Relative Contribution of G-protein and β-Arrestin-Mediated Signaling of the AT1 Receptor in Neurons

The Angiotensin II (ANG) Type 1A receptor (AT1R) is the main effector of the renin-angiotensin system. The AT1R plays a major role in blood pressure (BP) and fluid homeostasis, thus understanding its signaling is essential. Sustained activation of AT1R-Gq signaling is pathogenic, whereas β-arrestin signaling causes receptor internalization and also elicits cardioprotective effects. There is an emerging concept that activating AT1R-β-arrestin might be more beneficial than blocking AT1R. ICV administration of TRV027, an AT1R biased agonist that selectively activates β-arrestin, reduces BP and increases aversion to salt. This study aims to understand the downstream signaling mechanisms of G protein- and β-arrestin-mediated signaling in several neuronal cell types and cardiometabolic effects (BP and fluid intake) of selective activation of each arm in key cardiovascular neuronal circuits. To dissect G protein- and β-arrestin-mediated signaling we are employing two well characterized AT1R mutants: a) mutation of the conserved DRY motif to prevent G-protein binding while retaining the capability to recruit β-arrestin (AT1RDRY >AAY), and b) mutation of 4 conserved serine/threonine residues which block β-arrestin recruitment while preserving G-protein interaction (AT1RTSTS >AAAA). We tested internalization capability of these mutants in hypothalamic neuronal GT1-7 cells. We generated N-terminal HiBiT tagged AT1R WT and mutant constructs. Consistent with an inability to recruit β-arrestin, AT1RTSTS >AAAA exhibited significantly impaired internalization in response to 150 nM ANG at 30- and 60-minute time points (n=5, p=0.026, WT vs TSTS Mean difference is -0.453). In contrast, AT1RDRY >AAY internalization pattern was not significantly different from AT1RWT (n=5, p=0.23, WT vs DRY Mean difference is -0.164), suggesting that AT1RDRY >AAY is capable of recruiting β-arrestin and thus getting internalized from the extracellular surface. Our studies in GT1-7 cells revealed sustained phospho-ERK (pERK, phospho-p44/42 MAPK) response in AT1RTSTS >AAAA in response to ANG, which was inhibited by the AT1R antagonist losartan. Our current studies on AT1RTSTS >AAAA and AT1RDRY >AAY indicates we can dissect the G-protein and β-arrestin-mediated signaling in hypothalamic neurons. To further assess functional aspects AT1R-G protein interaction and β-arrestin recruitment capability of the mutants in neuronal cell lines we are generating BRET constructs. Finally, to understand the physiological effects of selective activation each arm of signaling in vivo we generated transgenic mice with Cre-inducible expression of AT1RWT, AT1RTSTS >AAAA, or AT1RDRY >AAY. To validate inducible expression, primary fibroblasts collected and cultured from these mice revealed robust ANG-induced ERK activation in the AT1RTSTS >AAAA, modest ERK activation in the AT1RWT, but minimal ERK activation in the AT1RDRY >AAY. Our future studies of inducible expression of these mutants in key cardiovascular neuronal circuits will inform us about the AT1R-G protein and β-arrestin mediated cardiometabolic effects. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.