或 Met1 连接的泛素链，这是一种以前只知道的连接类型 由 E3 连接酶复合体 LUBAC 在 NFkB 信号传导背景下形成。进一步的生化研究表明 b-arrestin1 是在 STAM1 上特定赖氨酸残基处形成 M1 连接的线性泛素链所必需的 通过 AIP4。根据初步数据，我们假设 b-arrestin1 协调 AIP4 的赖氨酸选择 介导 STAM1 的线性泛素化。为了检验这个假设，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 的位点特异性线性泛素化： b-arrestin1 通过 AIP4 和（目标 2）确定线性多聚泛素链对 GPCR 运输的作用和 发信号。在项目结束时，我们将确定 M1 相关泛素的意想不到的作用 GPCR 信号传导和运输中的链。该项目将在威斯康星医学院进行 在泛素对 GPCR 信号传导和作用方面的专家 Adriano Marchese 博士的指导下 贩运。威斯康星医学院的申办实验室和整个科学界提供了 成功完成拟议研究的良好环境。