Abstract 315: Mechanical Stretch Activates Biased AT1R Signaling and a Distinct β-arrestin Conformation

Abstract

Introduction: Angiotensin II Type 1 receptor (AT1R) is a member of the G protein-coupled receptors (GPCRs) family, playing an important role in several cardiovascular diseases. Depending on the stimulus, AT1R activates a cascade of signaling pathways including those mediated by G proteins and the multifunctional proteins β-arrestins. AT1R is also mechanosensor and respond to membrane stretch by activating ligand-independent β-arrestin-biased signaling. Objective: The aim of this study is to investigate the precise molecular mechanism for mechanoactivation of the AT1R. Methods and Results: Our previous work demonstrated that mechanical stretch induced by hypotonicity (osmotic-stretch, OSM) allosterically activates the AT1R to mediate β-arrestin signaling. Using Proximity Ligation Assays (PLA) and co-IP we now demonstrate that OSM uniquely promotes Gαi recruitment to AT1R to initiate β-arrestin signaling. In sharp contrast to the β-arrestin-biased ligand TRV120023, the Gαi inhibitor Pertussis Toxin (PTX) blocked the OSM-induced β-arrestin recruitment, EGFR transactivation and ERK signaling. To determine whether the two biased stimuli (OSM and TRV120023) can activate different β-arrestin conformations, we used β-arrestin FLAsH constructs and monitored BRET following AT1R activation by either ligand or stretch stimulation. A FlAsH constructs located in the N-domain domain and two located in the C-domain domain showed a distinct BRET pattern indicating that in response to OSM, β-arrestin assumes a distinct conformation from either the balanced ligand Angiontensin II and the TRV120023 (N=7; p<0.05). Current studies are underway using site-specific stable-isotope labeling strategies coupled with mass spectrometry-based quantitative analysis (CDSiL-MS) to obtain biophysical information regarding the precise AT1R conformations induced by these balanced and β-arrestin-biased stimuli. Conclusion: The principal driver for differential signaling is thought to be different receptor conformations stabilized by each ligand. Our data suggest that in response to two β-arrestin-biased stimuli, AT1R likely adopt distinct conformations indicating remarkable conformational heterogeneity in activating intracellular responses.