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

• Published in: Nature communications Vol. 9; no. 1; pp. 341 - 16
• Main Authors: Grundmann, Manuel, Merten, Nicole, Malfacini, Davide, Inoue, Asuka, Preis, Philip, Simon, Katharina, Rüttiger, Nelly, Ziegler, Nicole, Benkel, Tobias, Schmitt, Nina Katharina, Ishida, Satoru, Müller, Ines, Reher, Raphael, Kawakami, Kouki, Inoue, Ayumi, Rick, Ulrike, Kühl, Toni, Imhof, Diana, Aoki, Junken, König, Gabriele M., Hoffmann, Carsten, Gomeza, Jesus, Wess, Jürgen, Kostenis, Evi
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 23.01.2018; Nature Publishing Group; Nature Portfolio
• Subjects: 13; 45/41; 631/80/86; 631/92/609; 96; 96/10; 96/33; 96/95; Arrestin; beta-Arrestin 1 - metabolism; beta-Arrestin 2 - metabolism; beta-Arrestins - metabolism; Biological activity; Biosensors; CRISPR; CRISPR-Cas Systems; Deactivation; G protein-coupled receptors; Gene Knockout Techniques; 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; HEK293 Cells; Humanities and Social Sciences; Humans; Inactivation; Internalization; MAP Kinase Signaling System; multidisciplinary; Pertussis; Pertussis toxin; Phosphorylation; Proteins; Receptors; Receptors, G-Protein-Coupled - metabolism; Science; Science (multidisciplinary); Signal Transduction; Signaling
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-017-02661-3

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.