1. Gαs is dispensable for β-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by β2-adrenergic receptor
- Author
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Burghi, Valeria, Paradis, Justine S, Officer, Adam, Adame-Garcia, Sendi Rafael, Wu, Xingyu, Matthees, Edda SF, Barsi-Rhyne, Benjamin, Ramms, Dana J, Clubb, Lauren, Acosta, Monica, Tamayo, Pablo, Bouvier, Michel, Inoue, Asuka, von Zastrow, Mark, Hoffmann, Carsten, and Gutkind, J Silvio
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Genetics ,Biotechnology ,1.1 Normal biological development and functioning ,2.1 Biological and endogenous factors ,Underpinning research ,Aetiology ,Generic health relevance ,Humans ,beta-Arrestin 1 ,beta-Arrestin 2 ,beta-Arrestins ,Cyclic AMP-Dependent Protein Kinases ,Gene Expression Regulation ,GTP-Binding Proteins ,Mitogen-Activated Protein Kinases ,Phosphorylation ,Receptors ,Adrenergic ,beta-2 ,HEK293 Cells ,GTP-Binding Protein alpha Subunits ,Protein Structure ,Tertiary ,Protein Isoforms ,Enzyme Activation ,G protein ,G protein–coupled receptor ,gene expression ,signaling ,β-arrestin ,Chemical Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biological sciences ,Biomedical and clinical sciences ,Chemical sciences - Abstract
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.
- Published
- 2023