PhD Scientific Days 2025

Poster Session II. - B: Molecular Medicine

Investigating Biased Signaling of the AT1 Angiotensin Receptor Using Conformational- and Activation-Based Biosensors

Name of the presenter

Misák Ádám

Institute/workplace of the presenter

Department of Physiology, Faculty of Medicine, Semmelweis University

Authors

Ádám Misák¹, András Dávid Tóth^2,3, Gábor Turu^1,2, László Hunyady^1,4

1: Department of Physiology, Faculty of Medicine, Semmelweis University
2: Institute of Molecular Life Sciences, Centre of Excellence of the Hungarian Academy of Sciences, HUN-REN Research Centre for Natural Sciences
3: Department of Internal Medicine and Haematology, Semmelweis University
4: Institute of Molecular Life Sciences, Centre of Excellence of the Hungarian Academy of Sciences, HUN-REN Research Centre for Natural Sciences,

Text of the abstract

Investigating Biased Signaling of the AT1 Angiotensin Receptor Using Conformational- and Activation-Based Biosensors
The angiotensin II type 1 receptor (AT1R) is a key regulator of blood pressure and fluid homeostasis, and it is a major therapeutic target for hypertension. Recent research has focused on biased signaling – where ligands selectively activate certain pathways over others – as such agonists may offer therapeutic benefits. However, the mechanism of biased signaling of AT1R remains incompletely understood.
We employed a panel of bioluminescence resonance energy transfer-based biosensors to monitor AT1R signaling across multiple pathways in transiently transfected HEK 293T cells. These included a conformation-sensitive nanobody (Nb.AT110i1)-based and mini G protein biosensors, G protein activation and β-arrestin recruitment sensors, as well as sensors for PIP2 depletion and IP3 production. Various AT1R agonists were tested, with angiotensin II serving as a reference ligand.
We observed distinct receptor conformational- and signaling profiles across agonists, and the conformational signatures induced by the agonists could be linked to their signaling properties. Interestingly, stimulation with the non-peptide AT1R partial agonist L-162,313 led to a unique conformational profile. We found that L-162,313 was able to induce a partial effect in IP3 elevation and PIP2 cleavage, consistent with Gq/11 protein-mediated phospholipase C activation. However, direct measurement of Gq activity with the TRUPATH sensor did not show significant activation upon L-162,313 treatment, indicating that L-162,313 can only weakly stimulate G protein activity. In contrast, we detected robust β-arrestin-2 recruitment to the receptor.
Integrating conformational biosensors with functional assays is a powerful strategy to uncover the mechanistic basis of biased signaling. Our findings suggest that L-162,313 may induce a signaling profile characterized by low Gq/11 activation and maintained β-arrestin recruitment, with the observed IP3 elevation likely originating from the amplification by downstream signaling cascades. These results indicate that L-162,313 should be classified as a β-arrestin–biased AT1R agonist with a hitherto unknown mechanism, and they highlight its potential as a lead compound for the development of novel non-peptide biased ligands.