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

背景和目标。LGR 5属于富含亮氨酸的G蛋白偶联受体（GPCR）家族。通常，R-脊椎蛋白（RSPO）结合LGR 5以增强Wnt/β-连环蛋白信号传导。尽管存在经典的GPCR特征，例如保守的DRY和NPXXY基序，但RSPO与LGR 5的结合不会诱导经典的GPCR行为，例如与G蛋白的偶联。这表明GPCR反应可能涉及替代配体或信号效应物。事实上，确切的LGR 5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LGR 5信号传导网络的分子决定因素。这项研究计划的一般假设是，肠道微生物群代谢物或产物是替代LGR 5配体或活性调节剂。该研究计划的目的是：1）阐明LGR 5信号网络，2）识别潜在的微生物来源的LGR 5活性调节剂。目的1：阐明LGR 5信号网络。重组LGR 5将在HEK 293和肠上皮细胞系HCT 116中表达。用RSPO刺激后，将使用定量LC-MS/MS蛋白质组学分析来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。将使用与BioID邻近标记偶联的类似蛋白质组学方法分析LGR 5相互作用组，以检测活细胞中的蛋白质-蛋白质缔合以及邻近蛋白质。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、免疫沉淀试验等）对结果进行验证。目的2：鉴定潜在的微生物来源的LGR 5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LGR 5以诱导GPCR特征应答或调节β-连环蛋白应答。微生物群代谢组学分析已经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LGR 5配体。β-连环蛋白应答将通过TCF/LEF荧光素酶测定和GPCR应答，随后是Ca 2+动员和磷酸肌醇产生（Gq应答）和cAMP产生（Gs，Gi/o）来监测。将通过BRET 2测定监测G蛋白和β-抑制蛋白向LGR 5的募集。根据我们的假设，将鉴定的新配体的相互作用组和信号传导网络与响应RSPO鉴定的相互作用组和信号传导网络进行比较。 冲击在这个原始的研究计划中，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LGR 5信号传导及其相互作用组，而且还将提供令人兴奋的新领域和研究途径，以充分了解这种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.