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

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) 结合 LGR5 以增强 Wnt/ß-catenin 信号传导。尽管存在经典 GPCR 特征，例如保守的 DRY 和 NPXXY 基序，RSPO 与 LGR5 的结合不会诱导经典 GPCR 行为，例如与 G 蛋白的偶联。有人建议 GPCR 反应可能涉及替代配体或信号传导效应器。事实上，确切的LGR5信令网络仍然没有解决。基于我们在 GPCR 信号传导方面的专业知识，该研究计划的长期目标是确定形成 LGR5 信号传导网络的分子决定因素。该研究项目的总体假设是肠道微生物代谢物或产物是替代的 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，2) 识别潜在的微生物群衍生的 LGR5 活性调节剂。目标 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。用 RSPO 刺激后，定量 LC-MS/MS 蛋白质组分析将用于测量蛋白质表达的变化以及磷酸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-蛋白质关联以及邻近蛋白质。结果将使用已建立的细胞生物学方法（例如蛋白质印迹、免疫定位、免疫沉淀测定等）进行验证。目标 2：鉴定潜在的微生物群衍生的 LGR5 活性调节剂。 RSPO 是否是该受体的唯一配体值得怀疑。在目标 2 中，我们假设微生物产物将结合 LGR5 以诱导 GPCR 特征反应或调节 ß-连环蛋白反应。微生物群代谢组学分析已导致活性代谢物的鉴定，例如短链脂肪酸（例如丁酸盐）、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。将通过 TCF/LEF 荧光素酶测定和 GPCR 反应监测 ß-连环蛋白反应，然后监测 Ca2 动员和磷酸肌醇产生（Gq 反应）和 cAMP 产生（Gs、Gi/o）。 G 蛋白和 β-抑制蛋白向 LGR5 的募集将通过 BRET2 测定进行监测。根据我们的假设，所鉴定的新配体的相互作用组和信号网络将与响应 RSPO 所鉴定的相互作用组和信号网络进行比较。 影响。在这个原创研究计划中，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，而且还将提供令人兴奋的新研究领域和途径，以充分了解该受体在干细胞生物学中的功能。

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.