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Monitoring Signalling and Trafficking of Neurotensin Type 1 Receptor in Animal Model using Fluorescent‐based Methods
Author(s) -
Couvineau Pierre,
Ehrlich Aliza,
Semache Meriem,
Kieffer Brigitte,
Bouvier Michel
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.502.4
Subject(s) - neurotensin receptor , g protein coupled receptor , receptor , neurotensin , signalling , agonist , signal transduction , chemistry , biology , microbiology and biotechnology , biochemistry , neuropeptide
Neurotensin Type 1 Receptor (NTSR1) is a G Protein‐Coupled Receptor (GPCR) with proposed roles in cancer, neuropsychiatric and neurodegenerative diseases, eating disorders, pain, as well as inflammation. It therefore represents a potential therapeutic target for the treatment of these diseases. However, relatively little is known about the repertoire of signalling effectors that can be engaged by NTSR1 and about the functional selectivity of drug candidates targeting this receptor in native tissue. In a first step toward generating an animal model allowing to monitor the activation and regulation of NTSR1 in native tissues, we developed a DNA construct expressing NTSR1 tagged to vYFP at its C‐terminus. We investigated the signalling responses of NTSR1‐vYFP promoted by eight agonists and two antagonists, and compared them to those obtained for the unmodified NTSR1. For this purpose, we used a suite of BRET‐based sensors expressed in HEK‐293 cells. We showed that NTSR1‐vYFP has the same signalling profile as the endogenous NTSR1 as it activates both β‐Arrestin 1 and 2 and all G proteins except G 12 and G i3 . Moreover, we confirmed that ML314 is a biased agonist toward β‐Arrestin pathways and we showed that PD149163 does not activate G s protein. Because the vYFP fusion does not seem to modify the signalling profile of the NTSR1, we have embarked on developing a knock‐in mouse model expressing NTSR1‐vYFP instead of the native receptor which will be a useful tool to study receptor signaling and trafficking under physiological conditions. Support or Funding Information The project is funded by the Consortium de recherche biopharmaceutique (CQDM). Pierre Couvineau is funded by the INSERM‐FRQS Postdoctoral Exchange Program This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .