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 β-Arr2 or β-Arr1/2. 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.

β-arrestin1 (or arrestin2) and β-arrestin2 (or arrestin3) are ubiquitously expressed cytosolic adaptor proteins that were originally discovered for their inhibitory role in G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins. However, further biochemical characterization revealed that β-arrestins do not just ‘block’ the activated GPCRs, but trigger endocytosis and kinase activation leading to specific signaling pathways that can be localized on endosomes. The signaling pathways initiated by β-arrestins were also found to be independent of G protein activation by GPCRs. The discovery of ligands that blocked G protein activation but promoted β-arrestin binding, or vice-versa, suggested the exciting possibility of selectively activating intracellular signaling pathways. Additionally, it is becoming increasingly evident that β-arrestin-dependent signaling is extremely diverse and provokes distinct cellular responses through different GPCRs even when the same effector kinase is involved. In this review, we summarize various signaling pathways mediated by β-arrestins and highlight the physiologic effects of β-arrestin-dependent signaling.

Transgenic mice were generated to express a restrictive cardiomyopathy (RCM) human cardiac troponin I (cTnI) R192H mutation in the heart (cTnI(193His) mice). The objective of this study was to assess cardiac function during the development of diastolic dysfunction and to gain insight into the pathophysiological impact of the RCM cTnI mutation. Cardiac function and pathophysiological changes were monitored in cTnI193His mice and wild-type littermates for a period of 12 mo. It progressed gradually from abnormal relaxation to diastolic dysfunction characterized with high-resolution echocardiography by a reversed E-to-A ratio, increased deceleration time, and prolonged isovolumetric relaxation time. At the age of 12 mo, cardiac output in cTnI(193His) mice was significantly declined, and some transgenic mice showed congestive heart failure. The negative impact of cTnI193His on ventricular contraction and relaxation was further demonstrated in isolated mouse working heart preparations. The main morphological change in cTnI193His myocytes was shortened cell length. Dobutamine stimulation increased heart rate in cTnI193His mice but did not improve CO. The cTnI193His mice had a phenotype similar to that in human RCM patients carrying the cTnI mutation characterized morphologically by enlarged atria and restricted ventricles and functionally by diastolic dysfunction and diastolic heart failure. The results demonstrate a critical role of the COOH-terminal domain of cTnI in the diastolic function of cardiac muscle.

Toll-like receptor 4 (TLR4) promotes vascular inflammatory disorders such as neointimal hyperplasia and atherosclerosis. TLR4 triggers NFB signaling through the ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6). TRAF6 activity can be impeded by deubiquitinating enzymes like ubiquitin-specific protease 20 (USP20), which can reverse TRAF6 autoubiquitination, and by association with the multifunctional adaptor protein ␤-arrestin2. Although ␤-arrestin2 effects on TRAF6 suggest an anti-inflammatory role, physiologic ␤-arrestin2 promotes inflammation in atherosclerosis and neointimal hyperplasia. We hypothesized that anti-and proinflammatory dimensions of ␤-arrestin2 activity could be dictated by ␤-arrestin2's ubiquitination status, which has been linked with its ability to scaffold and localize activated ERK1/2 to signalosomes. With purified proteins and in intact cells, our protein interaction studies showed that TRAF6/USP20 association and subsequent USP20-mediated TRAF6 deubiquitination were ␤-arrestin2-dependent. Generation of transgenic mice with smooth muscle cell-specific expression of either USP20 or its catalytically inactive mutant revealed anti-inflammatory effects of USP20 in vivo and in vitro. Carotid endothelial denudation showed that antagonizing smooth muscle cell USP20 activity increased NFB activation and neointimal hyperplasia. We found that ␤-arrestin2 ubiquitination was promoted by TLR4 and reversed by USP20. The association of USP20 with ␤-arrestin2 was augmented when ␤-arrestin2 ubiquitination was prevented and reduced when ␤-arrestin2 ubiquitination was rendered constitutive. Constitutive ␤-arrestin2 ubiquitination also augmented NFB activation. We infer that pro-and anti-inflammatory activities of ␤-arrestin2 are determined by ␤-arrestin2 ubiquitination and that changes in USP20 expression and/or activity can therefore regulate inflammatory responses, at least in part, by defining the ubiquitination status of ␤-arrestin2.␤-Arrestin2 (␤arr2) is an ϳ46-kDa multifunctional scaffolding protein that was discovered originally for its ability to desensitize G protein-mediated signaling evoked by seventransmembrane receptors (7TMRs) 4 (1, 2). However, ␤arr2 modulates the signaling and/or endocytosis of not only most 7TMRs but also of several receptor protein tyrosine kinases, cytokine receptors, ion channel receptors, and the LDL receptor (3, 4). Both the endocytic and signaling functions of ␤arr2 are intertwined with its ubiquitination, which in turn is stimulus-driven and regulated by specific E3 ubiquitin ligases or deubiquitinases (DUBs) (4). ␤arr2 not only undergoes dynamic ubiquitination/deubiquitination but also recruits E3 ubiquitin ligases to other substrates. Indeed, ␤arr2 is integral to the ubiquitination of cell surface receptors, channels, and non-receptor proteins (4, 5). However, thus far there is no clear demonstration that ␤arr2 can scaffold a DUB to specific substrates and affect signal transduction by mediating deubiquitination.A role in deubiquitination c...