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

2025-04-24

AI Summary

This study addresses the unresolved mechanistic questions surrounding the dynamic activation process and β-arrestin–biased signaling of neurotensin receptor 1 (NTSR1). Combining elastic network-based molecular dynamics simulations, Markov state modeling, time-dependent communication network analysis, and experimental validation via site-directed mutagenesis and conformational biosensors, we elucidate, for the first time, the stepwise activation pathway of NTSR1. We reveal a temporally coordinated regulatory mechanism involving a polar network, a non-canonical ionic lock, and an aromatic cluster. Critically, we identify a cryptic allosteric site in the intracellular region—transiently exposed only in functionally relevant intermediate states and invisible in static crystal structures. At atomic resolution, our work maps the signal transduction network underlying NTSR1 activation and identifies novel druggable allosteric sites. These findings provide essential structural insights and theoretical foundations for developing NTSR1-biased or allosteric modulators targeting addiction disorders.

Technology Category

• Cognitive Modeling & Cognitive Systems: Neural Spike Coding
• Machine Learning: Active Learning
• Planning, Routing, and Scheduling: Activity and Plan Recognition

Application Category

• Graph Algorithms and Modeling for the Web: Representation, reconstruction, and subgraph or motif discovery in Web-related graphs
• Social Networks and Social Media: Media and governance, opinion dynamics, filter bubbles, polarization
• Economics, Online Markets and Human Computation: Incentives in network design for Web infrastructures and ecosystems

Abstract

Neurotensin receptor 1 (NTSR1), a member of the Class A G protein-coupled receptor superfamily, plays an important role in modulating dopaminergic neuronal activity and eliciting opioid-independent analgesia. Recent studies suggest that promoting {beta}-arrestin-biased 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 utilized 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 biased signaling. Our study reveals a dynamic stepwise transition mechanism and activated transmission network associated with NTSR1 activation. It also yields valuable insights into the complex interplay between the unique polar network, non-conserved ion locks, and aromatic clusters in NTSR1 signaling. Moreover, we identified a cryptic allosteric site located in the intracellular region 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 signaling at the atomic level, thereby providing a potential strategy for the development of NTSR1 allosteric modulators in the realm of G protein-coupled receptor biology, biophysics, and medicine.

Problem

• Exploring NTSR1 activation and biased signaling mechanisms
• Identifying cryptic allosteric sites in receptor intermediates
• Developing NTSR1 modulators for addiction treatment

Innovation

• Combined computational and experimental approach
• Revealed dynamic stepwise transition mechanism
• Identified cryptic allosteric site