Manuel Grundmann 1 , Nicole Merten 1 , Davide Malfacini 1 , Asuka Inoue 2 , 3 , Philip Preis 1 , Katharina Simon 1 , Nelly Rüttiger 4 , Nicole Ziegler 5 , Tobias Benkel 1 , Nina Katharina Schmitt 1 , Satoru Ishida 2 , Ines Müller 1 , Raphael Reher 6 , Kouki Kawakami 2 , Ayumi Inoue 2 , Ulrike Rick 1 , Toni Kühl 7 , Diana Imhof 7 , Junken Aoki 2 , 8 , Gabriele M. König 6 , Carsten Hoffmann 4 , Jesus Gomeza 1 , Jürgen Wess 9 , Evi Kostenis , 1
23 January 2018
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.
Arrestins terminate signaling from GPCRs, but several lines of evidence suggest that they are also able to transduce signals independently of G proteins. Here, the authors systematically ablate G proteins in cell lines, and show that arrestins are unable to act as genuine signal initiators.