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; 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，这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后心脏的新血管形成 梗塞，减少损害并导致恢复，但控制CXCR4信号的机制仍然存在 理解不佳。更好地了解CXCR4信号可以有助于开发改进的治疗剂 心血管疾病。 CXCR4丰度和信号传导受翻译后修饰的调节 被称为泛素化，但这些机制仍然很少理解。该项目的目的是改善 了解泛素化如何控制CXCR4信号和贩运。用CXCL12刺激会诱导 CXCR4上C末端赖氨酸残基的泛素化，用作分类信号到多维 运输（MVB/ESCRT）途径需要降解所需的车身/内体分选复合物（MVB/ESCRT） 在导致信号传导下调的溶酶体中。此外，CXCR4的激动剂激活促进 ESCRT-0复合物的组成部分Stam1的泛素化。 Stam1的泛素化需要 内吞衔接蛋白B- arrestin1与调节CXCR4运输和信号传导有关。尽管 关于stam1泛素化，这种关键作用鲜为人知。初步数据表明B-arrestin1 E3泛素连接酶AIP4增加Stam1的泛素化，与B-arrestin1一致 STAM1泛素化的适配器。质谱分析表明，stam1是通过泛素修饰的 几种赖氨酸残基，具有多种类型的泛素链接。值得注意的是，之一 泛素链接对应于线性或Met1连接的泛素链，链接类型仅先前已知 在NFKB信号传导的背景下，由E3连接酶复合物lubac形成。进一步的生化研究表明 在stam1上的特定赖氨酸残基处，M1连接线性泛素链形成需要B- arrestin1 由AIP4。基于初步数据，我们假设B-arrestin1通过AIP4协调赖氨酸的选择 介导STAM1的线性泛素化。为了检验该假设，我们将使用几种生化的分子 和生物物理方法（目标1）以识别和表征STAM1对位点特异性线性泛素化的特异性线性泛素化。 B-arrestin1通过AIP4和（AIM 2）确定线性聚泛素链在GPCR运输和 信号。在项目结束时，我们将确定M1连接的泛素的意外作用 GPCR信号和贩运的链条。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 贩运。威斯康星州医学院的赞助商实验室和整个科学界提供 成功完成拟议研究的杰出环境。