Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9

Louis M. Luttrell, Jialu Wang, Bianca Plouffe, Jeffrey S. Smith, Lama Yamani, Suneet Kaur, Pierre Yves Jean-Charles, Christophe Gauthier, Mi Hye Lee, Biswaranjan Pani, Jihee Kim, Seungkirl Ahn, Sudarshan Rajagopal, Eric Reiter, Michel Bouvier, Sudha K. Shenoy, Stéphane A. Laporte, Howard A. Rockman, Robert J. Lefkowitz*

*Corresponding author for this work

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Abstract

G protein-coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four G s -coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for βArr2 or βArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β 2 adrenergic receptors (β 2 ARs) and β 1 ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V 2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β 2 AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein-deleted cells to GPCR behavior in native systems.

Original languageEnglish
Article numbereaat7650
Number of pages23
JournalScience Signaling
Volume11
Issue number549
DOIs
Publication statusPublished - 25 Sep 2018
Externally publishedYes

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Luttrell, L. M., Wang, J., Plouffe, B., Smith, J. S., Yamani, L., Kaur, S., Jean-Charles, P. Y., Gauthier, C., Lee, M. H., Pani, B., Kim, J., Ahn, S., Rajagopal, S., Reiter, E., Bouvier, M., Shenoy, S. K., Laporte, S. A., Rockman, H. A., & Lefkowitz, R. J. (2018). Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9. Science Signaling, 11(549), [eaat7650]. https://doi.org/10.1126/scisignal.aat7650