A75 ON THE TRAIL OF ALTERNATIVE LIGANDS AND SIGNALING PATHWAYS FOR LGR5
Bibliographic record
Abstract
Abstract Background Leucine-rich G protein coupled receptor-5 (LGR5) is a GPCR originally identified as a marker for intestinal stem cells but is now associated to stemness in numerous other tissues. R-spondins (RSPO) are the only reported ligands of LGR5, which upon receptor binding, potentiates the canonical Wnt/b-catenin pathway. Surprisingly, despite the presence of classical GPCR features such as conserved DRY and NPXXY motifs RSPO binding to LGR5 does not induce classical GPCR behaviors such as coupling to heterotrimeric G-proteins or engagement of b-arrestins. For this reason, LGR5 is still considered to be an orphan receptor. Aims Aberrant expression of LGR5 is a common feature of many cancers, including gastrointestinal cancers, but it is still unclear why LGR5 is pro-tumorigenic in some cancers, while anti-tumorigenic in others. One potential explanation is that LGR5 does not only signals through the Wnt/b-catenin pathway, but also to alternative pathways. Thus, the goals of this study were 1), to elucidate LGR5 signaling networks and 2), to identify new LGR5 ligands and activity modulators. We suggested that the intestinal lumen content could be a source of bioactive molecules that could act as alternative LGR5 ligands Methods All experiments were performed on HEK293 cells expressing recombinant LGR5 (HEK293/LGR5) and compared to control cells expressing empty vector. Characterization of LGR5 signaling network was initiated with LC MS/MS spectrometric analyses using cells stimulated with Wnt3a and RSPO1. To identify new LGR5 modulators, commercially available bacteria-derived metabolites (BDM) were tested on HEK293/LGR5 cells stimulated with Wnt3a and RSPO1 and assessed using the TOP-Flash luciferase system. Finally, a mouse intestinal lumen extract (ILE) was investigated in whole-cell impedance assay to detect if it contained native BDM or potential host-derived LGR5 ligands. Results Mass spectrometric analyses revealed distinct protein expression and phosphorylation profiles in HEK293/LGR5 cells stimulated with Wnt3a and RSPO1. The TOP/Flash luciferase assays showed that the flavonoid derivative 3,4-dihydroxyphenylacetic acid (DOPAC) and lipopolysaccharide (LPS) acted as negative regulators of LGR5, while butyrate had no effect. Interestingly, stimulation of HEK293/LGR5 with ILE induced a Gαq-like response in the impedance assay, while control cells did not respond. Conclusions Stimulation of LGR5 with Wnt and RSPO triggered a complex signaling network. In agreement with our hypothesis, some BDM such as DOPAC and LPS were indeed activity modulators of LGR5. Moreover, a still unidentified compound present in the ILE triggered a selective GPCR-like response in HEK293/LGR5 cells. Future works aim at identifying this putative LGR5 ligand(s) and establish the LGR5 interactome using proximity labeling and mass spectrometry. Funding Agencies NSERC, Université de Sherbrooke, FRQS
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.007 | 0.002 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".