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Activation of human Mas-related G Protein-coupled Receptor F (MRGPRF) by the cysteine protease Cathepsin S : implication in neuro-immune communication within the gut

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Abstract
G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane protein receptors. These receptors regulate a wide array of physiological processes in both healthy and diseased conditions via G protein-dependent and –independent signaling pathways. Remarkably, a subfamily of class A GPCRs, Mas-related G protein-coupled receptors (MRGPRs), is predominantly expressed in sensory neurons of the dorsal root and trigeminal ganglia, as well as on mast cells. Interestingly, we recently discovered that Mas-related G protein-coupled receptor F (MRGPRF), which is known to be expressed by brain, spinal cord and enteric neurons, is also expressed by enteroendocrine cells in the gastrointestinal tract. MRGPRF expression was predominantly found at basal and basolateral sites, indicating a novel role in endocrine regulation. However, due to the orphan nature of MRGPRF, little is known about its role in the pathophysiology of intestinal inflammation. Additionally, increasing evidence suggests a prominent role of peptides and proteases, predominantly secreted by macrophages, microglia and enteroendocrine cells, in inflammatory conditions of the gut, but the therapeutic perspectives are still restricted because the underlying molecular mechanisms are unknown. Here, we report for the first time on the activation of human MRGPRF by the cysteine protease Cathepsin S (Cat-S). To this end, we first performed in-silico analysis of the N-terminal sequence of MRGPRF, which revealed consensus motif sites of cleavage by Cat-S. Furthermore, the N-terminal peptide of MRGPRF was synthesized, incubated with Cat-S and analyzed for cleaved sites using mass spectroscopy. The results clearly showed cleavage sites and as such corroborated our in-silico analysis. Moreover, luciferase tagged at the N-terminus of MRGPRF was expressed in HeLa cells incubated with Cat-S and receptor N-terminal cleavage was assessed by measuring luminescence activity in the supernatant. The high luciferase activity in the supernatant compared to its control (no Cat-S treatment) indicated cleavage of the N-terminus of MRGPRF. We next sought to determine receptor activation upon Cat-S treatment using live cell calcium imaging with Fluo-4. HeLa cells transiently transfected with MRGPRF and incubated with Cat-S, gave rise to protease-based receptor activation, as evident by a fluorescence trace when compared to controls (non-transfected HeLa cells). These results pave the way for deorphanization of MRGPRF and call for further pathological investigations related to MRGPRF, in particular with regard to its involvement in neuro-immune communication within the gut.

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MLA
Arora, Rohit, et al. “Activation of Human Mas-Related G Protein-Coupled Receptor F (MRGPRF) by the Cysteine Protease Cathepsin S : Implication in Neuro-Immune Communication within the Gut.” Experimental Biology Meeting, Abstracts, vol. 33, no. Suppl. 1, 2019, doi:10.1096/fasebj.2019.33.1_supplement.585.1.
APA
Arora, R., Van Remoortel, S., Van Raemdonck, G., Baggerman, G., Buckinx, R., Snyders, D. J., … Timmermans, J.-P. (2019). Activation of human Mas-related G Protein-coupled Receptor F (MRGPRF) by the cysteine protease Cathepsin S : implication in neuro-immune communication within the gut. In Experimental Biology Meeting, Abstracts (Vol. 33). Orlando, FL. https://doi.org/10.1096/fasebj.2019.33.1_supplement.585.1
Chicago author-date
Arora, Rohit, Samuel Van Remoortel, Geert Van Raemdonck, Geert Baggerman, Roeland Buckinx, Dirk J. Snyders, Alain J Labro, and Jean-Pierre Timmermans. 2019. “Activation of Human Mas-Related G Protein-Coupled Receptor F (MRGPRF) by the Cysteine Protease Cathepsin S : Implication in Neuro-Immune Communication within the Gut.” In Experimental Biology Meeting, Abstracts. Vol. 33. https://doi.org/10.1096/fasebj.2019.33.1_supplement.585.1.
Chicago author-date (all authors)
Arora, Rohit, Samuel Van Remoortel, Geert Van Raemdonck, Geert Baggerman, Roeland Buckinx, Dirk J. Snyders, Alain J Labro, and Jean-Pierre Timmermans. 2019. “Activation of Human Mas-Related G Protein-Coupled Receptor F (MRGPRF) by the Cysteine Protease Cathepsin S : Implication in Neuro-Immune Communication within the Gut.” In Experimental Biology Meeting, Abstracts. Vol. 33. doi:10.1096/fasebj.2019.33.1_supplement.585.1.
Vancouver
1.
Arora R, Van Remoortel S, Van Raemdonck G, Baggerman G, Buckinx R, Snyders DJ, et al. Activation of human Mas-related G Protein-coupled Receptor F (MRGPRF) by the cysteine protease Cathepsin S : implication in neuro-immune communication within the gut. In: Experimental Biology Meeting, Abstracts. 2019.
IEEE
[1]
R. Arora et al., “Activation of human Mas-related G Protein-coupled Receptor F (MRGPRF) by the cysteine protease Cathepsin S : implication in neuro-immune communication within the gut,” in Experimental Biology Meeting, Abstracts, Orlando, FL, 2019, vol. 33, no. Suppl. 1.
@inproceedings{8705541,
  abstract     = {{G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane protein receptors. These receptors regulate a wide array of physiological processes in both healthy and diseased conditions via G protein-dependent and –independent signaling pathways. Remarkably, a subfamily of class A GPCRs, Mas-related G protein-coupled receptors (MRGPRs), is predominantly expressed in sensory neurons of the dorsal root and trigeminal ganglia, as well as on mast cells. Interestingly, we recently discovered that Mas-related G protein-coupled receptor F (MRGPRF), which is known to be expressed by brain, spinal cord and enteric neurons, is also expressed by enteroendocrine cells in the gastrointestinal tract. MRGPRF expression was predominantly found at basal and basolateral sites, indicating a novel role in endocrine regulation. However, due to the orphan nature of MRGPRF, little is known about its role in the pathophysiology of intestinal inflammation. Additionally, increasing evidence suggests a prominent role of peptides and proteases, predominantly secreted by macrophages, microglia and enteroendocrine cells, in inflammatory conditions of the gut, but the therapeutic perspectives are still restricted because the underlying molecular mechanisms are unknown. Here, we report for the first time on the activation of human MRGPRF by the cysteine protease Cathepsin S (Cat-S). To this end, we first performed in-silico analysis of the N-terminal sequence of MRGPRF, which revealed consensus motif sites of cleavage by Cat-S. Furthermore, the N-terminal peptide of MRGPRF was synthesized, incubated with Cat-S and analyzed for cleaved sites using mass spectroscopy. The results clearly showed cleavage sites and as such corroborated our in-silico analysis. Moreover, luciferase tagged at the N-terminus of MRGPRF was expressed in HeLa cells incubated with Cat-S and receptor N-terminal cleavage was assessed by measuring luminescence activity in the supernatant. The high luciferase activity in the supernatant compared to its control (no Cat-S treatment) indicated cleavage of the N-terminus of MRGPRF. We next sought to determine receptor activation upon Cat-S treatment using live cell calcium imaging with Fluo-4. HeLa cells transiently transfected with MRGPRF and incubated with Cat-S, gave rise to protease-based receptor activation, as evident by a fluorescence trace when compared to controls (non-transfected HeLa cells). These results pave the way for deorphanization of MRGPRF and call for further pathological investigations related to MRGPRF, in particular with regard to its involvement in neuro-immune communication within the gut.}},
  articleno    = {{585.1}},
  author       = {{Arora, Rohit and Van Remoortel, Samuel and Van Raemdonck, Geert and Baggerman, Geert and Buckinx, Roeland and Snyders, Dirk J. and Labro, Alain J and Timmermans, Jean-Pierre}},
  booktitle    = {{Experimental Biology Meeting, Abstracts}},
  issn         = {{0892-6638}},
  language     = {{eng}},
  location     = {{Orlando, FL}},
  number       = {{Suppl. 1}},
  pages        = {{2}},
  title        = {{Activation of human Mas-related G Protein-coupled Receptor F (MRGPRF) by the cysteine protease Cathepsin S : implication in neuro-immune communication within the gut}},
  url          = {{http://dx.doi.org/10.1096/fasebj.2019.33.1_supplement.585.1}},
  volume       = {{33}},
  year         = {{2019}},
}

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