Distinct Spatial Conformations and Trafficking Patterns of β-arrestin Isoforms in AT1R Signaling

Excessive activation of the AT1R by the hormone Angiotensin II (AngII) is implicated in the pathogenesis of hypertension by promoting vascular smooth muscle cell migration, hypertrophy, and cardiac remodeling. A limitation of current drug development to target this receptor is the complex pattern of its downstream signaling pathways, the majority of which are mediated by G proteins and β-arrestins. Although the two β-arrestin isoforms, β-arrestin 1 and 2, share ~78% amino acid sequence homology, they have been shown to differentially regulate signaling pathways such as receptor internalization and the mitogen-activated protein kinase pathways, leading to distinct physiologic and pathophysiologic outcomes. In our study, we investigated the functional divergence of the β-arrestin isoforms at the AT1R and its link to physiologic function. We hypothesized that β-arrestin isoforms adopt distinct recruitment patterns and spatial conformations that lead to differential regulation of downstream signaling and physiologic outcomes and "biased responses" by different agonists. Using the split NanoLuciferase complementation assay (NanoBiT), we determined that β-arrestin 1 and 2 are recruited differentially to the receptor, the plasma membrane and the endosomes following AT1R stimulation with AngII and the β-arrestin-biased agonist TRV023. Compared to TRV023, AngII was found to be more potent and efficacious in inducing the recruitment of both β-arrestin isoforms to the receptor. We also developed a new conformational biosensor, called the complex Fluorescent Arsenical Hairpin (FlAsH) assay, to assess the spatial conformational signatures of the β-arrestin isoforms when they are bound to the AT1R or localized at the plasma membrane and endosomes. β-arrestins 1 and 2 were found to adopt distinct conformations at all three cellular locations following agonist treatment. Our finding provides new insights into the mechanisms underlying the functional differences between the β-arrestin isoforms at the AT1R. Further studies will be necessary to elucidate the link between the trafficking and conformational differences of the β-arrestin isoforms and their distinct physiological roles in AT1R signaling.

Duke Scholars

Author Sudarshan Rajagopal Medicine, Cardiology