β-Arrestin Isoform Conformations are Location-Biased and affect Downstream Signaling of G-protein Coupled Receptors

Abstract ID 101740 Poster Board 271 G Protein-Coupled Receptors (GPCRs) are the most commonly expressed receptors in the human body and serve as targets for around 30% percent of all FDA-approved drugs. GPCRs canonically signal through a variety of transducers including G proteins and β-arrestins, which connect to a variety of downstream signaling pathways including cyclic AMP (cAMP) and MAPK/ERK pathways. Previous research has demonstrated that an individual GPCR can adopt distinct signaling profiles, if stimulated by different biased ligands, in a process called 'biased agonism'. Furthermore, GPCRs from different cellular locations can elicit unique signaling pathways, a phenomenon called 'location bias'. However, the mechanisms underlying location bias in GPCR signaling remain unclear. To investigate the role of β-arrestins in facilitating location-biased signaling, intracellular trafficking of β-arrestins 1 and 2 was assessed using NanoBiT complementation assays. Angiotensin II type 1 Receptor (AT1R) stimulation by either Angiotensin II, the endogenous ligand (AngII) or by TRV023, a β-arrestin-biased ligand, resulted in different β-arrestin recruitment patterns at the receptor, plasma membrane, and endosomes. A pool of catalytically activated β-arrestins independent of the receptor-ligand complex was also observed at the plasma membrane. To assess whether β-arrestins adopt distinct location-specific conformations, novel NanoBiT Fluorescein Arsenical Hairpin (FlAsH) biosensors were constructed. We find that the AT1R ligands promote ligand-specific β-arrestin 1 and 2 conformations at the receptor and the endosomes but not at the plasma membrane and lipid rafts. Using ERK activity reporters targeted to different cellular locations, we found that the patterns of ERK signaling correlate well with β-arrestin conformations. Our data suggest that β-arrestin trafficking and location-specific conformations direct location bias in GPCR signaling.