Protein-coupled Receptor Desensitization Research Articles

Abstract 16000: β-arrestins Mediate Cardiac Infiltration of Cytotoxic CD8+ T Cells and Macrophages in Acute CVB3 Viral Myocarditis

Introduction: In response to Coxsackievirus B3 (CVB3) infection, immune cells infiltrate the heart affecting viral clearance, tissue damage, and cardiac remodeling. However, the molecular mechanisms regulating the immune response during acute CVB3-induced myocarditis are not well understood. β arrestins (βarrs), scaffolding proteins that mediate G protein-coupled receptor internalization, desensitization, and signaling are known to regulate immune responses. Hypothesis: We hypothesized that the immune populations infiltrating the heart during acute CVB3 infection are dependent on βarrs. Methods: To test our hypothesis, 5- to 7-week-old mice lacking βarr1 (βarr1 KO; n=8), βarr2 (βarr2 KO; n=4), or C57BL/6 controls (WT; n=7) were injected intraperitoneally with 1x10 4 plaque forming units (PFU) of CVB3 or saline and immune cell populations were measured in the heart at 7 days post infection (dpi) using flow cytometry. Results: At 7 dpi, there was a marked increase in cytotoxic CD8+ T cells in hearts of WT mice that positively correlated with the level of viral infection as assessed by heart tissue PFU (slope= 1.34, Y-intercept= -3.77). In contrast, the degree of infiltration of CD8+ T cells was significantly blunted in βarr KO hearts over a broad range of heart PFU (βarr1 KO: Slope=-0.3, Y-intercept=1.65; p=0.03 vs WT; βarr2 KO: Slope=-0.32, Y-intercept= 1.3; p=0.003 vs WT). Helper CD4+ T cell populations remained unaltered at 7 dpi among all groups. Macrophages at 7 dpi were significantly reduced in βarr KO hearts over a range of PFU (WT: Slope= 18.6, Y-intercept -52.32; βarr1 KO: Slope= -5, Y-intercept=32; βarr2 KO: Slope=3.46, Y-intercept=-8.4; p=0.04 vs WT). To assess cardiac injury, we measured the level of apoptosis by TUNEL staining and found that CVB3 infected βarr KO hearts showed significantly fewer TUNEL positive cells than WT CVB3 infected controls (WT 8.7%± 2.74; βarr1 KO 1.7%±0.51; βarr2 KO 1.7%±0.37; p=0.04 vs. WT). Conclusions: These data indicate that βarrs participate in the cardiac infiltration of CD8+ T cells and macrophages in response to CVB3 infection and that blocking the action of βarrs may diminish the cardiotoxicity with the acute phase of CVB3-induced myocarditis.

Plasma membrane preassociation drives β-arrestin coupling to receptors and activation

β-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-β-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in β-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that β-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, they indicate that, following receptor interaction, the plasma membrane stabilizes β-arrestin in a longer-lived, membrane-bound state, allowing it to diffuse to clathrin-coated pits separately from the activating receptor. These results expand our current understanding of β-arrestin function at the plasma membrane, revealing a critical role for β-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.

The Predominant Role of Arrestin3 in General GPCR Desensitization in Platelets.

Arrestins in concert with GPCR kinases (GRKs) function in G protein-coupled receptor (GPCR) desensitization in various cells. Therefore, we characterized the functional differences of arrestin3 versus arrestin2 in the regulation of GPCR signaling and its desensitization in platelets using mice lacking arrestin3 and arrestin2. In contrast to arrestin2, platelet aggregation and dense granule secretion induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in arrestin3-deficient platelets compared to wild-type (WT) platelets, while non-GPCR agonist CRP-induced platelet aggregation and secretion were not affected. Surprisingly, in contrast to GRK6, platelet aggregation induced by the co-stimulation of serotonin and epinephrine was significantly potentiated in arrestin3-deficient platelets, suggesting the central role of arrestin3 in general GPCR desensitization in platelets. In addition, the second challenge of ADP and AYPGKF restored platelet aggregation in arrestin3-deficient platelets but failed to do so in WT and arrestin2-deficient platelets, confirming that arrestin3 contributes to GPCR desensitization. Furthermore, ADP- and AYPGKF-induced Akt and ERK phosphorylation were significantly increased in arrestin3-deficient platelets. Finally, we found that arrestin3 is critical for thrombus formation in vivo. In conclusion, arrestin3, not arrestin2, plays a central role in the regulation of platelet functional responses and thrombus formation through general GPCR desensitization in platelets.

228-LB: ß-arrestin-2 Deletion Influences GLP-1 Receptor Signaling in Pancreatic ß Cells In Vivo

β-arrestin-2 (Barr2) is typically associated with G protein-coupled receptor desensitization and endocytosis, and reduced Barr2 recruitment is thought to enable prolongation of pharmacodynamic effects of biased glucagon-like peptide-1 receptor (GLP-1R) agonists. However, the precise role of Barr2 in GLP-1R function in β-cells is poorly understood. To study this role, a tamoxifen-inducible β-cell specific Barr2 knockout mouse model was generated (Pdx1CreERT/Barr2 fl/fl, referred to as β-Barr2 KO). β-Barr2 KO animals on high-fat high-sucrose diet were less glucose tolerant and tended to respond worse to acute exendin-4 (Ex4) treatment vs. controls (Barr2 fl/fl). However, Ex4 effects on blood glucose were more pronounced in KO mice 6h post-agonist injection [IPGTT AUC (6h) for Ex4/ vehicle: 0.84 ± 0.04 vs. 1.08 ± 0.05, p<0.001; ΔAUC (6h-0h): -49.8 ± 65.8 vs. 158.2 ± 43.6, p<0.05; KO vs. control]. This effect was not apparent with Ex-phe1, an Ex4 derivative biased away from Barr2 recruitment. Ex4-induced potentiation of insulin secretion in β-Barr2 KO islets was reduced acutely, but tended to increase overnight, resulting in significantly reduced desensitization vs. control islets. On chow diet, acute Ex4-stimulated calcium increases were unaffected, but acute cAMP responses were attenuated in KO islets [Emax (vs. baseline): 1.51 ± 0.17 vs. 1.72 ± 0.07, p<0.05; KO vs. control]. The latter was corroborated in INS1 832/3 Barr2 KO CRISPR cells, as NanoBiT™ functional assays showed reduced Ex4-induced Gs protein activation vs. control. GLP-1R trafficking profiles observed in vivo by imaging of Ex4-TMR uptake into islets implanted in the anterior chamber of the eye, as well as in vitro with GLP-1R-NLuc - KRAS/Rab5 NanoBRET assays were unaltered by Barr2 deletion. Thus, we provide evidence for opposing effects of Barr2 in acute vs. prolonged GLP-1R signaling, supporting a direct role for Barr2 in mediating the anti-hyperglycemic effects of biased GLP-1R agonists, but dispensability for GLP-1R trafficking. Disclosure S. Bitsi: None. K. Suba: None. N. Mohamed: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. G. A. Rutter: Advisory Panel; Self; Sun Pharmaceutical Industries Ltd. V. Salem: None. B. Jones: Research Support; Self; Sun Pharmaceutical Industries Ltd. A. Tomas: Other Relationship; Self; Sun Pharmaceutical Industries Ltd. Funding Medical Research Council (MR/R010676/1)

Host versus cell-dependent effects of β-arrestin 1 expression in prostate tumorigenesis.

Prostate cancer (PCa) constitutes a serious health challenge and remains one of the main causes of cancer-related death among men. The more aggressive form of the disease has been attributed to androgen independence, resulting in a lack of response to androgen deprivation therapy and sustained activation of other growth pathways. The scaffold proteins β-arrestin 1 and 2 (βarr1 and βarr2), which are known to mediate G protein-coupled receptor desensitization and internalization, were also shown to modulate prostate tumorigenesis. βarr1 is significantly overexpressed (>4-fold) in PCa cells relative to βarr2. In this study, we investigated the effect of βarr1 overexpression in PCa development and progression using the mouse and human PCa cell xenografts, and autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) models deficient in β-arrestin depletion of βarr1 in TRAMP mice (TRAMP/βarr1-/-) increased PCa growth and decreased overall survival relative to control TRAMP or TRAMP/βarr2-/- animals. Prostate tissues from TRAMP/βarr1-/- tumors displayed an increase in androgen receptor (AR) expression, whereas overexpression of βarr1 in TRAMP-C1 (TRAMP-C1-βarr1-GFP) which derived from TRAMP decreased AR expression, cell proliferation and tumor growth in nude mice xenografts, relative to control TRAMP-C1-GFP. Knockdown of βarr1 expression in human MDA PCa 2b cells (MDA PCa 2b-βarr1-/-) also decreased AR expression cell proliferation and tumor growth relative to control (MDA PCa 2b-Sham) cells. Interestingly, both TRAMP-C1-βarr1-GFP and MDA PCa 2b-βarr1-/- xenografts showed a decrease in AKT phosphorylation but an increase in MAPK activation. Altogether, the data indicate that the effect of βarr1 in modulating AR signaling to regulate PCa aggressiveness is cell and host autonomous.

The G Protein-Coupled Receptor Kinases (GRKs) in Chemokine Receptor-Mediated Immune Cell Migration: From Molecular Cues to Physiopathology.

Although G protein-coupled receptor kinases (GRKs) have long been known to regulate G protein-coupled receptor (GPCR) desensitization, their more recently characterized functions as scaffolds and signalling adapters underscore that this small family of proteins governs a larger array of physiological functions than originally suspected. This review explores how GRKs contribute to the complex signalling networks involved in the migration of immune cells along chemokine gradients sensed by cell surface GPCRs. We outline emerging evidence indicating that the coordinated docking of several GRKs on an active chemokine receptor determines a specific receptor phosphorylation barcode that will translate into distinct signalling and migration outcomes. The guidance cues for neutrophil migration are emphasized based on several alterations affecting GRKs or GPCRs reported to be involved in pathological conditions.

Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and α-granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6−/− platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6−/− platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled α2A adrenergic receptors, respectively, was not affected in GRK6−/− platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6−/− platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKCδ) phosphorylation were significantly potentiated in GRK6−/− platelets. Finally, GRK6−/− mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6−/− mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

A new inhibitor of the \u03b2-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases

G protein-coupled receptor kinases (GRKs) are members of the protein kinase A, G, and C families (AGC) and play a central role in mediating G protein-coupled receptor phosphorylation and desensitization. One member of the family, GRK5, has been implicated in several human pathologies, including heart failure, hypertension, cancer, diabetes, and Alzheimer disease. To gain mechanistic insight into GRK5 function, we determined a crystal structure of full-length human GRK5 at 1.8 Å resolution. GRK5 in complex with the ATP analog 5'-adenylyl β,γ-imidodiphosphate or the nucleoside sangivamycin crystallized as a monomer. The C-terminal tail (C-tail) of AGC kinase domains is a highly conserved feature that is divided into three segments as follows: the C-lobe tether, the active-site tether (AST), and the N-lobe tether (NLT). This domain is fully resolved in GRK5 and reveals novel interactions with the nucleotide and N-lobe. Similar to other AGC kinases, the GRK5 AST is an integral part of the nucleotide-binding pocket, a feature not observed in other GRKs. The AST also mediates contact between the kinase N- and C-lobes facilitating closure of the kinase domain. The GRK5 NLT is largely displaced from its previously observed position in other GRKs. Moreover, although the autophosphorylation sites in the NLT are >20 Å away from the catalytic cleft, they are capable of rapid cis-autophosphorylation suggesting high mobility of this region. In summary, we provide a snapshot of GRK5 in a partially closed state, where structural elements of the kinase domain C-tail are aligned to form novel interactions to the nucleotide and N-lobe not previously observed in other GRKs.

Mu opioid receptor stimulation activates c-Jun N-terminal kinase 2 by distinct arrestin-dependent and independent mechanisms

G protein-coupled receptor desensitization is typically mediated by receptor phosphorylation by G protein-coupled receptor kinase (GRK) and subsequent arrestin binding; morphine, however, was previously found to activate a c-Jun N-terminal kinase (JNK)-dependent, GRK/arrestin-independent pathway to produce mu opioid receptor (MOR) inactivation in spinally-mediated, acute anti-nociceptive responses [Melief et al.] [1]. In the current study, we determined that JNK2 was also required for centrally-mediated analgesic tolerance to morphine using the hotplate assay. We compared JNK activation by morphine and fentanyl in JNK1−/−, JNK2−/−, JNK3−/−, and GRK3−/− mice and found that both compounds specifically activate JNK2 in vivo; however, fentanyl activation of JNK2 was GRK3-dependent, whereas morphine activation of JNK2 was GRK3-independent. In MOR-GFP expressing HEK293 cells, treatment with either arrestin siRNA, the Src family kinase inhibitor PP2, or the protein kinase C (PKC) inhibitor Gö6976 indicated that morphine activated JNK2 through an arrestin-independent Src- and PKC-dependent mechanism, whereas fentanyl activated JNK2 through a Src-GRK3/arrestin-2-dependent and PKC-independent mechanism. This study resolves distinct ligand-directed mechanisms of JNK activation by mu opioid agonists and understanding ligand-directed signaling at MOR may improve opioid therapeutics.

G protein-coupled receptor kinase 2: a link between myocardial contractile function and cardiac metabolism.

Heart failure (HF) causes a tremendous burden on the worldwide healthcare system, affecting >23 million people. There are many cardiovascular disorders that contribute to the development of HF and multiple risk factors that accelerate its occurrence, but regardless of its underlying cause, HF is characterized by a marked decrease in myocardial contractility and loss of pump function. One biomarker molecule consistently shown to be upregulated in human HF and several animal models is G protein-coupled receptor kinase-2 (GRK2), a kinase originally discovered to be involved in G protein-coupled receptor desensitization, especially β-adrenergic receptors. Higher levels of GRK2 can impair β-adrenergic receptor-mediated inotropic reserve and its inhibition, or molecular reduction has shown to improve pump function in several animal models including a preclinical pig model of HF. Recently, nonclassical roles for GRK2 in cardiovascular disease have been described, including negative regulation of insulin signaling, a role in myocyte cell survival and apoptotic signaling, and it has been shown to be localized in/on mitochondria. These new roles of GRK2 suggest that GRK2 may be a nodal link in the myocyte, influencing both cardiac contractile function and cell metabolism and survival and contributing to HF independent of its canonical role in G protein-coupled receptor desensitization. In this review, classical and nonclassical roles for GRK2 will be discussed, focusing on recently discovered roles for GRK2 in cardiomyocyte metabolism and the effects that these roles may have on myocardial contractile function and HF development.

Coordinate-based co-localization-mediated analysis of arrestin clustering upon stimulation of the C–C chemokine receptor 5 with RANTES/CCL5 analogues

G protein-coupled receptor activation and desensitization leads to recruitment of arrestin proteins from cytosolic pools to the cell membrane where they form clusters difficult to characterize due to their small size and further mediate receptor internalization. We quantitatively investigated clustering of arrestin 3 induced by potent anti-HIV analogues of the chemokine RANTES after stimulation of the C-C chemokine receptor 5 using single-molecule localization-based super-resolution microscopy. We determined arrestin 3 cluster sizes and relative fractions of arrestin 3 molecules in each cluster through image-based analysis of the localization data by adapting a method originally developed for co-localization analysis from molecular coordinates. We found that only classical agonists in the set of tested ligands were able to efficiently recruit arrestin 3 to clusters mostly larger than 150 nm in size and compare our results with existing data on arrestin 2 clustering induced by the same chemokine analogues.

Multiple functions of G protein-coupled receptor kinases

Desensitization is a physiological feedback mechanism that blocks detrimental effects of persistent stimulation. G protein-coupled receptor kinase 2 (GRK2) was originally identified as the kinase that mediates G protein-coupled receptor (GPCR) desensitization. Subsequent studies revealed that GRK is a family composed of seven isoforms (GRK1–GRK7). Each GRK shows a differential expression pattern. GRK1, GRK4, and GRK7 are expressed in limited tissues. In contrast, GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed throughout the body. The roles of GRKs in GPCR desensitization are well established. When GPCRs are activated by their agonists, GRKs phosphorylate serine/threonine residues in the intracellular loops and the carboxyl-termini of GPCRs. Phosphorylation promotes translocation of β-arrestins to the receptors and inhibits further G protein activation by interrupting receptor-G protein coupling. The binding of β-arrestins to the receptors also helps to promote receptor internalization by clathrin-coated pits. Thus, the GRK-catalyzed phosphorylation and subsequent binding of β-arrestin to GPCRs are believed to be the common mechanism of GPCR desensitization and internalization. Recent studies have revealed that GRKs are also involved in the β-arrestin-mediated signaling pathway. The GRK-mediated phosphorylation of the receptors plays opposite roles in conventional G protein- and β-arrestin-mediated signaling. The GRK-catalyzed phosphorylation of the receptors results in decreased G protein-mediated signaling, but it is necessary for β-arrestin-mediated signaling. Agonists that selectively activate GRK/β-arrestin-dependent signaling without affecting G protein signaling are known as β-arrestin-biased agonists. Biased agonists are expected to have potential therapeutic benefits for various diseases due to their selective activation of favorable physiological responses or avoidance of the side effects of drugs. Furthermore, GRKs are recognized as signaling mediators that are independent of either G protein- or β-arrestin-mediated pathways. GRKs can phosphorylate non-GPCR substrates, and this is found to be involved in various physiological responses, such as cell motility, development, and inflammation. In addition to these effects, our group revealed that GRK6 expressed in macrophages mediates the removal of apoptotic cells (engulfment) in a kinase activity-dependent manner. These studies revealed that GRKs block excess stimulus and also induce cellular responses. Here, we summarized the involvement of GRKs in β-arrestin-mediated and G protein-independent signaling pathways.

Abstract 18109: β-Arrestins Regulate Collagen Synthesis in Human Cardiac Fibroblasts via GPCR-Dependent and Independent Signaling

End-stage heart failure (HF) is characterized by high levels of circulating catecholamines to increase cardiac output and uncoupling of cardiac β-adrenergic receptors (β-ARs). β-arrestins are scaffold signaling molecules that also play a key role in G protein-coupled receptor (GPCR) desensitization, but also have other G protein independent functions including activation of MAPK signaling. This study investigates a potential novel role of β-arrestins in regulating cardiac fibroblasts (CF) biology and myocardial fibrosis which can lead to HF. Both isoforms of β-arrestins expressed in the heart, β-arrestin1 and 2 are upregulated in failing CF versus normal controls. This is a primary mechanism for impaired β-AR signaling as basal and β-agonist-stimulated cAMP production is much lower in the failing CF and there is loss of the β-agonist-mediated inhibition of collagen synthesis in the failing cells. siRNA-mediated knockdown of β-arrestins in failing CF led to decreased α-SMA and fibronectin expression indicative of a reversal from the activated myofibroblast phenotype to the normal quiescent state. β-arrestin knockdown also restored CF β-AR signaling as measured by β-agonist-stimulated cAMP production as well as β-agonist-stimulated inhibition of collagen synthesis. Overexpression of either β-arrestin1 or 2 in normal control CF uncoupled β-AR signaling and blunted the inhibition of collagen synthesis by β-agonist stimulation. To investigate potential non-GPCR effects of β-arrestins in CF, cells were treated with TGF-β. Knockdown of β-arrestin1 or 2 in failing CF significantly inhibited TGF-β-stimulated collagen synthesis and expression of collagen I and III were decreased under both basal conditions and with TGF-β. Pre-treatment of these CF with the pharmacological ERK inhibitor, UO126, abolished Smad phosphorylation in the setting of β-arrestin overexpression and inhibited TGF-β-stimulated collagen synthesis. Both of these isoforms of β-arrestin play a significant role in CF transformation and regulating collagen synthesis via GPCR-dependent and independent signaling pathways. Targeted inhibition of β-arrestins in CF may represent a novel therapeutic strategy to prevent pathological myocardial fibrosis and progression to HF.

Overexpression of βarrestin-2 in prostate cancer cells from African American males may contribute to aggressive tumor growth (P2075)

Abstract Prostate cancer is the second leading cause of death in African American males. Its incidence is ~60% higher in African American males relative to Caucasian American males. More aggressive prostate cancer is attributed to cancer that is androgen independent resulting in lack of response to androgen replacement therapy. βarrestins are scaffold proteins that promote G protein coupled receptor desensitization and internalization. βarrestin-2 was recently shown to reduce Androgen Receptor expression in prostate cancer cells. The goal of this study was to determine the levels of expression of βarrestin-1/2 in prostate cancer cells from both racial groups and assess their roles in tumor aggressiveness. To that end, prostate cancer cell lines from African Americans (MDAPCa2B, E006AA), Caucasian Americans (LNCaP, PC3), and control (RWPE-1) were assayed for βarrestin-1/2 expression and cell proliferation. βarrestin-1 expression was elevated in all prostate cancer cell lines whereas βarrestin-2 overexpression was observed only in MDAPCa2B. Interestingly, MDAPCa2B showed greater cell proliferation relative to LNCaP and PC3. Suppression of βarrestin-2 in MDAPCa2B cells reduced cell proliferation and tumor growth in nude mice. Altogether the data indicate that 1) βarrestins modulate prostate cancer growth, and 2) overexpression of βarrestin-2 in prostate cancer cells from African Americans relative to Caucasian Americans may contribute to the prostate cancer disparity.

β‐arrestin2 expression is localized in cholinergic but not nitrergic motor neurons in the mouse longitudinal musclemyenteric plexus (LMMP)

Morphine inhibits intestinal peristalsis via its action on cholinergic enteric neurons. Tolerance to morphine does not develop in the colon, except upon deletion of β‐arrestin2 (β‐arr2), a key molecule involved in G protein–coupled receptor desensitization (Maguma et al., 2012). However, the specific localization of β‐arr2 in the myenteric plexus is not known. In this study we utilized dual‐labeling immunofluorescence to characterize myenteric neurons expressing β‐arr2 and investigated the impact of β‐arr2 abrogation on cholinergic function. Our results reveal robust expression of β‐arr2 in the soma of select neurons that stained positive for choline acetyltransferase and calretinin, a motor neuron marker. β‐arr2 was neither co‐localized with substance P, nitric oxide synthase nor calbindin. The density of β‐arr2 neurons was higher in the ileum (10.8%) vs. colon (4.3%). Deletion of β‐arr2 did not affect cholinergic neuron activation by nicotine in the isolated ileum (− log EC50: wild type = 5.8 vs β‐arr2 knockout = 5.9). Our findings suggest specificity in the localization of β‐arr2 within cholinergic motor neurons of the myenteric plexus. β‐arr2 deletion does not directly alter cholinergic neuronal activation. Studying β‐arr2 signaling in the cholinergic motor neurons would be critical to assess mechanisms involved in reversal of opioid effect in the colon. Support: NIH grant DA024009

Adrenergic Receptor Gene Variation and Selective Norepinephrine Reuptake Inhibitors

Adrenergic Receptor Gene Variation and Selective Norepinephrine Reuptake Inhibitors

β-Arrestin-1 directly interacts with Gαs and regulates its function

β-Arrestins function to mediate G protein-coupled receptor (GPCR) desensitization and internalization and to initiate G protein independent signaling of GPCRs. Elucidating how β-arrestin and G protein signal pathways coordinate with each other is important to fully understand GPCR signaling. Here we report that β-arrestin-1 directly interacts with Gαs. Purified β-arrestin-1 binds to Gαs in a rapid association and dissociation manner. β-Arrestin-1 promotes the binding and the release of GTPγS from Gαs in vitro. β-Arrestin-1 L33K mutant shows reduced interaction with Gαs and has no detectable effects on Gαs function. Our study thus reveals a direct crosstalk of β-arrestin-1 with Gαs. Structured summary of protein interactionsGαqphysically interacts with Beta-arrestin-1 by cross-linking study (View interaction).Gαqphysically interacts with Beta-arrestin-1 by anti tag coimmunoprecipitation (View interaction).Gαsbinds to Beta-arrestin-1 by biophysical (View Interaction: 1, 2).Gαsphysically interacts with Beta-arrestin-1 by anti tag coimmunoprecipitation (View interaction).Beta-arrestin-1physically interacts with Gαs by anti bait coimmunoprecipitation (View interaction).Gαsphysically interacts with Beta-arrestin-1 by cross-linking study (View interaction).

Expression of β-arrestin 1 in Gastric Cardiac Adenocarcinoma and its Relation with Progression

Arrestins act as mediators of G protein-coupled receptor (GPCR) desensitization and trafficking, also actin as a scaffold for many intracellular signaling network. The role that β-arrestin 1 plays in gastric cardiac adenocarcinoma (GCA) and its clinicopathologic significance are untouched. Fifty patients with gastric cardiac adenocarcinoma were retrospectively enrolled and β-arrestin 1 was detected using immunohistochemistry in tissue samples. Nuclear expression of β-arrestin 1 was observed in 78% of GCA samples (39/50) and cytoplasmic expression in 70% (35/50). β-arrestin 1 could be found in both nucleus and cytoplasm of 54% GCA (27/50) or in either of them in 94% (47/50). β-arrestin 1 protein positivity in well/ moderately differentiated carcinomas was significantly higher than that in poorly differentiated carcinomas (P=0.005). We found increased expression of β-arrestin 1 in cytoplasm was correlated with lymph nodal metastasis (P=0.002) and pathological lymph nodal staging (P=0.030). We also found β-arrestin 1 to be over-expressed in glandular epithelia cells of mucinous adenocarcinoma, a tumour type associated with an adverse outcome of gastric cardiac adenocarcinoma (P=0.022). β-arrestin 1 is over-expressed in the nucleus and/or cytoplasm of gastric cardiac adenocarcinoma. However, β-arrestin 1 has no relationship with the prognosis of gastric cardiac adenocarcinoma (P>0.05). Our data imply that β-arrestin 1 in cytoplasm may be involved in differentiation and metastasis of gastric cardiac adenocarcinoma.

Application of BRET technology in interaction of G protein-coupled receptor with β-arrestins: recent progress

G protein-coupled receptors constitute an important super family of cell surface proteins.β-arrestin 1and 2 are widely expressed intracellular adaptor and scaffolding proteins playing important roles in G protein-coupled receptor desensitization,internalization,intracellular trafficking,and G protein-independent signaling.Recent development in bioluminescence resonance energy transfer(BRET)technology makes it possible to monitor the GPCR-β-arrestin complexes in live cells in a real-time manner;and investigators can further elucidate the ever-expanding roles ofβ-arrestins in mediating GPCR function,which will help to develop the new generation of drugs targeting GPCRs.