Research ArticleGPCR SIGNALING

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

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Science Signaling  25 Sep 2018:
Vol. 11, Issue 549, eaat7650
DOI: 10.1126/scisignal.aat7650

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The balancing act of β-arrestins

G protein–coupled receptors (GPCRs) are thought to activate the kinases ERK1/2 through G protein– and β-arrestin–dependent pathways. The relative contribution of each is difficult to assess because β-arrestins prevent G protein coupling by GPCRs (see the Focus by Gurevich and Gurevich). Studies based on CRISPR/Cas9-generated cell lines suggested that β-arrestins are dispensable for ERK1/2 activation. Luttrell et al. compared the effects of siRNA-mediated and CRISPR/Cas9-mediated knockdown of β-arrestins on ERK1/2 activation by several GPCRs in independent clones. Their data showed that signaling rewiring in the CRISPR clones rendered GPCR-dependent ERK1/2 activation more G protein–dependent, which was reversed by reconstitution with β-arrestins. Together, these findings suggest that β-arrestins balance signaling through the different pathways from GPCRs to ERK1/2 and suggest that experiments with deletion of β-arrestins or G proteins should be interpreted with caution.


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 Gs-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 β2 adrenergic receptors (β2ARs) and β1ARs, 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 V2 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 β2AR-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.

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