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 (beta-arrestins. Using BRET-based biosensors, we found that neurotensin (NT), NT(8-13) and neuromedin N (NN) activated the G alpha(q)-, G alpha(OA)-, and G alpha(13)-protein signaling pathways as well as the recruitment of beta-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 alpha(i1) and Ga-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 Ga-s-dependent manner. Additionally, we demonstrated that the full activation of ERK1/2 required signaling through both a PTX-sensitive G(i/o)-c-Src signaling pathway and PLC beta-DAG-PKC-Raf-1-dependent pathway downstream of G(q). 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.