IRIC – Institut de recherche en immunologie et en cancérologie de l’Université de Montréal

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. Recent studies using genome editing approaches revealed that whereas G proteins are essential for ERK activation by GPCRs, β-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not be able to 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 HEK293 cells wild type or CRISPR/Cas9 gene edited for Gα_s, β-arrestin1/2, or GPCR kinases (GRK) 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gα_s and β-arrestins in signaling to the nucleus, thereby 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 GRKs isoforms involved in β-arrestin recruitment. By RNAseq analysis we found that PKA and MAPK gene signatures were activated by stimulation of β2AR in wild type and β-arrestin1/2-KO cells but absent in Gα_s-KO cells. These results were validated by rescue experiments expressing Gα_s in the corresponding KO cells and silencing β-arrestins in wild type cells. These findings were extended to cellular systems expressing endogenous levels of β2AR. Overall, our results support that Gs is essential for β2AR-promoted PKA and MAPK gene expression signatures, while β-arrestins initiates signaling events modulating Gα_s-driven nuclear transcriptional activity.