Abstract
G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of “zero functional G” at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking β-arrestins (“zero arrestin”), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at “zero functional G”: arrestin recruitment and internalization, but—unexpectedly—complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2.
Highlights
G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes
What happens when G protein-coupled receptors are active but their associated G proteins are all inactive or collectively eliminated? What does signaling at “zero functional G” look like on a cellular level and can we visualize arrestin-dependent, G protein-independent signaling using a label-free, real-time integrated readout for global cellular activity? Albeit intriguing, answers remained elusive in large part ascribed to the lack of tools to accomplish collective absence or functional inactivity of Gα isoforms from all four major G protein families, and arrestins, respectively
Arrestins have been viewed by some as core components of G protein-independent signaling to the extracellular signal-regulated kinase 1/2 (ERK1/2) MAP kinase cascade[12,42,57]
Summary
G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. We appreciate the large body of excellent experimental evidence addressing GPCR β-arrestin interaction up to atomic level resolution[17,18,19] as well as the sophisticated biophysical studies resolving the fine details of arrestin conformational changes imparted by activated receptors[20,21] and its functional consequences[22,23] Despite these enormous advances in understanding the biophysical facets of arrestin function, the role of heterotrimeric G proteins and how they interplay with arrestinmediated processes remains largely unclear, in part ascribed to the lack of tools for specific and quantitative elimination of all relevant G protein signaling routes
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