The signaling signature of the neurotensin type 1 receptor with endogenous ligands.
The human neurotensin 1 receptor (hNTS1) is a G protein-coupled receptor involved in many physiological functions, including analgesia, hypothermia, and hypotension. To gain a better understanding of which signaling pathways or combination of pathways are linked to NTS1 activation and function, we investigated the ability of activated hNTS1, which was stably expressed by CHO-K1 cells, to directly engage G proteins, activate second messenger cascades and recruit β-arrestins. Using BRET-based biosensors, we found that neurotensin (NT), NT(8-13) and neuromedin N (NN) activated the Gαq-, Gαi1-, GαoA-, and Gα13-protein signaling pathways as well as the recruitment of β-arrestins 1 and 2. Using pharmacological inhibitors, we further demonstrated that all three ligands stimulated the production of inositol phosphate and modulation of cAMP accumulation along with ERK1/2 activation. Interestingly, despite the functional coupling to Gαi1and GαoA, NT was found to produce higher levels of cAMP in the presence of pertussis toxin, supporting that hNTS1 activation leads to cAMP accumulation in a Gαs-dependent manner. Additionally, we demonstrated that the full activation of ERK1/2 required signaling through both a PTX-sensitive Gi/o-c-Src signaling pathway and PLCβ-DAG-PKC-Raf-1-dependent pathway downstream of Gq. Finally, the whole-cell integrated signatures monitored by the cell-based surface plasmon resonance and changes in the electrical impedance of a confluent cell monolayer led to identical phenotypic responses between the three ligands. The characterization of the hNTS1-mediated cellular signaling network will be helpful to accelerate the validation of potential NTS1 biased ligands with an improved therapeutic/adverse effect profile.
Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. Electronic address: Elie.Besserer-Offroy@USherbrooke.ca.
Élie
É
Besserer-Offroy
Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. Electronic address: Rebecca.Brouillette@USherbrooke.ca.
Rebecca L
RL
Brouillette
Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. Electronic address: Sandrine.Lavenus@USherbrooke.ca.
Sandrine
S
Lavenus
Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. Electronic address: Ulrike.Froehlich@USherbrooke.ca.
Ulrike
U
Froehlich
Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. Electronic address: Andrea.Brumwell@USherbrooke.ca.
Andrea
A
Brumwell
Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. Electronic address: Alexandre.Murza@USherbrooke.ca.
Alexandre
A
Murza
Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. Electronic address: Jean-Michel.Longpre@USherbrooke.ca.
Jean-Michel
JM
Longpré
Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. Electronic address: Eric.Marsault@USherbrooke.ca.
Éric
É
Marsault
Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. Electronic address: Michel.Grandbois@USherbrooke.ca.
Michel
M
Grandbois
Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. Electronic address: Philippe.Sarret@USherbrooke.ca.
Philippe
P
Sarret
Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. Electronic address: Richard.Leduc@USherbrooke.ca.
Richard
R
Leduc
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009496:Neurotensin / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D018028:Receptors, Neurotensin / Q000378:metabolism*; D015398:Signal Transduction; D000071559:beta-Arrestin 1 / Q000378:metabolism; D000071560:beta-Arrestin 2 / Q000378:metabolism
D008024:Ligands; D010446:Peptide Fragments; D018028:Receptors, Neurotensin; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; C114036:neurotensin type 1 receptor; C042090:neuromedin N; D009496:Neurotensin; C054381:neurotensin (8-13); D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019204:GTP-Binding Proteins
G protein; G protein-coupled receptor (GPCR); Neuromedin N; Neuromedin N (PubChem CID: 9940301); Neurotensin; Neurotensin (1-13) (PubChem CID: 25077406); Neurotensin (8-13) (PubChem CID: 5311318); Neurotensin receptor 1; SR48692 (PubChem CID: 119192); β-arrestin