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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=743415
• 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属于富含亮氨酸的G蛋白偶联受体家族。通常，R-Spindins(RSPO)与LGR5结合以增强Wnt/？-catenin信号转导。尽管存在经典的GPCR特征，如保守的Dry和NPXXY基序，但RSPO与LGR5的结合不会诱导典型的GPCR行为，如与G蛋白的偶联。提示GPCR反应可能涉及替代配体或信号效应器。事实上，确切的LGR5信令网络仍然没有解决。基于我们在GPCR信号方面的专业知识，这项研究计划的长期目标是确定形成LGR5信号网络的分子决定因素。这项研究计划的一般假设是，肠道微生物区系代谢物或产物是替代的LGR5配体或活性调节剂。本研究的目的是：1)阐明LGR5信号网络；2)寻找潜在的微生物来源的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蛋白质组的调节以及其他翻译后修饰。将使用类似的蛋白质组学方法结合BioID邻近标记来分析Lgr5相互作用组，以检测活细胞中的蛋白质-蛋白质结合以及邻近蛋白质。结果将使用已建立的细胞生物学方法(例如，免疫印迹、免疫定位、免疫沉淀分析等)进行验证。目的2：确定潜在的微生物来源的LGR5活性调节剂。RSPO是否是该受体的唯一配基是值得怀疑的。在目标2中，我们假设微生物产物将与LGR5结合，以诱导GPCR签名反应或调节？连环蛋白反应。微生物组代谢图谱有助于鉴定活性代谢物，如短链脂肪酸(如丁酸盐)、有机酸、胆盐和多酚(如类黄酮类)。我们将测试这些代谢物是否为潜在的LGR5配体。通过TCF/Lef荧光素酶分析和GPCR反应，以及随后的钙动员和肌醇磷酸生产(GQ反应)和cAMP产生(Gs，Gi/o)，将监测？连环蛋白的反应。将通过BRET2分析监测G蛋白和ü-arrestins向LGR5的募集。根据我们的假设，识别的新配体(S)的相互作用组和信号网络将与响应RSPO的识别的配基进行比较。冲击力。在这个最初的研究计划中，我们正在解决这些基本问题。所提出的方法和预期的结果不仅将以公正的方式表征LGR5信号及其相互作用组，而且还将提供令人兴奋的新的研究领域和途径，以充分了解该受体在干细胞生物学中的功能。