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Single-molecule force spectroscopy study of interactions between angiotensin II type 1 receptor and different biased ligands in living cells

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Abstract

Angiotensin II type 1 receptor (AT1R), a typical G protein-coupled receptor, plays a key role in regulating many cardiovascular functions. Different ligands can bind with AT1R to selectively activate either G protein (Gq) or β-arrestin (β-arr) pathway, or both pathways, but the molecular mechanism is not clear yet. In this work, we used, for the first time, atomic force microscopy-based single molecule force spectroscopy (SMFS) to study the interactions of AT1R with three types of ligands, balanced ligand, Gq-biased ligand, and β-arr-biased ligand, in living cells. The results revealed their difference in binding force and binding stability. The complex of the Gq-biased ligand-AT1R overcame two energy barriers with an intermediate state during dissociation, whereas that of β-arr-biased ligand–AT1R complex overcame one energy barrier. This indicated that AT1R had different ligand-binding conformational substates and underwent different structural changes to activate downstream signaling pathways with variable agonist efficacies. Quantitative analysis of AT1R-ligand binding in living cells at the single-molecule level offers a new tool to study the molecular mechanism of AT1R biased activation.

Single-molecule force measurement on the living cell expressing AT1R-eGFP with a ligand modified AFM tip (left), the dynamic force spectra of β-arrestin biased ligands-AT1R (middle), and Gq-biased ligands-AT1R (right). The complexes of β-arr-biased ligand-AT1R overcame one energy barrier, with one linear region in the spectra, whereas the Gq-biased ligand-AT1R complexes overcame two energy barriers with two linear regions

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Acknowledgments

This work was supported by National Basic Research Program of China (2013CB933701), National Natural Science Foundation of China (no. 21735006, 21127901, 91413119), and Chinese Academy of Sciences.

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Authors and Affiliations

  1. Beijing National Laboratory for Molecular Sciences, Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry, Chinese Academy of Sciences, 2 North First Street, Zhongguancun, Beijing, 100190, China

    Wenhui Li, Jiachao Xu, Xiaolong Kou, Rong Zhao, Wei Zhou & Xiaohong Fang

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Wenhui Li, Jiachao Xu, Xiaolong Kou, Rong Zhao, Wei Zhou & Xiaohong Fang

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  1. Wenhui Li
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  2. Jiachao Xu
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Correspondence to Xiaohong Fang.

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Published in the topical collection Euroanalysis XIX with guest editors Charlotta Turner and Jonas Bergquist.

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Li, W., Xu, J., Kou, X. et al. Single-molecule force spectroscopy study of interactions between angiotensin II type 1 receptor and different biased ligands in living cells. Anal Bioanal Chem 410, 3275–3284 (2018). https://doi.org/10.1007/s00216-018-0956-3

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  • DOI: https://doi.org/10.1007/s00216-018-0956-3

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