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

• DOI: 10.1016/j.jbc.2023.105293
• Publication Date: 2023-09-27T15:21:07Z
• Authors: Valeria Burghi; Justine S. Paradis; Adam Officer; Sendi Rafael Adam...; Xingyu Wu; Edda S.F. Matthees; Benjamin Barsi-Rhyne; Dana J. Ramms; Lauren Clubb; Monica Acosta; Pablo Tamayo; Michel Bouvier; Asuka Inoue; Mark von Zastrow; Carsten Hoffmann; J. Silvio Gutkind
• Keywords: 570; Protein Structure; Biochemistry & Molecular Biology; Biomedical and clinical sciences; 1.1 Normal biological development and functioning; beta-2; Medical and Health Sciences; GTP-Binding Proteins; Receptors; Genetics; 2.1 Biological and endogenous factors; Humans; Protein Isoforms; Phosphorylation; beta-Arrestins; β-arrestin; Inflammatory and immune system; G protein; Biological Sciences; beta-Arrestin 2; Cyclic AMP-Dependent Protein Kinases; GTP-Binding Protein alpha Subunits; Protein Structure, Tertiary; Enzyme Activation; Biological sciences; beta-Arrestin 1; HEK293 Cells; Chemical sciences; Gene Expression Regulation; Adrenergic; G protein–coupled receptor; Chemical Sciences; gene expression; Biochemistry and Cell Biology; Generic health relevance; Receptors, Adrenergic, beta-2; Mitogen-Activated Protein Kinases; signaling; Tertiary; Research Article

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 Gαs, β-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and β-arrestins in controlling gene expression. We found that Gαs is not required for β2AR and β-arrestin conformational changes, β-arrestin recruitment, and receptor internalization, but that Gαs 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 Gαs-KO cells. These results were validated by re-expressing Gαs 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 Gαs-driven nuclear transcriptional activity.