Location-Biased β-Arrestin Conformations Direct G Protein-Coupled Receptor Signaling

Abstract

G protein-coupled receptors (GPCRs) regulate diverse cellular processes by converting extracellular signals into specific intracellular responses. While biased agonism has traditionally been understood in terms of differential G protein and β-arrestin engagement, the role of subcellular localization in modulating GPCR signaling remains less defined. This dissertation explores how β-arrestin conformation and function are shaped by cellular location, with a focus on the angiotensin II type 1 receptor (AT1R). Using conformational biosensors and computational modeling, we demonstrated that agonist-dependent conformations of receptor-associated β-arrestin are dictated by their direct interaction with the receptor core. At the receptor and endosomes, biased agonists induce distinct β-arrestin conformational signatures, but at the plasma membrane, a catalytically activated pool of β-arrestins adopts a uniform conformation which depends on the lipid interactions, regardless of agonist identity. Additionally, we found that ERK activation is regulated by different transducers depending on the subcellular localization. At endosomes, cytosol, and the nucleus, ERK activity is largely driven by G proteins, with biased agonists inducing distinct signaling profiles. In contrast, at the plasma membrane, ERK activation is predominantly mediated by catalytically active β-arrestins, independent of G protein signaling. This highlights a previously unappreciated role for membrane-localized β-arrestins in promoting ERK activity, supporting the concept that β-arrestin function extends beyond receptor desensitization to active signal propagation. Overall, this work provides a mechanistic framework for location-biased GPCR signaling, highlighting how subcellular localization influences β-arrestin structure and function.