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The Frank–Starling law of the heart is a physiological phenomenon that describes an intrinsic property of heart muscle in which increased cardiac filling leads to enhanced cardiac contractility. Identified more than a century ago, the Frank–Starling relationship is currently known to involve length-dependent enhancement of cardiac myofilament Ca2+ sensitivity. However, the upstream molecular events that link cellular stretch to the length-dependent myofilament Ca2+ sensitivity are poorly understood. Because the angiotensin II type 1 receptor (AT1R) and the multifunctional transducer protein β-arrestin have been shown to mediate mechanosensitive cellular signaling, we tested the hypothesis that these two proteins are involved in the Frank–Starling mechanism of the heart. Using invasive hemodynamics, we found that mice lacking β-arrestin 1, β-arrestin 2, or AT1R were unable to generate a Frank–Starling force in response to changes in cardiac volume. Although wild-type mice pretreated with the conventional AT1R blocker losartan were unable to enhance cardiac contractility with volume loading, treatment with a β-arrestin–biased AT1R ligand to selectively activate β-arrestin signaling preserved the Frank–Starling relationship. Importantly, in skinned muscle fiber preparations, we found markedly impaired length-dependent myofilament Ca2+ sensitivity in β-arrestin 1, β-arrestin 2, and AT1R knockout mice. Our data reveal β-arrestin 1, β-arrestin 2, and AT1R as key regulatory molecules in the Frank–Starling mechanism, which potentially can be targeted therapeutically with β-arrestin–biased AT1R ligands.

Opioids such as morphine—acting at the mu opioid receptor—are the mainstay for treatment of moderate to severe pain and have good efficacy in these indications. However, these drugs produce a plethora of unwanted adverse effects including respiratory depression, constipation, immune suppression and with prolonged treatment, tolerance, dependence and abuse liability. Studies in β-arrestin 2 gene knockout (βarr2(−/−)) animals indicate that morphine analgesia is potentiated while side effects are reduced, suggesting that drugs biased away from arrestin may manifest with a reduced-side-effect profile. However, there is controversy in this area with improvement of morphine-induced constipation and reduced respiratory effects in βarr2(−/−) mice. Moreover, studies performed with mice genetically engineered with G-protein-biased mu receptors suggested increased sensitivity of these animals to both analgesic actions and side effects of opioid drugs. Several new molecules have been identified as mu receptor G-protein-biased agonists, including oliceridine (TRV130), PZM21 and SR–17018. These compounds have provided preclinical data with apparent support for bias toward G proteins and the genetic premise of effective and safer analgesics. There are clinical data for oliceridine that have been very recently approved for short term intravenous use in hospitals and other controlled settings. While these data are compelling and provide a potential new pathway-based target for drug discovery, a simpler explanation for the behavior of these biased agonists revolves around differences in intrinsic activity. A highly detailed study comparing oliceridine, PZM21 and SR–17018 (among others) in a range of assays showed that these molecules behave as partial agonists. Moreover, there was a correlation between their therapeutic indices and their efficacies, but not their bias factors. If there is amplification of G-protein, but not arrestin pathways, then agonists with reduced efficacy would show high levels of activity at G-protein and low or absent activity at arrestin; offering analgesia with reduced side effects or ‘apparent bias’. Overall, the current data suggests—and we support—caution in ascribing biased agonism to reduced-side-effect profiles for mu-agonist analgesics.

The Cannabis sativa plant contains more than 120 cannabinoids. With the exceptions of ∆ 9 -tetrahydrocannabinol (∆ 9 -THC) and cannabidiol (CBD), comparatively little is known about the pharmacology of the less-abundant plant-derived (phyto) cannabinoids. The best-studied transducers of cannabinoid-dependent effects are type 1 and type 2 cannabinoid receptors (CB1R, CB2R). Partial agonism of CB1R by ∆ 9 -THC is known to bring about the ‘high’ associated with Cannabis use, as well as the pain-, appetite-, and anxiety-modulating effects that are potentially therapeutic . CB2R activation by certain cannabinoids has been associated with anti-inflammatory activities. We assessed the activity of 8 phytocannabinoids at human CB1R, and CB2R in Chinese hamster ovary (CHO) cells stably expressing these receptors and in C57BL/6 mice in an attempt to better understand their pharmacodynamics. Specifically, ∆ 9 -THC, ∆ 9 -tetrahydrocannabinolic acid (∆ 9 -THCa), ∆ 9 -tetrahydrocannabivarin (THCV), CBD, cannabidiolic acid (CBDa), cannabidivarin (CBDV), cannabigerol (CBG), and cannabichromene (CBC) were evaluated. Compounds were assessed for their affinity to receptors, ability to inhibit cAMP accumulation, βarrestin2 recruitment, receptor selectivity, and ligand bias in cell culture; and cataleptic, hypothermic, anti-nociceptive, hypolocomotive, and anxiolytic effects in mice. Our data reveal partial agonist activity for many phytocannabinoids tested at CB1R and/or CB2R, as well as in vivo responses often associated with activation of CB1R. These data build on the growing body of literature showing cannabinoid receptor-dependent pharmacology for these less-abundant phytocannabinoids and are critical in understanding the complex and interactive pharmacology of Cannabis -derived molecules.

Cardiac fibroblasts (CF) play a critical role in post-infarction remodeling which can ultimately lead to pathological fibrosis and heart failure. Recent evidence demonstrates that remote (non-infarct) territory fibrosis is a major mechanism for ventricular dysfunction and arrhythmogenesis. β-arrestins are important signaling molecules involved in β-adrenergic receptor (β-AR) desensitization and can also mediate signaling in a G protein independent fashion. Recent work has provided evidence that β-arrestin signaling in the heart may be beneficial, however, these studies have primarily focused on cardiac myocytes and their role in adult CF biology has not been well studied. In this study, we show that β-arrestins can regulate CF biology and contribute to pathological fibrosis. Adult male rats underwent LAD ligation to induce infarction and were studied by echocardiography. There was a significant decline in LV function at 2-12 weeks post-MI with increased infarct and remote territory fibrosis by histology consistent with maladaptive remodeling. Collagen synthesis was upregulated 2.9-fold in CF isolated at 8 and 12 weeks post-MI and β-arrestin expression was significantly increased. β-adrenergic signaling was uncoupled in the post-MI CF and β-agonist-mediated inhibition of collagen synthesis was lost. Knockdown of β-arrestin1 or 2 in the post-MI CF inhibited transformation to myofibroblasts as well as basal and TGF-β-stimulated collagen synthesis. These data suggest that β-arrestins can regulate CF biology and that targeted inhibition of these signaling molecules may represent a novel approach to prevent post-infarction pathological fibrosis and the transition to HF.

Virus infection may induce excessive interferon (IFN) responses that can lead to host tissue injury or even death. β-arrestin 2 regulates multiple cellular events through the G protein-coupled receptor (GPCR) signaling pathways. Here we demonstrate that β-arrestin 2 also promotes virus-induced production of IFN-β and clearance of viruses in macrophages. β-arrestin 2 interacts with cyclic GMP-AMP synthase (cGAS) and increases the binding of dsDNA to cGAS to enhance cyclic GMP-AMP (cGAMP) production and the downstream stimulator of interferon genes (STING) and innate immune responses. Mechanistically, deacetylation of β-arrestin 2 at Lys171 facilitates the activation of the cGAS–STING signaling and the production of IFN-β. In vitro, viral infection induces the degradation of β-arrestin 2 to facilitate immune evasion, while a β-blocker, carvedilol, rescues β-arrestin 2 expression to maintain the antiviral immune response. Our results thus identify a viral immune-evasion pathway via the degradation of β-arrestin 2, and also hint that carvedilol, approved for treating heart failure, can potentially be repurposed as an antiviral drug candidate. Excessive interferon (IFN) responses often follow viral infection to induce pathology or even death. Here the authors show that a signaling adaptor, β-arrestin 2, enhances the cGAS/STING innate immunity signaling pathway to promote IFN-β production, but may be degraded in infected cells to serve as a target of viral immune evasion.

Bioluminescence resonance energy transfer (BRET) is a transfer of energy between a luminescence donor and a fluorescence acceptor. Because BRET occurs when the distance between the donor and acceptor is <10 nm, and its efficiency is inversely proportional to the sixth power of distance, it has gained popularity as a proximity-based assay to monitor protein–protein interactions and conformational rearrangements in live cells. In such assays, one protein of interest is fused to a bioluminescent energy donor (luciferases from Renilla reniformis or Oplophorus gracilirostris), and the other protein is fused to a fluorescent energy acceptor (such as GFP or YFP). Because the BRET donor does not require an external light source, it does not lead to phototoxicity or autofluorescence. It therefore represents an interesting alternative to fluorescence-based imaging such as FRET. However, the low signal output of BRET energy donors has limited the spatiotemporal resolution of BRET imaging. Here, we describe how recent improvements in detection devices and BRET probes can be used to markedly improve the resolution of BRET imaging, thus widening the field of BRET imaging applications. The protocol described herein involves three main stages. First, cell preparation and transfection require 3 d, including cell culture time. Second, image acquisition takes 10–120 min per sample, after an initial 60 min for microscope setup. Finally, image analysis typically takes 1–2 h. The choices of energy donor, acceptor, luminescent substrates, cameras and microscope setup, as well as acquisition modes to be used for different applications, are also discussed.This protocol describes the experimental design and procedures for bioluminescence resonance energy transfer (BRET) imaging. The authors discuss choices of energy donors and acceptors, luminescent substrates, microscope setup and cameras.

Chemokines are essential for guiding cell migration. Atypical chemokine receptors (ACKRs) contribute to the cell migration process by binding, internalizing and degrading local chemokines, which enables the formation of confined gradients. ACKRs are heptahelical membrane spanning molecules structurally related to G-protein coupled receptors (GPCRs), but seem to be unable to signal through G-proteins upon ligand binding. ACKR4 internalizes the chemokines CCL19, CCL21, and CCL25 and is best known for shaping functional CCL21 gradients. Ligand binding to ACKR4 has been shown to recruit β-arrestins that has led to the assumption that chemokine scavenging relies on β-arrestin-mediated ACKR4 trafficking, a common internalization route taken by class A GPCRs. Here, we show that CCL19, CCL21, and CCL25 readily recruited β-arrestin1 and β-arrestin2 to human ACKR4, but found no evidence for β-arrestin-dependent or independent ACKR4-mediated activation of the kinases Erk1/2, Akt, or Src. However, we demonstrate that β-arrestins interacted with ACKR4 in the steady-state and contributed to the spontaneous trafficking of the receptor in the absence of chemokines. Deleting the C-terminus of ACKR4 not only interfered with the interaction of β-arrestins, but also with the uptake of fluorescently labeled cognate chemokines. We identify the GPCR kinase GRK3, and to a lesser extent GRK2, but not GRK4, GRK5, and GRK6, to be recruited to chemokine-stimulated ACKR4. We show that GRK3 recruitment proceded the recruitment of β-arrestins upon ACKR4 engagement and that GRK2/3 inhibition partially interfered with steady-state interaction and chemokine-driven recruitment of β-arrestins to ACKR4. Overexpressing β-arrestin2 accelerated the uptake of fluorescently labeled CCL19, indicating that β-arrestins contribute to the chemokine scavenging activity of ACKR4. By contrast, cells lacking β-arrestins were still capable to take up fluorescently labeled CCL19 demonstrating that β-arrestins are dispensable for chemokine scavenging by ACKR4.

Background Atypical chemokine receptors (ACKRs) play an important role in regulating the availability of chemokines and are responsible for the formation of chemokine gradients required for the directed migration of immune cells in health and disease. ACKR4 shapes gradients of the chemokines CCL19 and CCL21, which are essential for guiding leukocyte homing to lymphoid organs where they initiate an adaptive immune response against invading pathogens. How ACKRs internalize and scavenge chemokines on the molecular level remains poorly understood. Current state-of the art methods to study βarrestin recruitment, signaling and trafficking of ACKRs - and G-protein-coupled receptors in general - rely heavily on C-terminally tagged receptors with unknown consequences for receptor functions. Methods Fluorescently labelled CCL19 was used to quantify chemokine internalization by native and tagged receptors as assessed by flow cytometry and live cell confocal microscopy. Steady-state interaction and chemokine-driven recruitment of βarrestins was determined by NanoBiT bystander assays. βarrestin-dependency for CCL19 internalization was determined in wild-type versus βarrestin1/2-double deficient cell lines. Statistical significance was determined by unpaired t-test or one-way ANOVA with Dunnett’s or Tukey’s multiple comparison tests. Results Addition of a C-terminal tag selectively affected the function of ACKR4, but not other ACKRs. Fusing a short peptide tag or a fluorescent protein to ACKR4 significantly augmented its ability to internalize its cognate ligand CCL19. In comparison to native ACKR4, its C-terminal tagging provoked an elevated pre-association of βarrestins with the plasma membrane, yet a reduction in chemokine-driven βarrestin recruitment. Furthermore, the addition of a C-terminal tag led to a shift from a βarrestin-dependent towards a βarrestin-independent endocytosis pathway. Similar results on chemokine uptake and on βarrestin-dependency were obtained with ACKR4 variants, in which a putative class II PDZ-binding domain located at the C-terminal tip of the receptor was mutated. Conclusion This study identifies that the integrity of the C-terminus of ACKR4 is critical for receptor function. The addition of a C-terminal tag to ACKR4 enhances chemokine uptake and alters the involvement of βarrestins in receptor trafficking.

Bitter taste receptor (TAS2R) agonists dilate airways by receptor-dependent smooth muscle relaxation. Besides their coupling to relaxation, we have found that human airway smooth muscle (HASM) cell TAS2Rs activate (phosphorylate) extracellular signal–related kinase 1/2 (ERK1/2), but the cellular effects are not known. In the present study, we show in HASM cells that TAS2R agonists initially stimulate phosphorylated ERK1/2 (pERK1/2) but by 24 hours cause a marked (50–70%) downregulation of pERK1/2 without a change in total ERK1/2. It was hypothesized that TAS2R agonists suppress cell growth through this pERK1/2 downregulation. Agonist-dependent inhibition of cell proliferation was indeed found in HASM cells derived from normal and asthmatic human lungs, as well as in an immortalized HASM cell line. pERK1/2 downregulation was linked to downregulation of the upstream kinase MEK1/2 (mitogen-activated protein kinase/extracellular signal–regulated kinase). Various structurally diverse TAS2R agonists evoked a range of inhibition of HASM proliferation, the magnitude of which directly correlated with the downregulation of pERK1/2 (R  2 = 0.86). Some TAS2R agonists were as effective as pharmacological inhibitors of Raf1 and MEK1/2 in suppressing growth. siRNA silencing of TAS2Rs (subtypes 10, 14, and 31) ablated the pERK1/2 and growth-inhibitory effects of TAS2R agonists. These phenotypes were attenuated by inhibiting the TAS2R G protein Gαi and by knocking down β-arrestin 1/2, indicating a dual pathway, although there may be additional mechanisms involved in this HASM TAS2R multidimensional signaling. Thus, TAS2R agonist structure can be manipulated to maintain the relaxation response and can be biased toward suppression of HASM growth. The latter response is of potential therapeutic benefit in asthma, in which an increase in smooth muscle mass contributes to airway obstruction.

Background Macrophages play an important role in the pathogenesis of ulcerative colitis (UC). We will explore the effects of sodium butyrate (SB) on macrophage function. Methods The targets of butyric acid were identified using SwissTargetPrediction database and surface plasmon resonance (SPR). Limited proteolysis mass spectrometry (Lip-MS) was used to further investigate the binding sites of butyric acid with its targets and molecular docking was employed to simulate their binding modes. Macrophage polarization model was established with lipopolysaccharide (LPS) in vitro. Takeda G protein-coupled receptor 5 (TGR5) and β-arrestin2 expression and macrophage polarization markers were detected with or without SB. Results TGR5 was identified as the target of butyric acid. Moreover, the amino acid regions 275–286 and 321–330 of TGR5 (GPBAR1 [275–286] and GPBAR1 [321–330]) were the potential binding regions for butyric acid. Based on molecular docking analysis, butyric acid formed effective hydrogen-bonding interactions with ASP-284 and TYR-287 of TGR5. In cell experiments, LPS inhibited the expression of TGR5, β-arrestin2, IL-10, ARG1, and CD206 and increased the expression of IL-1β, iNOS, and CD86, while SB reversed the effect of LPS. SBI-115, a TGR5 antagonist, and knockdown of β-arrestin2 inhibited the effect of sodium butyrate. INT-777, a TGR5 agonist, reversed the inhibitory effect of knockdown of β-arrestin2. Conclusion SB inhibited M1-like polarization and promoted M2-like polarization induced by LPS via TGR5/β-arrestin2 in RAW264.7 cells and TGR5 was the target of SB.