Elucidating alternative leucine-rich G protein coupled receptor-5 (Lgr5) signaling

阐明替代的富含亮氨酸的 G 蛋白偶联受体 5 (Lgr5) 信号传导

• 批准号: RGPIN-2019-05294
• 负责人: Gendron, FernandPierre
• 金额: $ 2.33万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=738176
• 关键词: Elucidating; alternative; leucine; rich; protein

Background and objectives. LGR5 belongs to the family of leucine-rich G protein-coupled receptors (GPCR). Typically, R-spondins (RSPO) bind LGR5 to potentiate Wnt/ß-catenin signaling. Despite the presence of classical GPCR features such as conserved DRY and NPXXY motifs, RSPO binding to LGR5 do not induce classical GPCR behaviors such a coupling to G-proteins. It was suggested that GPCR responses might involve alternative ligand or signaling effectors. In fact, the exact LGR5 signaling networks is still unsolved. Based on our expertise in GPCR signaling, the long-term objective of this research program is to identify the molecular determinants that forge LGR5 signaling networks. The general hypothesis of this research program is that intestinal microbiota metabolites or products are alternative LGR5 ligands or activity modulators. The aims of this research program are: 1) Elucidate LGR5 signaling networks, and 2) Identify potential microbiota-derived LGR5 activity modulators. AIM 1: Elucidate LGR5 signaling networks. Recombinant LGR5 will be express in HEK293 and the intestinal epithelial cell line HCT116. Following stimulation with RSPO quantitative LC-MS/MS proteomic analysis will be used to measure variation in protein expression and modulations of the phosphoproteome amongst other posttranslational modifications. LGR5 interactome will be analyzed using a similar proteomic approach coupled to BioID proximity labeling to detect protein-protein associations as well as proximate proteins in live cells. Results will be validated using established cell biology approaches (e.g. Western blots, immunolocalisation, immunoprecipitation assays, etc.). AIM 2: Identify potential microbiota-derived LGR5 activity modulators. It is doubtful that RSPO is the only ligand for this receptor. In aim 2, we hypothesize that microbial products will bind LGR5 to induce GPCR signature responses or modulate ß-catenin responses. Microbiota metabolomic profiling has led to the identification of active metabolites such as short-chain fatty acids (e.g. butyrate), organic acids, bile salts and polyphenol (e.g. flavonoids). We will test if these metabolites are potential LGR5 ligands. ß-catenin responses will be monitored by TCF/LEF luciferase assays and GPCR responses followed by Ca2+ mobilization and inositol phosphate production (Gq responses) and cAMP production (Gs, Gi/o). G-proteins and ß-arrestins recruitment to LGR5 will be monitored by BRET2 assays. In accordance with our hypothesis, the interactome and signaling networks of the identified new ligand(s) will be compared to the one identified in response to RSPO. IMPACT. In this original research program, we are addressing these fundamental questions. The proposed methodology and expected results will not only characterize in an unbiased manner LGR5 signaling and its interactome, but will also provide exciting new areas and avenues of research to fully understand the function of this receptor in stem cell biology.

背景和目标。LGR5属于富亮氨酸G蛋白偶联受体（GPCR）家族。通常，R-spondins （RSPO）结合LGR5增强Wnt/ß-catenin信号传导。尽管存在经典的GPCR特征，如保守的DRY和NPXXY基序，但RSPO结合LGR5不会诱导经典的GPCR行为，如与g蛋白的偶联。这表明GPCR反应可能涉及其他配体或信号效应体。事实上，确切的LGR5信号网络仍未得到解决。基于我们在GPCR信号方面的专业知识，本研究项目的长期目标是确定形成LGR5信号网络的分子决定因素。本研究项目的一般假设是肠道菌群代谢物或产物是替代LGR5配体或活性调节剂。本研究计划的目的是：1)阐明LGR5信号网络；2)鉴定潜在的微生物衍生的LGR5活性调节剂。目的1：阐明LGR5信号网络。重组LGR5将在HEK293和肠上皮细胞系HCT116中表达。在RSPO刺激后，定量LC-MS/MS蛋白质组学分析将用于测量蛋白质表达的变化和磷酸化蛋白质组的调节以及其他翻译后修饰。LGR5相互作用组将使用类似的蛋白质组学方法进行分析，结合BioID接近标记来检测活细胞中的蛋白质蛋白质关联以及近似蛋白质。结果将使用已建立的细胞生物学方法（如Western blots、免疫定位、免疫沉淀测定等）进行验证。目标2：鉴定潜在的微生物衍生的LGR5活性调节剂。值得怀疑的是，RSPO是否是该受体的唯一配体。在目标2中，我们假设微生物产物将结合LGR5诱导GPCR特征反应或调节ß-catenin反应。微生物群代谢组学分析已经鉴定出活性代谢物，如短链脂肪酸（如丁酸盐）、有机酸、胆盐和多酚（如类黄酮）。我们将测试这些代谢物是否是潜在的LGR5配体。ß-catenin反应将通过TCF/LEF荧光素酶测定和GPCR反应监测，随后是Ca2+动员和肌醇磷酸产生（Gq反应）和cAMP产生（Gs, Gi/o）。g蛋白和ß-阻滞蛋白向LGR5的募集将通过BRET2检测进行监测。根据我们的假设，鉴定的新配体的相互作用组和信号网络将与响应RSPO鉴定的配体进行比较。的影响。在这个原创的研究项目中，我们正在解决这些基本问题。所提出的方法和预期结果不仅将以公正的方式表征LGR5信号传导及其相互作用组，而且将为充分了解该受体在干细胞生物学中的功能提供令人兴奋的新领域和研究途径。