Role of the Go/i signaling network in the regulation of neurite outgrowthThis paper is one of a selection of papers published in this Special issue, entitled Second Messengers and Phosphoproteins—12th International Conference.

Neurite outgrowth is a complex differentiation process stimulated by many neuronal growth factors and transmitters and by electrical activity. Among these stimuli are ligands for G-protein-coupled receptors (GPCR) that function as neurotransmitters. The pathways involved in GPCR-triggered neurite outgrowth are not fully understood. Many of these receptors couple to Gαo, one of the most abundant proteins in the neuronal growth cones. We have studied the Go signaling network involved in neurite outgrowth in Neuro2A cells. Gαo can induce neurite outgrowth. The CB1 cannabinoid receptor, a Go/i-coupled receptor expressed endogenously in Neuro2A cells, triggers neurite outgrowth by activating Rap1, which promotes the Gαo-stimulated proteasomal degradation of Rap1GAPII. CB1-receptor-mediated Rap1 activation leads to the activation of a signaling network that includes the small guanosine triphosphate (GTP)ases Ral and Rac, the protein kinases Src, and c-Jun N-terminal kinase (JNK), which converge onto the activation of signal transducer and activator of transcription 3 (Stat3), a key transcription factor that mediates the gene expression process of neurite outgrowth in Neuro2A cells. This review describes current findings from our laboratory and also discusses alternative pathways that Go/i might mediate to trigger neurite outgrowth. We also analyze the role neurotransmitters, which stimulate Go/i to activate a complex signaling network controlling neurite outgrowth, play in regeneration after neuronal injury.

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A75 ON THE TRAIL OF ALTERNATIVE LIGANDS AND SIGNALING PATHWAYS FOR LGR5

Journal of the Canadian Association of Gastroenterology ◽

10.1093/jcag/gwz047.074 ◽

2020 ◽

Vol 3 (Supplement_1) ◽

pp. 89-90

Author(s):

S Dagenais Bellefeuille ◽

G Arguin ◽

F Gendron

Keyword(s):

Signaling Network ◽

Bioactive Molecules ◽

Orphan Receptor ◽

Hek293 Cells ◽

Intestinal Lumen ◽

Aberrant Expression ◽

Alternative Pathways ◽

Canonical Wnt ◽

Complex Signaling ◽

Stimulation Of

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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