Fine-tuning an intracellular allosteric pocket programs GPCR signaling bias

Abstract

Biased signaling of G protein-coupled receptors (GPCRs) offers a route for precise therapeutics with fewer adverse effects. Intracellular allosteric ligands can impose such bias, yet how subtle chemical changes within these pockets reshape receptor-transducer coupling and downstream signaling remains unclear. Here, we systematically fine-tuned the intracellular allosteric pocket of neurotensin receptor 1 (NTSR1) through chemical modification of the {beta}-arrestin-biased ligand SBI-553. Minor changes to the ligand scaffold altered transducer-coupling preferences, generating distinct profiles of G-protein and {beta}-arrestin signaling. Cryo-electron microscopy structures of NTSR1-{beta}-arrestin2 complexes bound to designed allosteric probes revealed ligand-dependent remodeling of the intracellular cavity that reshapes the receptor-effector interface. Alanine scanning identified transducer-sensitive residues within and beyond the immediate ligand-contact region. In mice, SBI-553 and N065 produced distinct physiological profiles, with N065 improving survival in a lethal endotoxemia model. Finally, phosphoproteomic profiling revealed partially overlapping but distinct downstream signaling profiles among the three modulators. Together, these findings establish the intracellular allosteric pocket as a tunable determinant of transducer coupling and show that closely related modulators can generate distinct signaling and physiological profiles.

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BibTeX RIS

BibTeX

@misc{shi2026finetuning,
  title = {{Fine-tuning an intracellular allosteric pocket programs GPCR signaling bias}},
  author = {X. Shi and R. Zhu and K. Chang and P. Yu and Q. Yuan and Y. Zhang and X. Lu and Z. Zhou and C. Zhu and X. Lan and M. Li and R. Li and X. Qiao and W. Xie and Y. Zhuang and H. E. Xu and W. Yin and M. Zhao and J. Zhang},
  howpublished = {bioRxiv},
  year = {2026},
  month = oct,
  doi = {10.64898/2026.10.22.760766},
  url = {https://www.biorxiv.org/content/10.64898/2026.10.22.760766v1},
  note = {CC BY-NC-ND},
}

RIS

TY  - UNPB
TI  - Fine-tuning an intracellular allosteric pocket programs GPCR signaling bias
AU  - X. Shi
AU  - R. Zhu
AU  - K. Chang
AU  - P. Yu
AU  - Q. Yuan
AU  - Y. Zhang
AU  - X. Lu
AU  - Z. Zhou
AU  - C. Zhu
AU  - X. Lan
AU  - M. Li
AU  - R. Li
AU  - X. Qiao
AU  - W. Xie
AU  - Y. Zhuang
AU  - H. E. Xu
AU  - W. Yin
AU  - M. Zhao
AU  - J. Zhang
PY  - 2026
DA  - 2026/10/22
PB  - bioRxiv
DO  - 10.64898/2026.10.22.760766
UR  - https://www.biorxiv.org/content/10.64898/2026.10.22.760766v1
AB  - Biased signaling of G protein-coupled receptors (GPCRs) offers a route for precise therapeutics with fewer adverse effects. Intracellular allosteric ligands can impose such bias, yet how subtle chemical changes within these pockets reshape receptor-transducer coupling and downstream signaling remains unclear. Here, we systematically fine-tuned the intracellular allosteric pocket of neurotensin receptor 1 (NTSR1) through chemical modification of the {beta}-arrestin-biased ligand SBI-553. Minor changes to the ligand scaffold altered transducer-coupling preferences, generating distinct profiles of G-protein and {beta}-arrestin signaling. Cryo-electron microscopy structures of NTSR1-{beta}-arrestin2 complexes bound to designed allosteric probes revealed ligand-dependent remodeling of the intracellular cavity that reshapes the receptor-effector interface. Alanine scanning identified transducer-sensitive residues within and beyond the immediate ligand-contact region. In mice, SBI-553 and N065 produced distinct physiological profiles, with N065 improving survival in a lethal endotoxemia model. Finally, phosphoproteomic profiling revealed partially overlapping but distinct downstream signaling profiles among the three modulators. Together, these findings establish the intracellular allosteric pocket as a tunable determinant of transducer coupling and show that closely related modulators can generate distinct signaling and physiological profiles.
ER  -

Abstract

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