The signaling signature of the neurotensin type 1 receptor with endogenous ligands

&NA; 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 &bgr;‐arrestins. Using BRET‐based biosensors, we found that neurotensin (NT), NT(8‐13) and neuromedin N (NN) activated the G&agr;q‐, G&agr;i1‐, G&agr;oA‐, and G&agr;13‐protein signaling pathways as well as the recruitment of &bgr;‐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 G&agr;i1 and G&agr;oA, 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 G&agr;s‐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 PLC&bgr;‐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.