Lack of beta-arrestin signaling in the absence of active G proteins

Nat Commun. 2018 Jan 23;9(1):341. doi: 10.1038/s41467-017-02661-3.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems
  • GTP-Binding Protein alpha Subunits / genetics
  • GTP-Binding Protein alpha Subunits, G12-G13 / genetics
  • GTP-Binding Protein alpha Subunits, Gs / genetics
  • GTP-Binding Proteins / genetics*
  • GTP-Binding Proteins / metabolism
  • Gene Knockout Techniques
  • HEK293 Cells
  • Humans
  • MAP Kinase Signaling System*
  • Phosphorylation
  • Receptors, G-Protein-Coupled / metabolism*
  • Signal Transduction
  • beta-Arrestin 1 / metabolism*
  • beta-Arrestin 2 / metabolism*
  • beta-Arrestins / metabolism

Substances

  • GTP-Binding Protein alpha Subunits
  • Receptors, G-Protein-Coupled
  • beta-Arrestin 1
  • beta-Arrestin 2
  • beta-Arrestins
  • olfactory G protein subunit alpha olf
  • GTP-Binding Proteins
  • GTP-Binding Protein alpha Subunits, G12-G13
  • GTP-Binding Protein alpha Subunits, Gs