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Project

The study of the possible interactions between human Mas-related G Protein-coupled receptors.

Mas-related G-Protein coupled receptors (Mrgpr's) have a putative role in sensory processes, including itch and pain. Previous studies on Mrgpr's in intestinal inflammatory conditions revealed marked expressional changes in them, specifically in sensory neurons of enteric nervous system (ENS). The fact that most of Mrgpr's are still classified as orphan, has hampered the study of their functions and regulatory mechanisms. This project develops novel approaches to explore the function of Mrgpr despite their current orphan status and explores protease-linked activation mechanisms of these Mrgprs. MrgprE and MrgprF are of special interest since these receptors are co-expressed in both enteric plexusses (myenteric and submucosal), and co-regulated in Schistosoma mansoni and TNBS-induced ileitis murine models. Mouse and human MrgprE and MrgprF are orthologous, and ongoing studies on gut biopsies in our laboratory confirm expression of MrgprE and MrgprF in the human gastrointestinal tract. This has prompted us to elucitade the interaction of human (h-) MrgprE and MrgprF. Therefore, we employed various biophysical and biochemical techniques including a novel luciferase complementation technique (NanoBiT), Bioluminescence resonance energy transfer (BRET), Fluorescence resonance energy transfer (FRET) and co-immunoprecipitation to validate our hypothesis. Results gave concrete evidence of h-MrgprE/h-MrgprF heteromerization, as well as h-MrgprE and h-MrgprF homomerization respectively.Over the last decade examples of GPCR's homo- and hetero-merizations are increasing at accelerating pace. Homo- and heteromeric states of GPCR's modulate the signal transductions capabilities and, therefore, regulate G-protein-dependent or -independent signalling associated with them. Nevertheless, biophysical tools used for detecting homo- and heteromerization of GPCR's account only for the oligomerization state but do not define their affinity and the competition among interacting partners for oligomerization. Therefore, there is need of novel protein-protein interaction (PPI) tools to ascertain affinity and competition among GPCR's. Starting from our obtained results, we are developing a quantitative NanoBiT assay, which will expand the current PPI tools and provide a better tool to study affinity, competition, biased agonism and transient protein interactions.
Date:15 Jul 2017  →  14 Jul 2018
Keywords:PROTEIN-PROTEIN INTERACTIONS, INTESTINAL INFLAMMATION, PROTEASES, RECEPTORS
Disciplines:Medicinal and biomolecular chemistry, Biochemistry and metabolism, Molecular and cell biology, Plant biology, Systems biology, Biophysics, Gastro-enterology and hepatology, Medical biochemistry and metabolism