The role of ubiquitin in GPCR signaling

泛素在 GPCR 信号传导中的作用

• 批准号: 10700805
• 负责人: Chandler J McElrath
• 金额: $ 4.77万
• 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/10700805
• 关键词: Adaptor Signaling Protein; Address; Agonist; Arginine; Automobile Driving; Biochemical; Biological Assay; Biology; Biophysics; C-terminal; CXC Chemokines; 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; Ligation; Link; Lysine; Lysosomes; Mass Spectrum Analysis; Mediating; Medicine; Mentorship; Methionine; Molecular; Multivesicular Body; Mutate; Myocardial Infarction; NF-kappa B; Pathway interactions; Peptides; Play; Polyubiquitin; Polyubiquitination; Post-Translational Protein Processing; Proteins; Recovery; Role; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Sorting; Stromal Cell-Derived Factor 1; System; Testing; Therapeutic; Translations; Ubiquitin; Ubiquitination; Wisconsin; amino group; angiogenesis; beta-arrestin; biophysical techniques; cardiac repair; cell motility; chemokine receptor; improved; insight; medical schools; neovascularization; novel; protein purification; 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.

摘要 G蛋白偶联受体（GPCR）C-X-C趋化因子受体4（CXCR 4）及其同源配体CXCL 12 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。 具体地说，CXCR 4的动脉表达促进心肌缺血后心脏的新血管形成。 梗死，减少损伤并导致恢复，但控制CXCR 4信号传导的机制仍然存在 不太了解。更好地了解CXCR 4信号传导可以帮助开发改善的治疗方法， 心血管疾病CXCR 4丰度和信号传导受翻译后修饰的调节 称为泛素化，但其机制仍然知之甚少。该项目的目标是更好地 了解泛素化如何控制CXCR 4信号传导和运输。用CXCL 12刺激诱导 CXCR 4上C-末端赖氨酸残基的泛素化，作为进入多囊泡的分选信号， 运输（MVB/ESCRT）途径所需的体/内体分选复合物，用于随后的降解 导致信号转导下调。此外，CXCR 4的激动剂激活促进了 STAM 1的泛素化，其是ESCRT-0复合物的组分。STAM 1的泛素化需要 内吞衔接蛋白b-抑制蛋白1，并参与调节CXCR 4运输和信号传导。尽管 关于STAM 1泛素化的这种关键作用知之甚少。初步数据表明，b-抑制蛋白1 通过E3泛素连接酶AIP 4增加STAM 1的泛素化，与b-抑制蛋白1作为一种蛋白酶一致。 STAM 1泛素化的衔接子。质谱分析显示，STAM 1被泛素修饰， 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， 泛素连接对应于线性或Met 1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连接酶复合物LUBAC形成。进一步的生化研究表明 b-抑制蛋白1是在STAM 1上的特定赖氨酸残基处形成M1连接的线性泛素链所必需的 AIP 4的基于初步数据，我们假设b-抑制蛋白1与AIP 4的赖氨酸选择相协调 介导STAM 1的线性泛素化。为了验证这一假设，我们将使用几种生物化学，分子， 和生物物理方法（目的1），以确定和表征位点特异性线性泛素化的STAM 1， b-抑制蛋白1通过AIP 4和（目的2），以确定线性多聚泛素链对GPCR运输的作用， 信号在项目结束时，我们将确定一个意想不到的作用M1连接的泛素 GPCR信号传导和运输链。该项目将在威斯康星州医学院进行 在Adriano Marchese博士的指导下，他是泛素对GPCR信号传导作用的专家， 贩卖人口赞助实验室和整个科学界在威斯康星州医学院提供 一个出色的环境，以成功地完成拟议的研究。