Gαs is dispensable for β-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by β2-adrenergic receptor.

β-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether β-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., β-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate β-arrestins, thereby limiting the ability to distinguish G protein from β-arrestin-mediated signaling events. We used β2-adrenergic receptor (β2AR) and its β2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gα _s , β-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gα _s and β-arrestins in controlling gene expression. We found that Gα _s is not required for β2AR and β-arrestin conformational changes, β-arrestin recruitment, and receptor internalization, but that Gα _s dictates the GPCR kinase isoforms involved in β-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of β2AR in wildtype and β-arrestin1/2-KO cells but absent in Gα _s -KO cells. These results were validated by re-expressing Gα _s in the corresponding KO cells and silencing β-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of β2AR. Overall, our results support that Gs is essential for β2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas β-arrestins initiate signaling events modulating Gα _s -driven nuclear transcriptional activity.
Competing Interests: Conflict of interest J. S. G. is consultant for Domain Therapeutics, Pangea Therapeutics, and io9, and founder of Kadima Pharmaceuticals, outside the submitted work. M. B. is the president of Domain Therapeutics Scientific Advisory Board. The authors declare that they have no conflicts of interest with the contents of this article.
(Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)