Deciphering the unique dynamic activation pathway in a G protein-coupled receptor enables unveiling biased signaling and identifying cryptic allosteric sites in conformational intermediates

N eurotensin receptor 1 (NTSR1), a member of the C lass A G protein - coupled receptor superfamily, plays a n important role in modulating dopamine rgic neuronal activity and eliciting opioid - independent analgesia. Recent studies suggest that promoting β - arrestin - bias ed signaling in NTSR1 may diminish drugs of abuse, such as psychostimulants, thereby offering a potential avenue for treating human addiction - related disorders . In this study, we utiliz e d a novel computational and experimental approach that combined nudged elastic band - based molecular dynamics simulations, Markov state models, temporal communication network analysis, site - directed mutagenesis, and conformational biosensors, to explore the intricate mechanisms underlying NTSR1 activation and bias ed signal ing . Our study reveal s a dynamic stepwise transition mechanism and activat ed transmission network associated with NTSR1 activation. It also yield s valuable insights into the complex interplay between the unique polar network, non - conserved ion locks, and aromatic clusters in NTSR1 signaling. Moreover, we identif ied a cryptic allosteric site located in the intracellular r egion of the receptor that exists in an intermediate state within the activation pathway. Collectively, these findings contribute to a more profound understanding of NTSR1 activation and biased signal ing at the atomic level, thereby providing a potential strateg y for the development of NTSR1 allosteric modulators in the realm of G protein - coupled receptor biology, biophysics, and medicine.