%0 Journal Article
%T Manifold roles of 汕-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9
%J -
%D 2018
%R 10.1126/scisignal.aat7650
%X G protein每coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. 汕-Arrestins (汕Arr1/2) 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 汕Arr1/2 and G proteins have cast doubt on the role of 汕-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of 汕Arr1/2 and reconstitution with 汕Arr1/2 in three different parental and CRISPR-derived 汕Arr1/2 knockout HEK293 cell pairs to assess the effect of 汕Arr1/2 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 汕Arr2 or 汕Arr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For 汕2 adrenergic receptors (汕2ARs) and 汕1ARs, 汕Arr1/2 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 汕Arr1/2. Loss of desensitization and receptor internalization in CRISPR 汕Arr1/2 knockout cells caused 汕2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing 汕Arr1/2. These data suggest that 汕Arr1/2 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 汕Arr1/2- or G protein每deleted cells to GPCR behavior in native systems
%U http://stke.sciencemag.org/content/11/549/eaat7650