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 over... More
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. Graphical Abstract 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.
