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

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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.