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（GPCR）家族。通常，R-Spondins（RSPO）将LGR5与潜在的Wnt/ß-catenin信号结合。尽管存在经典的GPCR特征，例如配置干燥和NPXXY基序，但RSPO与LGR5结合并不诱导经典的GPCR行为，例如与G蛋白的耦合。有人提出，GPCR反应可能涉及替代配体或信号传导效应。实际上，精确的LGR5信号网络仍未解决。基于我们在GPCR信号传导方面的专家，该研究计划的长期目标是确定伪造LGR5信号网络的分子确定器。该研究计划的一般假设是肠道微生物群代谢物或产品是替代性LGR5配体或活性调节剂。该研究计划的目的是：1）阐明LGR5信号网络，以及2）确定潜在的微生物群衍生的LGR5活性调节剂。 AIM 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。在用RSPO定量LC-MS/MS刺激后，蛋白质组学分析将用于测量其他翻译后修饰的蛋白质表达和磷酸蛋白酶调节的变化。 LGR5 Intercontome将使用类似的蛋白质组学方法与生物体接近标记结合以检测活细胞中的蛋白质 - 蛋白质关联以及近端蛋白质。结果将通过已建立的细胞生物学方法（例如蛋白质印迹，免疫定位，免疫沉淀测定等）进行验证。 AIM 2：确定潜在的微生物源LGR5活性调节剂。 RSPO是该接收器的唯一配体令人怀疑。在AIM 2中，我们假设微生物产物将结合LGR5以诱导GPCR签名反应或调节ß-catenin响应。微生物群代谢组分析已导致鉴定活性代谢产物，例如短链脂肪酸（例如丁酸酯），有机酸，胆汁盐和多酚（例如类黄酮）。我们将测试这些代谢物是否是潜在的LGR5配体。 TCF/LEF荧光素酶测定和GPCR反应将监测β-catenin的反应，然后进行CA2+动员和肌醇磷酸盐产生（GQ反应）（GQ反应）和CAMP生产（GS，GI/O）。 G蛋白和β-arrestins招募到LGR5将通过BRET2测定法监测。根据我们的假设，将将已鉴定出的新配体的相互作用组和信号网络与响应RSPO识别的配体进行比较。影响。在这个原始研究计划中，我们正在解决这些基本问题。提出的方法和预期结果将不仅以公正的方式表征LGR5信号及其相互作用组，而且还将提供令人兴奋的新领域和研究途径，以充分了解该受体在干细胞生物学中的功能。