Fine-tuning an intracellular allosteric pocket programs GPCR signaling bias

Shi, X.; Zhu, R.; Chang, K.; Yu, P.; Yuan, Q.; Zhang, Y.; Lu, X.; Zhou, Z.; Zhu, C.; Lan, X.; Li, M.; Li, R.; Qiao, X.; Xie, W.; Zhuang, Y.; Xu, H. E.; Yin, W.; Zhao, M.; Zhang, J.

doi:10.64898/2026.10.22.760766

bioRxiv · 10.64898/2026.10.22.760766

Fine-tuning an intracellular allosteric pocket programs GPCR signaling bias

Shi, X.·Zhu, R.·Chang, K.·Yu, P.·Yuan, Q.·Zhang, Y.·Lu, X.·Zhou, Z.·Zhu, C.·Lan, X.·Li, M.·Li, R.·Qiao, X.·Xie, W.·Zhuang, Y.·Xu, H. E.·Yin, W.·Zhao, M.·Zhang, J.

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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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Shi, X., Zhu, R., Chang, K., Yu, P., Yuan, Q., Zhang, Y., Lu, X., Zhou, Z., Zhu, C., Lan, X., Li, M., Li, R., Qiao, X., Xie, W., Zhuang, Y., Xu, H. E., Yin, W., Zhao, M., Zhang, J.. 2026-10-27. Fine-tuning an intracellular allosteric pocket programs GPCR signaling bias. https://doi.org/10.64898/2026.10.22.760766

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