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Sample records for arrestin content studied

  1. Different conformational dynamics of β-arrestin1 and β-arrestin2 analyzed by hydrogen/deuterium exchange mass spectrometry

    SciTech Connect

    Yun, Youngjoo; Kim, Dong Kyun; Seo, Min-Duk; Kim, Kyeong-Man; Chung, Ka Young

    2015-01-30

    Highlights: • The conformational dynamics of β-arrestin1 or β-arrestin2 were analyzed by HDX-MS. • β-Strands II through IV were more dynamic in β-arrestin2 than in β-arrestin1. • The middle loop was less dynamic in β-arrestin2 than in β-arrestin1. • Upon pre-activation by the R169E mutation, β-arrestins became more dynamic. • Pre-activation affected a wider region of β-arrestin1 compared to β-arrestin2. - Abstract: Arrestins have important roles in G protein-coupled receptor (GPCR) signaling including desensitization of GPCRs and G protein-independent signaling. There have been four arrestins identified: arrestin1, arrestin2 (e.g. β-arrestin1), arrestin3 (e.g. β-arrestin2), and arrestin4. β-Arrestin1 and β-arrestin2 are ubiquitously expressed and regulate a broad range of GPCRs, while arrestin1 and arrestin4 are expressed in the visual system. Although the functions of β-arrestin1 and β-arrestin2 widely overlap, β-arrestin2 has broader receptor selectivity, and a few studies have suggested that β-arrestin1 and β-arrestin2 have distinct cellular functions. Here, we compared the conformational dynamics of β-arrestin1 and β-arrestin2 by hydrogen/deuterium exchange mass spectrometry (HDX-MS). We also used the R169E mutant as a pre-activation model system. HDX-MS data revealed that β-strands II through IV were more dynamic in β-arrestin2 in the basal state, while the middle loop was more dynamic in β-arrestin1. With pre-activation, both β-arrestin1 and β-arrestin2 became more flexible, but broader regions of β-arrestin1 became flexible compared to β-arrestin2. The conformational differences between β-arrestin1 and β-arrestin2 in both the basal and pre-activated states might determine their different receptor selectivities and different cellular functions.

  2. Structure-activity relationship study of angiotensin II analogs in terms of β-arrestin-dependent signaling to aldosterone production.

    PubMed

    Valero, Thairy Reyes; Sturchler, Emmanuel; Jafferjee, Malika; Rengo, Giuseppe; Magafa, Vassiliki; Cordopatis, Paul; McDonald, Patricia; Koch, Walter J; Lymperopoulos, Anastasios

    2016-04-01

    The known angiotensin II (AngII) physiological effect of aldosterone synthesis and secretion induction, a steroid hormone that contributes to the pathology of postmyocardial infarction (MI) heart failure (HF), is mediated by both Gq/11 proteins and β-arrestins, both of which couple to the AngII type 1 receptors (AT1Rs) of adrenocortical zona glomerulosa (AZG) cells. Over the past several years, AngII analogs with increased selectivity ("bias") toward β-arrestin-dependent signaling at the AT1R have been designed and described, starting with SII, the gold-standard β-arrestin-"biased" AngII analog. In this study, we examined the relative potencies of an extensive series of AngII peptide analogs at relative activation of G proteins versus β-arrestins by the AT1R. The major structural difference of these peptides from SII was their varied substitutions at position 5, rather than position 4 of native AngII. Three of them were found biased for β-arrestin activation and extremely potent at stimulating aldosterone secretion in AZG cells in vitro, much more potent than SII in that regard. Finally, the most potent of these three ([Sar(1), Cys(Et)(5), Leu(8)]-AngII, CORET) was further examined in post-MI rats progressing to HF and overexpressing adrenal β-arrestin1 in vivo. Consistent with the in vitro studies, CORET was found to exacerbate the post-MI hyperaldosteronism, and, consequently, cardiac function of the post-MI animals in vivo. Finally, our data suggest that increasing the size of position 5 of the AngII peptide sequence results in directly proportional increases in AT1R-dependent β-arrestin activation. These findings provide important insights for AT1R pharmacology and future AngII-targeted drug development. PMID:27069636

  3. β-arrestins in the Immune System

    PubMed Central

    Xie, Ting; Liang, Jiurong

    2015-01-01

    Summary β-arrestins regulate G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) through receptor desensitization while also acting as signaling scaffolds to facilitate numerous effector pathways. Recent studies have provided evidence that β-arrestins play a key role in inflammatory responses. We here summarize these advances on the roles of β-arrestins in immune regulation and inflammatory responses under physiological and pathological conditions, with an emphasis on translational implications of β-arrestins on human diseases. PMID:23764061

  4. GPCR Signaling: β-arrestins Kiss and Remember.

    PubMed

    Ranjan, Ravi; Gupta, Pragya; Shukla, Arun K

    2016-04-01

    β-arrestin-dependent activation of the ERK MAP kinase downstream of GPCRs typically originates from internalized receptor-β-arrestin-ERK complexes. A new study now reports that β-arrestin 2 is sufficiently 'primed' after 'kissing' the β1-adrenergic receptor to initiate ERK activation even in the absence of formation of the receptor-β-arrestin-ERK complex and receptor internalization. PMID:27046816

  5. Protocol to Study β-Arrestin Recruitment by CB1 and CB2 Cannabinoid Receptors.

    PubMed

    Soethoudt, Marjolein; van Gils, Noortje; van der Stelt, Mario; Heitman, Laura H

    2016-01-01

    Cannabinoid CB1 and CB2 receptors are G-protein-coupled receptors (GPCRs) that recruit β-arrestins upon activation by (partial) agonists. β-Arrestin recruitment is induced by phosphorylation of their C-terminal tails, and is associated with the termination of GPCR signaling; yet, it may also activate cellular signaling pathways independent of G-proteins. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB1 and CB2 receptors, by using the PathHunter(®) assay. The latter is a cellular assay that can be performed in plates with 384-wells. The PathHunter(®) assay makes use of β-galactosidase complementation, and has a chemiluminescent readout. We used this assay to characterize a set of reference ligands (both agonists and antagonists) on human CB1 and CB2 receptors. PMID:27245896

  6. β-Arrestins promote podocyte injury by inhibition of autophagy in diabetic nephropathy

    PubMed Central

    Liu, J; Li, Q X; Wang, X J; Zhang, C; Duan, Y Q; Wang, Z Y; Zhang, Y; Yu, X; Li, N J; Sun, J P; Yi, F

    2016-01-01

    β-Arrestins are multifunctional proteins originally identified as negative adaptors of G protein-coupled receptors (GPCRs). Emerging evidence has also indicated that β-arrestins can activate signaling pathways independent of GPCR activation. This study was to elucidate the role of β-arrestins in diabetic nephropathy (DN) and hypothesized that β-arrestins contribute to diabetic renal injury by mediating podocyte autophagic process. We first found that both β-arrestin-1 and β-arrestin-2 were upregulated in the kidney from streptozotocin-induced diabetic mice, diabetic db/db mice and kidney biopsies from diabetic patients. We further revealed that either β-arrestin-1 or β-arrestin-2 deficiency (Arrb1−/− or Arrb2−/−) ameliorated renal injury in diabetic mice. In vitro, we observed that podocytes increased both β-arrestin-1 and β-arrestin-2 expression levels under hyperglycemia condition and further demonstrated that β-arrestin-1 and β-arrestin-2 shared common mechanisms to suppress podocyte autophagy by negative regulation of ATG12–ATG5 conjugation. Collectively, this study for the first time demonstrates that β-arrestin-1 and β-arrestin-2 mediate podocyte autophagic activity, indicating that β-arrestins are critical components of signal transduction pathways that link renal injury to reduce autophagy in DN. Modulation of these pathways may be an innovative therapeutic strategy for treating patients with DN. PMID:27054338

  7. Arrestin2/Clathrin Interaction is Regulated by Key N- and C-terminal Regions in Arrestin2+

    PubMed Central

    Kern, Ronald C.; Kang, Dong Soo; Benovic, Jeffrey L.

    2009-01-01

    The interaction of non-visual arrestins with clathrin is an important step in mediating the endocytosis of cell surface receptors. Previous studies have shown that mutation of the clathrin-binding box in arrestin leads to severe defects in arrestin mediated trafficking. However, little is known about how arrestin/clathrin interaction is regulated. Here we show that both the N- and C-terminal regions of arrestin2 function to inhibit basal interaction with clathrin. Truncation analysis revealed that clathrin binding increases as the C-tail of arrestin2 is shortened while site-directed mutagenesis identified Glu-404, Glu-405, and Glu-406 as being primarily responsible for this inhibition. Mutagenesis also identified Lys-4, Arg-7, Lys-10, and Lys-11 within the N-terminus as playing a key role regulating clathrin binding. Based on similarities with visual arrestin, Lys-10 and Lys-11 likely function as phospho-sensors in arrestin2 to initially discriminate the phosphorylation status of target receptors. Analysis of the arrestin2 structure reveals that Arg-7, Lys-10 and Lys-11 are in close proximity to Glu-389 and Asp-390, suggesting that these residues may form intramolecular interactions. In fact, simultaneous mutation of Glu-389 and Asp-390 also leads to enhanced clathrin binding. These results reveal that multiple intramolecular interactions coordinately regulate arrestin2 interaction with clathrin, highlighting this interaction as a critical step in regulating receptor trafficking. PMID:19555118

  8. The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system.

    PubMed

    Robinson, Kelly A; Ou, Wei-Lin; Guan, Xinyu; Sugamori, Kim S; Bandyopadhyay, Abhishek; Ernst, Oliver P; Mitchell, Jane

    2015-12-01

    Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to Gq and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G protein-coupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (Kd 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3- to 5-fold (Kd 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system. Invertebrate visual systems are inactivated by arrestin binding to metarhodopsin that does not require receptor phosphorylation. Here we show that squid rhodopsin kinase phosphorylates arrestin on two serines (S392,S397) in the C-terminus and phosphorylation decreases the affinity of arrestin for squid metarhodopsin. Metarhodopsin phosphorylation has very little effect on arrestin binding but does increase arrestin dissociation. PMID:26375013

  9. Physiological and Pharmacological Implications of Beta-Arrestin Regulation

    PubMed Central

    Schmid, Cullen L.; Bohn, Laura M.

    2008-01-01

    G protein-coupled receptor-targeted drug discovery as well as “compound reassessment” requires the utilization of diverse screens to determine agonist efficacies and potencies beyond the scope of ligand binding and G protein coupling. Such efforts have arisen from extensive studies, both in cellular and animal models, demonstrating that these seven transmembrane domain-spanning, G protein-coupled receptors may engage in more diverse functions than their name suggests and particular focus is drawn to their interactions with beta-arrestinsarrestins). As regulators, βarrestins are involved in dampening G protein-coupling pathways. βArrestins can also play pro-signaling roles in receptor mediated events and the coupling of receptors to βarrestins may be as important as their potential to couple to G proteins in the physiological setting. In the last decade, the development of βarrestin deficient mouse models has allowed for the assessment of the contribution of individual βarrestins to receptor function in vivo. This review will discuss the current literature that implicates βarrestins in receptor function in respect to physiological and behavioral responses observed in the live animal model. PMID:19100766

  10. FUNCTION OF NON-VISUAL ARRESTINS IN SIGNALING AND ENDOCYTOSIS OF THE GASTRIN-RELEASING PEPTIDE RECEPTOR (GRP RECEPTOR)

    PubMed Central

    Schumann, Michael; Nakagawa, Tomoo; Mantey, Samuel A.; Howell, Brian; Jensen, Robert T.

    2008-01-01

    Little is known about the role of arrestins in gastrointestinal hormone/neurotransmitter receptor endocytosis. With other G protein-coupled receptors, arrestins induce G protein-uncoupling and receptor endocytosis. In this study, we used arrestin wild-type and dominant-negative mutant constructs to analyze the arrestin dependence of endocytosis and desensitization of the gastrin- releasing peptide receptor (GRP-R). Co-expression of the GRP-R with wild-type arrestin-2 and arrestin-3 increased not only GRP-R endocytosis but also GRP-R desensitization in arrestin-overexpressing cells. Co-expression of the dominant-negative mutants V53D-arrestin-2 or V54D-arrestin-3 reduced GRP-R endocytosis. Notably, different trafficking routes for agonist-activated GRPR-arrestin-2 and GRPR-arrestin-3 complexes were found. Arrestin-3 internalizes with GRP-R to intracellular vesicles, arrestin-2 splits from the GRP-R and localizes to the cell membrane. Also, the recycling pathway of the GRP-R was different if co-expressed with arrestin-2 or arrestin-3. Using different GRP-R mutants, the C-terminus and the 2nd intracellular loop of the GRP-R were found to be important for the GRPR-arrestin interaction and for the difference in GRP receptor trafficking with the two arrestin subtypes. Our results show that both non-visual arrestins play an important role in GRP-R internalization and desensitization. PMID:18199425

  11. CO2 Pneumoperitoneum Preserves β-Arrestin 2 Content and Reduces High Mobility Group Box-1 (HMGB-1) Expression in an Animal Model of Peritonitis

    PubMed Central

    Montalto, Angela Simona; Minutoli, Letteria; Impellizzeri, Pietro; Costa, Gaetano; Squadrito, Francesco

    2015-01-01

    Laparoscopy (LS) has been shown to decrease the inflammatory sequelae of endotoxemia. β-arrestin 2 plays an important function in signal transduction pathway of TLR4. High mobility group box-1 (HMGB-1) is involved in the delayed systemic inflammatory response. We investigated the effects of CO2 insufflation on liver, lung, and kidney expression of both β-arrestin 2 and HMGB-1 during sepsis. Cecal ligation and puncture (CLP) was performed in male rats and 6 h later the animals were randomly assigned to receive a CO2 pneumoperitoneum or laparotomy. Animals were euthanized; liver, lung, and kidney were removed for the evaluation of β-arrestin 2 and HMGB-1 expression. Immunohistochemical detection of myeloperoxidase (MPO) was investigated in lung and liver and bacterial load was determined in the peritoneal fluid. CO2 pneumoperitoneum reduced peritoneal bacterial load, increased the expression of β-arrestin 2, and blunted the expression of the potent proinflammatory HMGB-1 in liver, lung, and kidney compared with laparotomy. Liver and lung MPO was markedly reduced in rats subjected to LS compared with laparotomy. We believe that CO2 exerts an early protective effect by reducing bacterial load and likely toll-like receptor activation which in turn leads to a preserved β-arrestin 2 expression and a reduced HMGB-1 expression. PMID:25810808

  12. The functional cycle of visual arrestins in photoreceptor cells

    PubMed Central

    Gurevich, Vsevolod V.; Hanson, Susan M.; Song, Xiufeng; Vishnivetskiy, Sergey A.; Gurevich, Eugenia V.

    2011-01-01

    Visual arrestin-1 plays a key role in the rapid and reproducible shutoff of rhodopsin signaling. Its highly selective binding to light-activated phosphorylated rhodopsin is an integral part of the functional perfection of rod photoreceptors. Structure-function studies revealed key elements of the sophisticated molecular mechanism ensuring arrestin-1 selectivity and paved the way to the targeted manipulation of the arrestin-1 molecule to design mutants that can compensate for congenital defects in rhodopsin phosphorylation. Arrestin-1 self-association and light-dependent translocation in photoreceptor cells work together to keep a constant supply of active rhodopsin-binding arrestin-1 monomer in the outer segment. Recent discoveries of arrestin-1 interaction with other signaling proteins suggest that it is a much more versatile signaling regulator than previously thought, affecting the function of the synaptic terminals and rod survival. Elucidation of the fine molecular mechanisms of arrestin-1 interactions with rhodopsin and other binding partners is necessary for the comprehensive understanding of rod function and for devising novel molecular tools and therapeutic approaches to the treatment of visual disorders. PMID:21824527

  13. Multifaceted role of β-arrestins in inflammation and disease

    PubMed Central

    Sharma, Deepika; Parameswaran, Narayanan

    2015-01-01

    Arrestins are intracellular scaffolding proteins known to regulate a range of biochemical processes including G-protein coupled receptor (GPCR) desensitization, signal attenuation, receptor turnover and downstream signaling cascades. Their roles in regulation of signaling network have lately been extended to receptors outside of the GPCR family, demonstrating their roles as important scaffolding proteins in various physiological processes including proliferation, differentiation, and apoptosis. Recent studies have demonstrated a critical role for arrestins in immunological processes including key functions in inflammatory signaling pathways. In this review, we provide a comprehensive analysis of the different functions of the arrestin family of proteins especially related to immunity and inflammatory diseases. PMID:26378652

  14. Light-Dependent Translocation of Arrestin in Rod Photoreceptors is Signaled Through a Phospholipase C Cascade and Requires ATP

    PubMed Central

    Orisme, Wilda; Li, Jian; Goldmann, Tobias; Bolch, Susan; Wolfrum, Uwe; Smith, W. Clay

    2009-01-01

    Partitioning of cellular components is a critical mechanism by which cells can regulate their activity. In rod photoreceptors, light induces a large-scale translocation of arrestin from the inner segments to the outer segments. The purpose of this project is to elucidate the signaling pathway necessary to initiate arrestin translocation to the outer segments and the mechanism for arrestin translocation. Mouse retinal organotypic cultures and eyes from transgenic Xenopus tadpoles expressing a fusion of GFP and rod arrestin were treated with both activators and inhibitors of proteins in the phosphoinositide pathway. Confocal microscopy was used to image the effects of the pharmacological agents on arrestin translocation in rod photoreceptors. Retinas were also depleted of ATP using potassium cyanide to assess the requirement for ATP in arrestin translocation. In this study, we demonstrate that components of the G-protein-linked phospholipase C (PLC) pathway play a role in initiating arrestin translocation. Our results show that arrestin translocation can be stimulated by activators of PLC and protein kinase C (PKC), and by cholera toxin in the absence of light. Arrestin translocation to the outer segments is significantly reduced by inhibitors of PLC and PKC. Importantly, we find that treatment with potassium cyanide inhibits arrestin translocation in response to light. Collectively, our results suggest that arrestin translocation is initiated by a G-protein-coupled cascade through PLC and PKC signaling. Furthermore, our results demonstrate that at least the initiation of arrestin translocation requires energy input. PMID:19887106

  15. Structural Determinants of Arrestin Functions

    PubMed Central

    Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2015-01-01

    Arrestins are a small protein family with only four members in mammals. Arrestins demonstrate an amazing versatility, interacting with hundreds of different G protein-coupled receptor (GPCR) subtypes, numerous nonreceptor signaling proteins, and components of the internalization machinery, as well as cytoskeletal elements, including regular microtubules and centrosomes. Here, we focus on the structural determinants that mediate various arrestin functions. The receptor-binding elements in arrestins were mapped fairly comprehensively, which set the stage for the construction of mutants targeting particular GPCRs. The elements engaged by other binding partners are only now being elucidated and in most cases we have more questions than answers. Interestingly, even very limited and imprecise identification of structural requirements for the interaction with very few other proteins has enabled the development of signaling-biased arrestin mutants. More comprehensive understanding of the structural underpinning of different arrestin functions will pave the way for the construction of arrestins that can link the receptor we want to the signaling pathway of our choosing. PMID:23764050

  16. Association study of the β-arrestin 2 gene (ARRB2) with opioid and cocaine dependence in a European American population

    PubMed Central

    Ambrose-Lanci, Lisa M.; Vaswani, Meera; Clarke, Toni-Kim; Zeng, Angela; Lohoff, Falk W.; Ferraro, Thomas N.; Berrettini, Wade H.

    2014-01-01

    The rewarding properties of drugs of abuse are mediated by the Mu-Opioid Receptor (MOR). Genetic variation in MOR and MOR interacting proteins (MORIPs) involved in MOR signaling may increase risk for drug dependence. The MORIP, B-arrestin, plays an important role in the regulation of MOR trafficking thereby highlighting it as a candidate gene for addiction phenotypes. In this case-control association study, DNA samples from cocaine (n=336) and opioid-dependent (n=335) patients and controls (n=656) were genotyped for 7 single nucleotide polymorphisms (SNPs) (rs11868227, rs3786047, rs4522461, rs1045280, rs2271167, rs2036657, and rs4790694) across ARRB2, the gene encoding the B-arrestin 2 protein. No significant differences were observed in genotype or allele frequency between drug dependent and control individuals for any of the SNPs analyzed. Haplotype analysis was similarly negative. Further studies are needed to determine whether variation in ARRB2 (or other MORIPs) are relevant to cocaine or opioid dependence in different ethnic populations or if they confer risk that is specific to dependence on other drugs of abuse. PMID:22472784

  17. β-Arrestin-1 deficiency protects mice from experimental colitis.

    PubMed

    Lee, Taehyung; Lee, Eunhee; Irwin, Regina; Lucas, Peter C; McCabe, Laura R; Parameswaran, Narayanan

    2013-04-01

    β-Arrestins are intracellular scaffolding proteins that modulate specific cell signaling pathways. Recent studies, in both cell culture and in vivo models, have demonstrated an important role for β-arrestin-1 in inflammation. However, the role of β-arrestin-1 in the pathogenesis of inflammatory bowel disease (IBD) is not known. Our goal was to investigate the role of β-arrestin-1 in IBD using mouse models of colitis. To this end, we subjected wild-type (WT) and β-arrestin-1 knockout (β-arr-1(-/-)) mice to colitis induced by trinitrobenzenesulfonic acid or dextran sulfate sodium and examined the clinical signs, gross pathology, and histopathology of the colon, as well as inflammatory components. The β-arr-1(-/-) mice displayed significantly attenuated colitis, compared with WT mice, in both models. Consistent with the phenotypic observations, histological examination of the colon revealed attenuated disease pathology in the β-arr-1(-/-) mice. Our results further demonstrate that β-arr-1(-/-) mice are deficient in IL-6 expression in the colon, but have higher expression of the anti-inflammatory IL-10 family of cytokines. Our results also demonstrate diminished ERK and NFκB pathways in the colons of β-arr-1(-/-) mice, compared with WT mice. Taken together, our results demonstrate that decreased IL-6 production and enhanced IL-10 and IL-22 production in β-arrestin-1-deficient mice likely lead to attenuated gut inflammation. PMID:23395087

  18. Structure of an Arrestin2-clathrin Complex Reveals a Novel Clathrin Binding Domain that Modulates Receptor Trafficking

    SciTech Connect

    Kang, D.; Kern, R; Puthenveedu, M; von Zastrow, M; Williams, J; Benovic, J

    2009-01-01

    Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin {beta}-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formed by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I){sub 2}GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.

  19. β-arrestin-1 contributes to brown fat function and directly interacts with PPARα and PPARγ

    PubMed Central

    Wang, Congcong; Zeng, Xianglu; Zhou, Zhaocai; Zhao, Jian; Pei, Gang

    2016-01-01

    The peroxisome proliferator-activated receptor (PPAR) family plays central roles in brown adipose tissue (BAT) adipogenesis and contributes to body temperature maintenance. The transcriptional activity of PPAR family has been shown to be tightly controlled by cellular signal networks. β-arrestins function as major secondary messengers of G protein-coupled receptors (GPCR) signaling by functional interactions with diverse proteins. Here, we report that β-arrestin-1 knock-out mice show enhanced cold tolerance. We found that β-arrestin-1 directly interacts with PPARα and PPARγ through a LXXXLXXXL motif, while D371 in PPARα and L311/N312/D380 in PPARγ are required for their interactions with β-arrestin-1. Further mechanistic studies showed that β-arrestin-1 promotes PPARα- but represses PPARγ-mediated transcriptional activities, providing potential regulatory pathway for BAT function. PMID:27301785

  20. β-arrestin-1 contributes to brown fat function and directly interacts with PPARα and PPARγ.

    PubMed

    Wang, Congcong; Zeng, Xianglu; Zhou, Zhaocai; Zhao, Jian; Pei, Gang

    2016-01-01

    The peroxisome proliferator-activated receptor (PPAR) family plays central roles in brown adipose tissue (BAT) adipogenesis and contributes to body temperature maintenance. The transcriptional activity of PPAR family has been shown to be tightly controlled by cellular signal networks. β-arrestins function as major secondary messengers of G protein-coupled receptors (GPCR) signaling by functional interactions with diverse proteins. Here, we report that β-arrestin-1 knock-out mice show enhanced cold tolerance. We found that β-arrestin-1 directly interacts with PPARα and PPARγ through a LXXXLXXXL motif, while D371 in PPARα and L311/N312/D380 in PPARγ are required for their interactions with β-arrestin-1. Further mechanistic studies showed that β-arrestin-1 promotes PPARα- but represses PPARγ-mediated transcriptional activities, providing potential regulatory pathway for BAT function. PMID:27301785

  1. Ubiquitin-Related Roles of β-Arrestins in Endocytic Trafficking and Signal Transduction.

    PubMed

    Jean-Charles, Pierre-Yves; Rajiv, Vishwaesh; Shenoy, Sudha K

    2016-10-01

    The non-visual arrestins, β-arrestin1, and β-arrestin2 were originally identified as proteins that bind to seven-transmembrane receptors (7TMRs, also called G protein-coupled receptors, GPCRs) and block heterotrimeric G protein activation, thus leading to desensitization of transmembrane signaling. However, as subsequent discoveries have continually demonstrated, their functionality is not constrained to desensitization. They are now recognized for their critical roles in mediating intracellular trafficking of 7TMRs, growth factor receptors, ion transporters, ion channels, nuclear receptors, and non-receptor proteins. Additionally, they function as crucial mediators of ubiquitination of 7TMRs as well as other receptors and non-receptor proteins. Recently, emerging studies suggest that a class of proteins with predicted structural features of β-arrestins regulate substrate ubiquitination in yeast and higher mammals, lending support to the idea that the adaptor role of β-arrestins in protein ubiquitination is evolutionarily conserved. β-arrestins also function as scaffolds for kinases and transduce signals from 7TMRs through pathways that do not require G protein activation. Remarkably, the endocytic and scaffolding functions of β-arrestin are intertwined with its ubiquitination status; the dynamic and site specific ubiquitination on β-arrestin plays a critical role in stabilizing β-arrestin-7TMR association and the formation of signalosomes. This review summarizes the current findings on ubiquitin-dependent regulation of 7TMRs as well as β-arrestins and the potential role of reversible ubiquitination as a "biological switch" in signal transduction. J. Cell. Physiol. 231: 2071-2080, 2016. © 2016 Wiley Periodicals, Inc. PMID:26790995

  2. β-Arrestin1 promotes the progression of chronic myeloid leukaemia by regulating BCR/ABL H4 acetylation

    PubMed Central

    Qin, R; Li, K; Qi, X; Zhou, X; Wang, L; Zhang, P; Zou, L

    2014-01-01

    Background: β-Arrestins are scaffold proteins that interact with various cellular signals. Although β-arrestin2 mediates the initiation and progression of myeloid leukaemia, the critical role of β-arrestin1 in the chronic myeloid leukaemia (CML) is still unknown. The aim of this study is to investigate the essential function of β-arrestin1 in CML. Methods: The expressions of β-arrestin1 and BCR/ABL in CML patients, animal models and K562 cells were measured by RT–PCR, immunofluorescence and western blotting. The effect of β-arrestin1 on CML animal models and K562 cells by colony formation, MTT and survival analysis were assessed. BCR/ABL H4 acetylation was analysed through the use of Chromatin-immunoprecipitation (ChIP) -on-chip and confirmed by ChIP respectively. Co-immunoprecipitation and confocal were examined for the binding of β-arrestin1 with enhancer of zeste homologue 2 (EZH2). Results: The higher expression of β-arrestin1 is positively correlated with clinical phases of CML patients. Depletion of β-arrestin1 decelerates progression of K562 and primary cells, and increases survival of CML mice. Importantly, silenced β-arrestin1 results in the decrease of BCR/ABL H4 acetylation level in K562 cells. Further data illustrate that nuclear β-arrestin1 binds to EZH2 to mediate BCR/ABL acetylation and thus regulates cell progression in K562 cells and the survival of CML mice. Conclusions: Our findings reveal a novel function of β-arrestin1 binding to EZH2 to promote CML progression by regulating BCR/ABL H4 acetylation. PMID:24937675

  3. Mechanisms of Biased β-Arrestin-Mediated Signaling Downstream from the Cannabinoid 1 Receptor

    PubMed Central

    Delgado-Peraza, Francheska; Ahn, Kwang H.; Nogueras-Ortiz, Carlos; Mungrue, Imran N.; Mackie, Ken; Kendall, Debra A.

    2016-01-01

    Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins β-arrestin 1/2. Ligands can elicit full or subsets of cellular responses, a concept defined as ligand bias or functional selectivity. However, our current understanding of β-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of β-arrestin-mediated signaling from the cannabinoid 1 receptor (CB1R). By using a signaling biased receptor, we define the cascades, specific receptor kinases, and molecular mechanism underlying β-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and β-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of β-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control β-arrestin-mediated signaling. PMID:27009233

  4. Mechanisms of Biased β-Arrestin-Mediated Signaling Downstream from the Cannabinoid 1 Receptor.

    PubMed

    Delgado-Peraza, Francheska; Ahn, Kwang H; Nogueras-Ortiz, Carlos; Mungrue, Imran N; Mackie, Ken; Kendall, Debra A; Yudowski, Guillermo A

    2016-06-01

    Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins β-arrestin 1/2. Ligands can elicit full or subsets of cellular responses, a concept defined as ligand bias or functional selectivity. However, our current understanding of β-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of β-arrestin-mediated signaling from the cannabinoid 1 receptor (CB1R). By using a signaling biased receptor, we define the cascades, specific receptor kinases, and molecular mechanism underlying β-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and β-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of β-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control β-arrestin-mediated signaling. PMID:27009233

  5. Roles of Dopamine D2 Receptor Subregions in Interactions with β-Arrestin2

    PubMed Central

    Zhang, Xiaohan; Choi, Bo-Gil; Kim, Kyeong-Man

    2016-01-01

    β-Arrestins are one of the protein families that interact with G protein-coupled receptors (GPCRs). The roles of β-arrestins are multifaceted, as they mediate different processes including receptor desensitization, endocytosis, and G protein-independent signaling. Thus, determining the GPCR regions involved in the interactions with β-arrestins would be a preliminary step in understanding the molecular mechanisms involved in the selective direction of each function. In the current study, we determined the roles of the N-terminus, intracellular loops, and C-terminal tail of a representative GPCR in the interaction with β-arrestin2. For this, we employed dopamine D2 and D3 receptors (D2R and D3R, respectively), since they display distinct agonist-induced interactions with β-arrestins. Our results showed that the second and third intracellular loops of D2R are involved in the agonist-induced translocation of β-arrestins toward plasma membranes. In contrast, the N- and C-termini of D2R exerted negative effects on the basal interaction with β-arrestins. PMID:27068263

  6. The Chemokine Receptor CCR1 Is Constitutively Active, Which Leads to G Protein-independent, β-Arrestin-mediated Internalization*

    PubMed Central

    Gilliland, C. Taylor; Salanga, Catherina L.; Kawamura, Tetsuya; Trejo, JoAnn; Handel, Tracy M.

    2013-01-01

    Activation of G protein-coupled receptors by their associated ligands has been extensively studied, and increasing structural information about the molecular mechanisms underlying ligand-dependent receptor activation is beginning to emerge with the recent expansion in GPCR crystal structures. However, some GPCRs are also able to adopt active conformations in the absence of agonist binding that result in the initiation of signal transduction and receptor down-modulation. In this report, we show that the CC-type chemokine receptor 1 (CCR1) exhibits significant constitutive activity leading to a variety of cellular responses. CCR1 expression is sufficient to induce inhibition of cAMP formation, increased F-actin content, and basal migration of human and murine leukocytes. The constitutive activity leads to basal phosphorylation of the receptor, recruitment of β-arrestin-2, and subsequent receptor internalization. CCR1 concurrently engages Gαi and β-arrestin-2 in a multiprotein complex, which may be accommodated by homo-oligomerization or receptor clustering. The data suggest the presence of two functional states for CCR1; whereas receptor coupled to Gαi functions as a canonical GPCR, albeit with high constitutive activity, the CCR1·β-arrestin-2 complex is required for G protein-independent constitutive receptor internalization. The pertussis toxin-insensitive uptake of chemokine by the receptor suggests that the CCR1·β-arrestin-2 complex may be related to a potential scavenging function of the receptor, which may be important for maintenance of chemokine gradients and receptor responsiveness in complex fields of chemokines during inflammation. PMID:24056371

  7. Beta-Arrestin1 Levels in Mononuclear Leukocytes Support Depression Scores for Women with Premenstrual Dysphoric Disorder

    PubMed Central

    Alam, Farzana; Nayyar, Sanket; Richie, William; Archibong, Anthony; Nayyar, Tultul

    2015-01-01

    Depression is very common in reproductive women particularly with premenstrual dysphoric disorder (PMDD), which is a severe form of premenstrual syndrome (PMS). Beta-arrestins were previously implicated in the pathophysiology, diagnosis and treatment for mood disorders. This study examined whether a measurement for beta-arrestin1 levels in peripheral blood mononuclear leukocytes (PBMC), could aid to distinguish between PMDD and PMS. Study participants (n = 25) were non-pregnant women between 18–42 years of age with the symptoms of PMS/PMDD, but not taking any antidepressants/therapy and at the luteal phase of menstruation. The levels of beta-arrestin1 protein in the PBMCs were determined by ELISA using human beta-arrestin1 kit. The beta-arrestin1 levels were compared with the Hamilton Depression Rating Scale scores among these women. The magnitude of the different parameters for Axis 1 mental disorders were significantly higher and beta arrestin1 protein levels in PBMCs were significantly lower in women with PMDD as compared to PMS women. The reduction in beta arrestin1 protein levels was significantly correlated with the severity of depressive symptoms. Beta-arrestin1 measurements in women may potentially serve for biochemical diagnostic purposes for PMDD and might be useful as evidence-based support for questionnaires. PMID:26703643

  8. Beta-Arrestin1 Levels in Mononuclear Leukocytes Support Depression Scores for Women with Premenstrual Dysphoric Disorder.

    PubMed

    Alam, Farzana; Nayyar, Sanket; Richie, William; Archibong, Anthony; Nayyar, Tultul

    2016-01-01

    Depression is very common in reproductive women particularly with premenstrual dysphoric disorder (PMDD), which is a severe form of premenstrual syndrome (PMS). Beta-arrestins were previously implicated in the pathophysiology, diagnosis and treatment for mood disorders. This study examined whether a measurement for beta-arrestin1 levels in peripheral blood mononuclear leukocytes (PBMC), could aid to distinguish between PMDD and PMS. Study participants (n = 25) were non-pregnant women between 18-42 years of age with the symptoms of PMS/PMDD, but not taking any antidepressants/therapy and at the luteal phase of menstruation. The levels of beta-arrestin1 protein in the PBMCs were determined by ELISA using human beta-arrestin1 kit. The beta-arrestin1 levels were compared with the Hamilton Depression Rating Scale scores among these women. The magnitude of the different parameters for Axis 1 mental disorders were significantly higher and beta arrestin1 protein levels in PBMCs were significantly lower in women with PMDD as compared to PMS women. The reduction in beta arrestin1 protein levels was significantly correlated with the severity of depressive symptoms. Beta-arrestin1 measurements in women may potentially serve for biochemical diagnostic purposes for PMDD and might be useful as evidence-based support for questionnaires. PMID:26703643

  9. Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

    PubMed Central

    Ahmed, M. Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2011-01-01

    Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmuno-precipitation of endogenous proteins from brain tissue, and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both non-visual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology. PMID:21466165

  10. β-Arrestins Negatively Regulate the Toll Pathway in Shrimp by Preventing Dorsal Translocation and Inhibiting Dorsal Transcriptional Activity.

    PubMed

    Sun, Jie-Jie; Lan, Jiang-Feng; Shi, Xiu-Zhen; Yang, Ming-Chong; Niu, Guo-Juan; Ding, Ding; Zhao, Xiao-Fan; Yu, Xiao-Qiang; Wang, Jin-Xing

    2016-04-01

    The Toll signaling pathway plays an important role in the innate immunity ofDrosophila melanogasterand mammals. The activation and termination of Toll signaling are finely regulated in these animals. Although the primary components of the Toll pathway were identified in shrimp, the functions and regulation of the pathway are seldom studied. We first demonstrated that the Toll signaling pathway plays a central role in host defense againstStaphylococcus aureusby regulating expression of antimicrobial peptides in shrimp. We then found that β-arrestins negatively regulate Toll signaling in two different ways. β-Arrestins interact with the C-terminal PEST domain of Cactus through the arrestin-N domain, and Cactus interacts with the RHD domain of Dorsal via the ankyrin repeats domain, forming a heterotrimeric complex of β-arrestin·Cactus·Dorsal, with Cactus as the bridge. This complex prevents Cactus phosphorylation and degradation, as well as Dorsal translocation into the nucleus, thus inhibiting activation of the Toll signaling pathway. β-Arrestins also interact with non-phosphorylated ERK (extracellular signal-regulated protein kinase) through the arrestin-C domain to inhibit ERK phosphorylation, which affects Dorsal translocation into the nucleus and phosphorylation of Dorsal at Ser(276)that impairs Dorsal transcriptional activity. Our study suggests that β-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity. PMID:26846853

  11. Triphenylmethane Dye Activation of Beta-Arrestin

    PubMed Central

    2013-01-01

    β-Arrestins regulate G protein-coupled receptor signaling as competitive inhibitors and protein adaptors. Low molecular weight biased ligands that bind receptors and discriminate between the G protein dependent arm and β-arrestin, clathrin-associated arm of receptor signaling are considered therapeutically valuable as a result of this distinctive pharmacological behavior. Other than receptor agonists, compounds that activate β-arrestins are not available. We show that within minutes of exposure to the cationic triphenylmethane dyes malachite green and brilliant green, tissue culture cells recruit β-arrestins to clathrin scaffolds in a receptor-activation independent manner. In the presence of these compounds, G protein signaling is inhibited, ERK and GSK3β signaling are preserved, and the recruitment of the beta2-adaptin, AP2 adaptor complex to clathrin as well as transferrin internalization is reduced. Moreover, malachite green binds β-arrestin2-GFP coated immunotrap beads relative to GFP only coated beads. Triphenylmethane dyes are FDA approved for topical use on newborns as components of triple-dye preparations and are not approved but used effectively as aqueous antibiotics in fish husbandry. As possible carcinogens, their chronic ingestion in food preparations, particularly through farmed fish, is discouraged in the U.S. and Europe. Our results indicate triphenylmethane dyes as a result of novel pharmacology may have additional roles as β-arrestin/clathrin pathway signaling modulators in both pharmacology research and clinical therapy. PMID:23865508

  12. α-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling

    PubMed Central

    O'Donnell, Allyson F.; Apffel, Alex; Gardner, Richard G.

    2010-01-01

    Extracellular signals regulate trafficking events to reorganize proteins at the plasma membrane (PM); however, few effectors of this regulation have been identified. β-Arrestins relay signaling cues to the trafficking machinery by controlling agonist-stimulated endocytosis of G-protein–coupled receptors. In contrast, we show that yeast α-arrestins, Aly1 and Aly2, control intracellular sorting of Gap1, the general amino acid permease, in response to nutrients. These studies are the first to demonstrate association of α-arrestins with clathrin and clathrin adaptor proteins (AP) and show that Aly1 and Aly2 interact directly with the γ-subunit of AP-1, Apl4. Aly2-dependent trafficking of Gap1 requires AP-1, which mediates endosome-to-Golgi transport, and the nutrient-regulated kinase, Npr1, which phosphorylates Aly2. During nitrogen starvation, Npr1 phosphorylation of Aly2 may stimulate Gap1 incorporation into AP-1/clathrin-coated vesicles to promote Gap1 trafficking from endosomes to the trans-Golgi network. Ultimately, increased Aly1-/Aly2-mediated recycling of Gap1 from endosomes results in higher Gap1 levels within cells and at the PM by diverting Gap away from trafficking pathways that lead to vacuolar degradation. This work defines a new role for arrestins in membrane trafficking and offers insight into how α-arrestins coordinate signaling events with protein trafficking. PMID:20739461

  13. Defining the roles of arrestin2 and arrestin3 in vasoconstrictor receptor desensitization in hypertension

    PubMed Central

    Nash, Craig A.; Rainbow, Richard D.; Nelson, Carl P.; Challiss, R. A. John

    2015-01-01

    Prolonged vasoconstrictor-stimulated phospholipase C activity can induce arterial constriction, hypertension, and smooth muscle hypertrophy/hyperplasia. Arrestin proteins are recruited by agonist-occupied G protein-coupled receptors to terminate signaling and counteract changes in vascular tone. Here we determine whether the development of hypertension affects arrestin expression in resistance arteries and how such changes alter arterial contractile signaling and function. Arrestin2/3 expression was increased in mesenteric arteries of 12-wk-old spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto (WKY) controls, while no differences in arrestin expression were observed between 6-wk-old SHR and WKY animals. In mesenteric artery myography experiments, high extracellular K+-stimulated contractions were increased in both 6- and 12-wk-old SHR animals. Concentration-response experiments for uridine 5′-triphosphate (UTP) acting through P2Y receptors displayed a leftward shift in 12-wk, but not 6-wk-old animals. Desensitization of UTP-stimulated vessel contractions was increased in 12-wk-old (but not 6-wk-old) SHR animals. Dual IP3/Ca2+ imaging in mesenteric arterial cells showed that desensitization of UTP and endothelin-1 (ET1) responses was enhanced in 12-wk-old (but not 6-wk-old) SHR compared with WKY rats. siRNA-mediated depletion of arrestin2 for UTP and arrestin3 for ET1, reversed the desensitization of PLC signaling. In conclusion, arrestin2 and 3 expression is elevated in resistance arteries during the emergence of the early hypertensive phenotype, which underlies an enhanced ability to desensitize vasoconstrictor signaling and vessel contraction. Such regulatory changes may act to compensate for increased vasoconstrictor-induced vessel contraction. PMID:25972452

  14. Arrestin 1 and Cone Arrestin 4 Have Unique Roles in Visual Function in an All-Cone Mouse Retina

    PubMed Central

    Deming, Janise D.; Pak, Joseph S.; Shin, Jung-a; Brown, Bruce M.; Kim, Moon K.; Aung, Moe H.; Lee, Eun-Jin; Pardue, Machelle T.; Craft, Cheryl Mae

    2015-01-01

    Purpose Previous studies discovered cone phototransduction shutoff occurs normally for Arr1−/− and Arr4−/−; however, it is defective when both visual arrestins are simultaneously not expressed (Arr1−/−Arr4−/−). We investigated the roles of visual arrestins in an all-cone retina (Nrl−/−) since each arrestin has differential effects on visual function, including ARR1 for normal light adaptation, and ARR4 for normal contrast sensitivity and visual acuity. Methods We examined Nrl−/−, Nrl−/−Arr1−/−, Nrl−/−Arr4−/−, and Nrl−/−Arr1−/−Arr4−/− mice with photopic electroretinography (ERG) to assess light adaptation and retinal responses, immunoblot and immunohistochemical localization analysis to measure retinal expression levels of M- and S-opsin, and optokinetic tracking (OKT) to measure the visual acuity and contrast sensitivity. Results Study results indicated that Nrl−/− and Nrl−/−Arr4−/− mice light adapted normally, while Nrl−/−Arr1−/− and Nrl−/−Arr1−/−Arr4−/− mice did not. Photopic ERG a-wave, b-wave, and flicker amplitudes followed a general pattern in which Nrl−/−Arr4−/− amplitudes were higher than the amplitudes of Nrl−/−, while the amplitudes of Nrl−/−Arr1−/− and Nrl−/−Arr1−/−Arr4−/− were lower. All three visual arrestin knockouts had faster implicit times than Nrl−/− mice. M-opsin expression is lower when ARR1 is not expressed, while S-opsin expression is lower when ARR4 is not expressed. Although M-opsin expression is mislocalized throughout the photoreceptor cells, S-opsin is confined to the outer segments in all genotypes. Contrast sensitivity is decreased when ARR4 is not expressed, while visual acuity was normal except in Nrl−/−Arr1−/−Arr4−/−. Conclusions Based on the opposite visual phenotypes in an all-cone retina in the Nrl−/−Arr1−/− and Nrl−/−Arr4−/− mice, we conclude that ARR1 and ARR4 perform unique

  15. β-Arrestin-dependent deactivation of mouse melanopsin.

    PubMed

    Cameron, Evan G; Robinson, Phyllis R

    2014-01-01

    In mammals, the expression of the unusual visual pigment, melanopsin, is restricted to a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs), whose signaling regulate numerous non-visual functions including sleep, circadian photoentrainment and pupillary constriction. IpRGCs exhibit attenuated electrical responses following sequential and prolonged light exposures indicative of an adaptational response. The molecular mechanisms underlying deactivation and adaptation in ipRGCs however, have yet to be fully elucidated. The role of melanopsin phosphorylation and β-arrestin binding in this adaptive process is suggested by the phosphorylation-dependent reduction of melanopsin signaling in vitro and the ubiquitous expression of β-arrestin in the retina. These observations, along with the conspicuous absence of visual arrestin in ipRGCs, suggest that a β-arrestin terminates melanopsin signaling. Here, we describe a light- and phosphorylation- dependent reduction in melanopsin signaling mediated by both β-arrestin 1 and β-arrestin 2. Using an in vitro calcium imaging assay, we demonstrate that increasing the cellular concentration of β-arrestin 1 and β-arrestin 2 significantly increases the rate of deactivation of light-activated melanopsin in HEK293 cells. Furthermore, we show that this response is dependent on melanopsin carboxyl-tail phosphorylation. Crosslinking and co-immunoprecipitation experiments confirm β-arrestin 1 and β-arrestin 2 bind to melanopsin in a light- and phosphorylation- dependent manner. These data are further supported by proximity ligation assays (PLA), which demonstrate a melanopsin/β-arrestin interaction in HEK293 cells and ipRGCs. Together, these results suggest that melanopsin signaling is terminated in a light- and phosphorylation-dependent manner through the binding of a β-arrestin within the retina. PMID:25401926

  16. Caenorhabditus elegans arrestin regulates neural G protein signaling and olfactory adaptation and recovery.

    PubMed

    Palmitessa, Aimee; Hess, Heather A; Bany, I Amy; Kim, You-Me; Koelle, Michael R; Benovic, Jeffrey L

    2005-07-01

    Although regulation of G protein-coupled receptor signaling by receptor kinases and arrestins is a well established biochemical process, the physiological significance of such regulation remains poorly understood. To better understand the in vivo consequences of arrestin function, we have examined the function of the sole arrestin in Caenorhabditis elegans (ARR-1). ARR-1 is primarily expressed in the nervous system, including the HSN neuron and various chemosensory neurons involved in detecting soluble and volatile odorants. arr-1 null mutants exhibit normal chemotaxis but have significant defects in olfactory adaptation and recovery to volatile odorants. In contrast, adaptation is enhanced in animals overexpressing ARR-1. Both the adaptation and recovery defects of arr-1 mutants are rescued by transgenic expression of wild-type ARR-1, whereas expression of a C-terminally truncated ARR-1 effectively rescues only the adaptation defect. A potential mechanistic basis for these findings is revealed by in vitro studies demonstrating that wild-type ARR-1 binds proteins of the endocytic machinery and promotes receptor endocytosis, whereas C-terminally truncated ARR-1 does not. These results demonstrate that ARR-1 functions to regulate chemosensory signaling, enabling organisms to adapt to a variety of environmental cues, and provide an in vivo link between arrestin, receptor endocytosis, and temporal recovery from adaptation. PMID:15878875

  17. Arrestin development: emerging roles for beta-arrestins in developmental signaling pathways.

    PubMed

    Kovacs, Jeffrey J; Hara, Makoto R; Davenport, Chandra L; Kim, Jihee; Lefkowitz, Robert J

    2009-10-01

    Arrestins were identified as mediators of G protein-coupled receptor (GPCR) desensitization and endocytosis. However, it is now clear that they scaffold many intracellular signaling networks to modulate the strength and duration of signaling by diverse types of receptors--including those relevant to the Hedgehog, Wnt, Notch, and TGFbeta pathways--and downstream kinases such as the MAPK and Akt/PI3K cascades. The involvement of arrestins in many discrete developmental signaling events suggests an indispensable role for these multifaceted molecular scaffolds. PMID:19853559

  18. β-Arrestin Regulates Estradiol Membrane-Initiated Signaling in Hypothalamic Neurons

    PubMed Central

    Wong, Angela M.; Abrams, Matthew C.; Micevych, Paul E.

    2015-01-01

    Estradiol (E2) action in the nervous system is the result of both direct nuclear and membrane-initiated signaling (EMS). E2 regulates membrane estrogen receptor-α (ERα) levels through opposing mechanisms of EMS-mediated trafficking and internalization. While ß-arrestin-mediated mERα internalization has been described in the cortex, a role of ß-arrestin in EMS, which underlies multiple physiological processes, remains undefined. In the arcuate nucleus of the hypothalamus (ARH), membrane-initiated E2 signaling modulates lordosis behavior, a measure of female sexually receptivity. To better understand EMS and regulation of ERα membrane levels, we examined the role of ß-arrestin, a molecule associated with internalization following agonist stimulation. In the present study, we used an immortalized neuronal cell line derived from embryonic hypothalamic neurons, the N-38 line, to examine whether ß-arrestins mediate internalization of mERα. β-arrestin-1 (Arrb1) was found in the ARH and in N-38 neurons. In vitro, E2 increased trafficking and internalization of full-length ERα and ERαΔ4, an alternatively spliced isoform of ERα, which predominates in the membrane. Treatment with E2 also increased phosphorylation of extracellular-signal regulated kinases 1/2 (ERK1/2) in N-38 neurons. Arrb1 siRNA knockdown prevented E2-induced ERαΔ4 internalization and ERK1/2 phosphorylation. In vivo, microinfusions of Arrb1 antisense oligodeoxynucleotides (ODN) into female rat ARH knocked down Arrb1 and prevented estradiol benzoate-induced lordosis behavior compared with nonsense scrambled ODN (lordosis quotient: 3 ± 2.1 vs. 85.0 ± 6.0; p < 0.0001). These results indicate a role for Arrb1 in both EMS and internalization of mERα, which are required for the E2-induction of female sexual receptivity. PMID:25803606

  19. Investigation of the role of βarrestin2 in kappa opioid receptor modulation in a mouse model of pruritus.

    PubMed

    Morgenweck, Jenny; Frankowski, Kevin J; Prisinzano, Thomas E; Aubé, Jeffrey; Bohn, Laura M

    2015-12-01

    The kappa opioid receptor (KOR) is involved in mediating pruritus; agonists targeting this receptor have been used to treat chronic intractable itch. Conversely, antagonists induce an itch response at the site of injection. As a G protein-coupled receptor (GPCR), the KOR has potential for signaling via G proteins and βarrestins, however, it is not clear which of these pathways are involved in the KOR modulation of itch. In this study asked whether the actions of KOR in pruritus involve βarrestins by using βarrestin2 knockout (βarr2-KO) mice as well as a recently described biased KOR agonist that biases receptor signaling toward G protein pathways over βarrestin2 recruitment. We find that the KOR antagonists nor-binaltorphimine (NorBNI) and 5'-guanidinonaltrindole (5'GNTI) induce acute pruritus in C57BL/6J mice, with reduced effects in KOR-KO mice. βArr2-KO mice display less of a response to KOR antagonist-induced itch compared to wild types, however no genotype differences are observed from chloroquine phosphate (CP)-induced itch, suggesting that the antagonists may utilize a KOR-βarrestin2 dependent mechanism. The KOR agonist U50,488H was equally effective in both WT and βarr2-KO mice in suppressing CP-induced itch. Furthermore, the G protein biased agonist, Isoquinolinone 2.1 was as effective as U50,488H in suppressing the itch response induced by KOR antagonist NorBNI or CP in C57BL/6J mice. Together these data suggest that the antipruritic effects of KOR agonists may not require βarrestins. PMID:26318102

  20. β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle.

    PubMed

    Nuber, Susanne; Zabel, Ulrike; Lorenz, Kristina; Nuber, Andreas; Milligan, Graeme; Tobin, Andrew B; Lohse, Martin J; Hoffmann, Carsten

    2016-03-31

    (β-)Arrestins are important regulators of G-protein-coupled receptors (GPCRs). They bind to active, phosphorylated GPCRs and thereby shut off 'classical' signalling to G proteins, trigger internalization of GPCRs via interaction with the clathrin machinery and mediate signalling via 'non-classical' pathways. In addition to two visual arrestins that bind to rod and cone photoreceptors (termed arrestin1 and arrestin4), there are only two (non-visual) β-arrestin proteins (β-arrestin1 and β-arrestin2, also termed arrestin2 and arrestin3), which regulate hundreds of different (non-visual) GPCRs. Binding of these proteins to GPCRs usually requires the active form of the receptors plus their phosphorylation by G-protein-coupled receptor kinases (GRKs). The binding of receptors or their carboxy terminus as well as certain truncations induce active conformations of (β-)arrestins that have recently been solved by X-ray crystallography. Here we investigate both the interaction of β-arrestin with GPCRs, and the β-arrestin conformational changes in real time and in living human cells, using a series of fluorescence resonance energy transfer (FRET)-based β-arrestin2 biosensors. We observe receptor-specific patterns of conformational changes in β-arrestin2 that occur rapidly after the receptor-β-arrestin2 interaction. After agonist removal, these changes persist for longer than the direct receptor interaction. Our data indicate a rapid, receptor-type-specific, two-step binding and activation process between GPCRs and β-arrestins. They further indicate that β-arrestins remain active after dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. Thus, GPCRs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signalling. PMID:27007855

  1. Chromosome mapping of the human arrestin (SAG), {beta}-arrestin 2 (ARRB2), and {beta}-adrenergic receptor kinase 2 (ADRBK2) genes

    SciTech Connect

    Calabrese, G.; Sallese, M.; Stornaiuolo, A.

    1994-09-01

    Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.

  2. Mapping binding sites for the PDE4D5 cAMP-specific phosphodiesterase to the N- and C-domains of beta-arrestin using spot-immobilized peptide arrays.

    PubMed

    Baillie, George S; Adams, David R; Bhari, Narinder; Houslay, Thomas M; Vadrevu, Suryakiran; Meng, Dong; Li, Xiang; Dunlop, Allan; Milligan, Graeme; Bolger, Graeme B; Klussmann, Enno; Houslay, Miles D

    2007-05-15

    Beta2-ARs (beta2-adrenoceptors) become desensitized rapidly upon recruitment of cytosolic beta-arrestin. PDE4D5 (family 4 cAMP-specific phosphodiesterase, subfamily D, isoform 5) can be recruited in complex with beta-arrestin, whereupon it regulates PKA (cAMP-dependent protein kinase) phosphorylation of the beta2-AR. In the present study, we have used novel technology, employing a library of overlapping peptides (25-mers) immobilized on cellulose membranes that scan the entire sequence of beta-arrestin 2, to define the interaction sites on beta-arrestin 2 for binding of PDE4D5 and the cognate long isoform, PDE4D3. We have identified a binding site in the beta-arrestin 2 N-domain for the common PDE4D catalytic unit and two regions in the beta-arrestin 2 C-domain that confer specificity for PDE4D5 binding. Alanine-scanning peptide array analysis of the N-domain binding region identified severely reduced interaction with PDE4D5 upon R26A substitution, and reduced interaction upon either K18A or T20A substitution. Similar analysis of the beta-arrestin 2 C-domain identified Arg286 and Asp291, together with the Leu215-His220 region, as being important for binding PDE4D5, but not PDE4D3. Transfection with wild-type beta-arrestin 2 profoundly decreased isoprenaline-stimulated PKA phosphorylation of the beta2-AR in MEFs (mouse embryo fibroblasts) lacking both beta-arrestin 1 and beta-arrestin 2. This effect was negated using either the R26A or the R286A mutant form of beta-arrestin 2 or a mutant with substitution of an alanine cassette for Leu215-His220, which showed little or no PDE4D5 binding, but was still recruited to the beta2-AR upon isoprenaline challenge. These data show that the interaction of PDE4D5 with both the N- and C-domains of beta-arrestin 2 are essential for beta2-AR regulation. PMID:17288540

  3. Arrestin-3 is essential for the activation of Fyn by the luteinizing hormone receptor (LHR) in MA-10 cells*

    PubMed Central

    Galet, Colette; Ascoli, Mario

    2008-01-01

    Recent studies showed that Fyn is a mediator of the LHR-induced activation of the ERK1/2 cascade in MA-10 cells. Since the LHR is a G protein-coupled receptor and the Src family of kinases can be activated by some Gα subunits and by the non-visual arrestins we investigated the role of these signaling molecules in the LHR-provoked activation of Fyn. Small interfering RNAs (siRNAs) that target two Gα subunits that participate in LHR signaling (Gαs and Gα11) and one that targets arrestin-3 were co-transfected with the hLHR in MA-10 cells. We then determined the effects of these siRNAs on the LHR-provoked activation of Fyn, the phosphorylation of FAK (a prominent Fyn substrate) and the release of EGF-like growth factors (a Fyn-mediated process). Expression of the siRNA against Gαs decreased the level of Gαs and LHR-stimulated cAMP production by ~50% but did not affect LHR-stimulated Fyn activation or FAK phosphorylation. Likewise, expression of the siRNA against Gα11 decreased the level of Gα11 and LHR-stimulated inositol phosphate production by ~50% but did not affect LHR-stimulated Fyn activation or FAK phosphorylation. Expression of the siRNA against arrestin-3 decreased the level of arrestin-3 and the rate of internalization of hCG by ~50% and it also inhibited the LHR-provoked stimulation of Fyn, the phosphorylation of FAK and the release of EGF-like growth factors. These results show that, in MA-10 cells, the hLHR activates Fyn through and arrestin-3-dependent pathway and that this pathway is a mediator of the hLHR-provoked release of EGF-like growth factors. PMID:18647647

  4. Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and β-Arrestins 2

    PubMed Central

    Malfacini, D.; Ambrosio, C.; Gro’, M. C.; Sbraccia, M.; Trapella, C.; Guerrini, R.; Bonora, M.; Pinton, P.; Costa, T.; Calo’, G.

    2015-01-01

    Nociceptin/orphanin FQ (N/OFQ) controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP). Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes) allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells). In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified. PMID:26248189

  5. Beta-arrestin-2 negatively modulates inflammation response in mouse chondrocytes induced by 4-mer hyaluronan oligosaccharide.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-01-01

    Beta-arrestin-2 is an adaptor protein that terminates G protein activation and seems to be involved in the modulation of the inflammatory response. Small hyaluronan (HA) fragments, such as 4-mer HA oligosaccharides, are known to interact with the toll-like receptor-4 (TLR-4) with consequent activation of the nuclear factor kappaB (NF-kB) that in turn stimulates the inflammation response. NF-kB activation is mediated by different pathways, in particular by the transforming growth factor-activated kinase-1 (TAK-1). Conversely, increased levels of protein kinase A (PKA), induced by cyclic adenosine monophosphate (cAMP), seem to inhibit NF-kB activation. We studied the involvement and role of beta-arrestin-2 in mouse chondrocytes stimulated with 4-mer HA fragments. The exposure of chondrocytes to 4-mer HA produced a significant up-regulation in TLR-4, cAMP, beta-arrestin-2, TAK-1, protein 38 mitogen-activated protein kinase (p38MAPK), and PKA, both in terms of mRNA expression and of the related protein levels. NF-kB was significantly activated, thereby producing the transcription of pro-inflammatory mediators, including tumor necrosis factor alpha, interleukin-6, and interleukin-17. The treatment of 4-mer HA-stimulated chondrocytes with antibodies against beta-arrestin-2 and/or a specific PKA inhibitor, significantly increased the inflammatory response, while the treatment with a specific p38MAPK inhibitor significantly reduced the inflammatory response. Interestingly, the anti-inflammatory action exerted by beta-arrestin-2 appeared to be mediated in part through the direct inhibition of p38MAPK, preventing NF-kB activation, and in part through cAMP and PKA activation primed by G protein signaling, which exerted an inhibitory effect on NF-kB. Taken together, these results could be useful for future anti-inflammatory strategies. PMID:25318610

  6. The conformational signature of β-arrestin2 predicts its trafficking and signalling functions.

    PubMed

    Lee, Mi-Hye; Appleton, Kathryn M; Strungs, Erik G; Kwon, Joshua Y; Morinelli, Thomas A; Peterson, Yuri K; Laporte, Stephane A; Luttrell, Louis M

    2016-03-31

    Arrestins are cytosolic proteins that regulate G-protein-coupled receptor (GPCR) desensitization, internalization, trafficking and signalling. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets the proteins for internalization via clathrin-coated pits. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G-protein effectors into GPCR-based 'signalsomes'. Although the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify these divergent functions is unclear. Using a panel of intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters to monitor conformational changes in β-arrestin2, here we show that GPCRs impose distinctive arrestin 'conformational signatures' that reflect the stability of the receptor-arrestin complex and role of β-arrestin2 in activating or dampening downstream signalling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in β-arrestin2 conformation. Our findings demonstrate that information about ligand-receptor conformation is encoded within the population average β-arrestin2 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands and in identifying factors that dictate arrestin conformation and function. PMID:27007854

  7. Differential Regulation of Endosomal GPCR/β-Arrestin Complexes and Trafficking by MAPK*

    PubMed Central

    Khoury, Etienne; Nikolajev, Ljiljana; Simaan, May; Namkung, Yoon; Laporte, Stéphane A.

    2014-01-01

    β-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/β-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either β-arrestin-1 or -2. We find a novel role for MAPK in the B2R/β-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat β-arrestin-2 (PET178P), but not rat β-arrestin-1 (PER177P). While the ERK1/2 regulatory motif is conserved between rat and mouse β-arrestin-2, it is surprisingly not conserved in human β-arrestin-2 (PEK178P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human β-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human β-arrestin-2 (T/K178D) significantly stabilizes B2R/β-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of β2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the β-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of β-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/β-arrestin-2 signaling roles among species. PMID:25016018

  8. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors

    PubMed Central

    Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands. PMID:26030739

  9. Co-purification of arrestin like proteins with alpha-enolase from bovine myocardial tissues and the possible role in heart diseases as an autoantigen

    SciTech Connect

    Mirshahi, M. Le Marchand, S.

    2015-05-08

    Aim: Previously, we reported that visual arrestin co-purified with glycolytic enzymes. The aim of this study was to analyze the co-purification of arrestin like proteins (ALP) in bovine cardiac tissues with enolases. Methods: The soluble extract of bovine myocardial tissues from different regions such as left and right atriums and ventricles of the bovine heart (n = 3) was analyzed by ACA-34 gel filtration, immuno-affinity column, SDS-PAGE, ELISA, western blot and a sandwich immune assay for quantification of ALP and sequence analysis. Results: We observed that; 1) The cardiac muscle contained a 50 kDa ALP at a concentration of 751 pg/mg of soluble protein extract, 2) ALP purified, by immunoaffinity, contained alpha-enolase of 48 kDa confirmed by protein sequence analysis; 3) Cardiomyocyte cells exposed to anti arrestin and anti enolase monoclonal antibodies showed decreased proliferation in vitro, 4) High level of autoantibodies were detected by ELISA (3.57% for arrestin and 9.12% for α-enolase) in serum of patients with infarcted heart disease. Conclusion: We suggest a possible interaction between ALP and alpha-enolases yielding a complex that may be involved in the induction of cardiac autoimmune diseases. - Highlights: • We examine a possible interaction between arrestin like protein and alpha-enolases in cardiomyocyte. • We demonstrated the effect of antibodies against arrestin and enolase on cardiomyocyte cell proliferation. • We suggest that this proteins complex may be involved in the induction of cardiac autoimmune diseases.

  10. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

    PubMed

    Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands. PMID:26030739

  11. β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation.

    PubMed

    Eichel, K; Jullié, D; von Zastrow, M

    2016-03-01

    β-Arrestins critically regulate G-protein-coupled receptor (GPCR) signalling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G-protein-independent signal through MAP kinase. Despite enormous recent progress towards understanding biophysical aspects of arrestin function, arrestin cell biology remains relatively poorly understood. Two key tenets underlie the prevailing current view: β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and MAP kinase activation requires endocytosis of formed GPCR-β-arrestin complexes. We show here, using β1-adrenergic receptors, that β-arrestin-2 (arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin- and β-arrestin-dependent MAP kinase signal. These results demonstrate β-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signalling from CCSs. We propose a β-arrestin signalling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery. PMID:26829388

  12. UBIQUITINATION OF β-ARRESTIN LINKS 7-TRANSMEMBRANE RECEPTOR ENDOCYTOSIS AND ERK ACTIVATION

    PubMed Central

    Shenoy, Sudha K.; Barak, Larry S.; Xiao, Kunhong; Ahn, Seungkirl; Berthouze, Magali; Shukla, Arun K.; Luttrell, Louis M.; Lefkowitz, Robert J.

    2007-01-01

    β-arrestin2 and its ubiquitination play crucial roles in both internalization and signaling of seven-transmembrane receptors (7TMRs). To understand the connection between ubiquitination and β-arrestin’s endocytic and signaling functions, we generated a β-arrestin2 mutant that is defective in ubiquitination (β-arrestin20K), by mutating all the ubiquitin acceptor lysines to arginines and compared its properties with the wild type and a stably ubiquitinated β-arrestin2-Ub chimera. In vitro translated β-arrestin2 and β-arrestin20K displayed equivalent binding to recombinant β2 adrenergic receptor (β2AR) reconstituted in vesicles, while β-arrestin2-Ub bound approximately four fold more. In cellular coimmunoprecipitation assays, β-arrestin20K bound non-receptor partners such as AP-2 and c-Raf and scaffolded pERK robustly, but displayed weak binding to clathrin. Moreover, β-arrestin20K was recruited only transiently to activated receptors at the membrane, did not enhance receptor internalization and decreased the amount of pERK assimilated into isolated β2AR complexes. While the wild type β-arrestin2 formed ERK signaling complexes with the β2AR at the membrane, a stably ubiquitinated β-arrestin2-Ub chimera, not only stabilized the ERK signalosomes but also led to their endosomal targeting. Interestingly, in cellular fractionation assays the ubiquitination state of β-arrestin2 favors its distribution in membrane fractions suggesting that ubiquitination increases β-arrestin’s propensity for membrane association. Our findings suggest that although β-arrestin ubiquitination is dispensable for β-arrestin’s cytosol to membrane translocation and its ‘constitutive’ interactions with some cytosolic proteins, it nevertheless is a prerequisite both for the formation of tight complexes with 7TMRs in vivo as well as for membrane compartment interactions that are crucial for downstream endocytic and signaling processes. PMID:17666399

  13. KISS1R signals independently of Gαq/11 and triggers LH secretion via the β-arrestin pathway in the male mouse.

    PubMed

    Ahow, Maryse; Min, Le; Pampillo, Macarena; Nash, Connor; Wen, Junping; Soltis, Kathleen; Carroll, Rona S; Glidewell-Kenney, Christine A; Mellon, Pamela L; Bhattacharya, Moshmi; Tobet, Stuart A; Kaiser, Ursula B; Babwah, Andy V

    2014-11-01

    Hypothalamic GnRH is the master regulator of the neuroendocrine reproductive axis, and its secretion is regulated by many factors. Among these is kisspeptin (Kp), a potent trigger of GnRH secretion. Kp signals via the Kp receptor (KISS1R), a Gαq/11-coupled 7-transmembrane-spanning receptor. Until this study, it was understood that KISS1R mediates GnRH secretion via the Gαq/11-coupled pathway in an ERK1/2-dependent manner. We recently demonstrated that KISS1R also signals independently of Gαq/11 via β-arrestin and that this pathway also mediates ERK1/2 activation. Because GnRH secretion is ERK1/2-dependent, we hypothesized that KISS1R regulates GnRH secretion via both the Gαq/11- and β-arrestin-coupled pathways. To test this hypothesis, we measured LH secretion, a surrogate marker of GnRH secretion, in mice lacking either β-arrestin-1 or β-arrestin-2. Results revealed that Kp-dependent LH secretion was significantly diminished relative to wild-type mice (P < .001), thus supporting that β-arrestin mediates Kp-induced GnRH secretion. Based on this, we hypothesized that Gαq/11-uncoupled KISS1R mutants, like L148S, will display Gαq/11-independent signaling. To test this hypothesis, L148S was expressed in HEK 293 cells. and results confirmed that, although strongly uncoupled from Gαq/11, L148S retained the ability to trigger significant Kp-dependent ERK1/2 phosphorylation (P < .05). Furthermore, using mouse embryonic fibroblasts lacking β-arrestin-1 and -2, we demonstrated that L148S-mediated ERK1/2 phosphorylation is β-arrestin-dependent. Overall, we conclude that KISS1R signals via Gαq/11 and β-arrestin to regulate GnRH secretion. This novel and important finding could explain why patients bearing some types of Gαq/11-uncoupled KISS1R mutants display partial gonadotropic deficiency and even a reversal of the condition, idiopathic hypogonadotropic hypogonadism. PMID:25147978

  14. Characterization of methadone as a β-arrestin-biased μ-opioid receptor agonist

    PubMed Central

    Doi, Seira; Mori, Tomohisa; Uzawa, Naoki; Arima, Takamichi; Takahashi, Tomoyuki; Uchida, Masashi; Yawata, Ayaka; Narita, Michiko; Uezono, Yasuhito; Suzuki, Tsutomu

    2016-01-01

    Background Methadone is a unique µ-opioid receptor agonist. Although several researchers have insisted that the pharmacological effects of methadone are mediated through the blockade of NMDA receptor, the underlying mechanism by which methadone exerts its distinct pharmacological effects compared to those of other µ-opioid receptor agonists is still controversial. In the present study, we further investigated the pharmacological profile of methadone compared to those of fentanyl and morphine as measured mainly by the discriminative stimulus effect and in vitro assays for NMDA receptor binding, µ-opioid receptor-internalization, and µ-opioid receptor-mediated β-arrestin recruitment. Results We found that fentanyl substituted for the discriminative stimulus effects of methadone, whereas a relatively high dose of morphine was required to substitute for the discriminative stimulus effects of methadone in rats. Under these conditions, the non-competitive NMDA receptor antagonist MK-801 did not substitute for the discriminative stimulus effects of methadone. In association with its discriminative stimulus effect, methadone failed to displace the receptor binding of MK801 using mouse brain membrane. Methadone and fentanyl, but not morphine, induced potent µ-opioid receptor internalization accompanied by the strong recruitment of β-arrestin-2 in µ-opioid receptor-overexpressing cells. Conclusions These results suggest that methadone may, at least partly, produce its pharmacological effect as a β-arrestin-biased µ-opioid receptor agonist, similar to fentanyl, and NMDA receptor blockade is not the main contributor to the pharmacological profile of methadone. PMID:27317580

  15. Visual arrestins in olfactory pathways of Drosophila and the malaria vector mosquito Anopheles gambiae

    PubMed Central

    Merrill, C. E.; Riesgo-Escovar, J.; Pitts, R. J.; Kafatos, F. C.; Carlson, J. R.; Zwiebel, L. J.

    2002-01-01

    Arrestins are important components for desensitization of G protein-coupled receptor cascades that mediate neurotransmission as well as olfactory and visual sensory reception. We have isolated AgArr1, an arrestin-encoding cDNA from the malaria vector mosquito, Anopheles gambiae, where olfaction is critical for vectorial capacity. Analysis of AgArr1 expression revealed an overlap between chemosensory and photoreceptor neurons. Furthermore, an examination of previously identified arrestins from Drosophila melanogaster exposed similar bimodal expression, and Drosophila arrestin mutants demonstrate impaired electrophysiological responses to olfactory stimuli. Thus, we show that arrestins in Drosophila are required for normal olfactory physiology in addition to their previously described role in visual signaling. These findings suggest that individual arrestins function in both olfactory and visual pathways in Dipteran insects; these genes may prove useful in the design of control strategies that target olfactory-dependent behaviors of insect disease vectors. PMID:11792843

  16. RACK1 and β-arrestin2 attenuate dimerization of PDE4 cAMP phosphodiesterase PDE4D5.

    PubMed

    Bolger, Graeme B

    2016-07-01

    PDE4 family cAMP-selective cyclic nucleotide phosphodiesterases are important in the regulation of cAMP abundance in numerous systems, and thereby play an important role in the regulation of PKA and EPAC activity and the phosphorylation of CREB. We have used the yeast 2-hybrid system to demonstrate recently that long PDE4 isoforms form homodimers, consistent with data obtained recently by structural studies. The long PDE4 isoform PDE4D5 interacts selectively with β-arrestin2, implicated in the regulation of G-protein-coupled receptors and other cell signaling components, and also with the β-propeller protein RACK1. In the present study, we use 2-hybrid approaches to demonstrate that RACK1 and β-arrestin2 inhibit the dimerization of PDE4D5. We also show that serine-to-alanine mutations at PKA and ERK1/2 phosphorylation sites on PDE4D5 detectably ablate dimerization. Conversely, phospho-mimic serine-to-aspartate mutations at the MK2 and oxidative stress kinase sites ablate dimerization. Analysis of PDE4D5 that is locked into the dimeric configuration by the formation of a trans disulfide bond between Ser261 and Ser602 shows that RACK1 interacts strongly with both the monomeric and dimeric forms, but that β-arrestin2 interacts exclusively with the monomeric form. This is consistent with the concept that β-arrestin2 can preferentially recruit the monomeric, or "open," form of PDE4D5 to β2-adrenergic receptors, where it can regulate cAMP signaling. PMID:26257302

  17. Assignment of the {beta}-arrestin 1 gene (ARRB1) to human chromosome 11q13

    SciTech Connect

    Calabrese, G.; Morizio, E.; Palka, G.

    1994-11-01

    Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor, and its functional cofactor, {beta}-arrestin. {beta}ARK is a member of a multigene family, consisting of six known subtypes, which have also been named G-protein-coupled receptor kinases (GRK 1 to 6) due to the apparently unique functional association of such kinases with this receptor family. The gene for {beta}ARK1 has been localized to human chromosome 11q13. The four members of the arrestin/{beta}-arrestin gene family identified so far are arrestin, X-arrestin, {beta}-arrestin 1, and {beta}-arrestin 2. Here the authors report the chromosome mapping of the human gene for {beta}-arrestin 1 (ARRB1) to chromosome 11q13 by fluorescence in situ hybridization (FISH). Two-color FISH confirmed that the two genes coding for the functionally related proteins {beta}ARK1 and {beta}arrestin 1 both map to 11q13. 16 refs., 1 fig., 1 tab.

  18. β-arrestin drives MAP kinase signaling from clathrin-coated structures after GPCR dissociation

    PubMed Central

    Eichel, K.; Jullié, D.

    2016-01-01

    β-arrestins critically regulate G protein-coupled receptor (GPCR) signaling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G protein-independent signal via MAP kinase1–3. Despite enormous recent progress toward understanding biophysical aspects of arrestin function4,5, its cell biology remains relatively poorly understood. Two key tenets underlie the present dogma: (1) β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and (2) MAP kinase activation requires endocytosis of formed GPCR - β-arrestin complexes6–9. We show here, using β1-adrenergic receptors, that β-arrestin-2 (Arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin and β-arrestin -dependent MAP kinase signal. These results demonstrate β-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signaling from CCSs. We propose a β-arrestin signaling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery. PMID:26829388

  19. α-Arrestins participate in cargo selection for both clathrin-independent and clathrin-mediated endocytosis

    PubMed Central

    Prosser, Derek C.; Pannunzio, Anthony E.; Brodsky, Jeffrey L.; Thorner, Jeremy; Wendland, Beverly; O'Donnell, Allyson F.

    2015-01-01

    ABSTRACT Clathrin-mediated endocytosis (CME) is a well-studied mechanism to internalize plasma membrane proteins; however, to endocytose such cargo, most eukaryotic cells also use alternative clathrin-independent endocytic (CIE) pathways, which are less well characterized. The budding yeast Saccharomyces cerevisiae, a widely used model for studying CME, was recently shown to have a CIE pathway that requires the GTPase Rho1, the formin Bni1, and their regulators. Nevertheless, in both yeast and mammalian cells, the mechanisms underlying cargo selection in CME and CIE are only beginning to be understood. For CME in yeast, particular α-arrestins contribute to recognition of specific cargos and promote their ubiquitylation by recruiting the E3 ubiquitin protein ligase Rsp5. Here, we show that the same α-arrestin–cargo pairs promote internalization through the CIE pathway by interacting with CIE components. Notably, neither expression of Rsp5 nor its binding to α-arrestins is required for CIE. Thus, α-arrestins are important for cargo selection in both the CME and CIE pathways, but function by distinct mechanisms in each pathway. PMID:26459639

  20. Proximal tubule NHE3 activity is inhibited by beta-arrestin-biased angiotensin II type 1 receptor signaling.

    PubMed

    Carneiro de Morais, Carla P; Polidoro, Juliano Z; Ralph, Donna L; Pessoa, Thaissa D; Oliveira-Souza, Maria; Barauna, Valério G; Rebouças, Nancy A; Malnic, Gerhard; McDonough, Alicia A; Girardi, Adriana C C

    2015-10-15

    Physiological concentrations of angiotensin II (ANG II) upregulate the activity of Na(+)/H(+) exchanger isoform 3 (NHE3) in the renal proximal tubule through activation of the ANG II type I (AT1) receptor/G protein-coupled signaling. This effect is key for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the beta-arrestin-biased AT1 receptor signaling pathway induces diuresis and natriuresis independent of G protein-mediated signaling. This study tested the hypothesis that activation of this AT1 receptor/beta-arrestin signaling inhibits NHE3 activity in proximal tubule. To this end, we determined the effects of the compound TRV120023, which binds to the AT1R, blocks G-protein coupling, and stimulates beta-arrestin signaling on NHE3 function in vivo and in vitro. NHE3 activity was measured in both native proximal tubules, by stationary microperfusion, and in opossum proximal tubule (OKP) cells, by Na(+)-dependent intracellular pH recovery. We found that 10(-7) M TRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro. Additionally, stimulation of NHE3 by ANG II was completely suppressed by TRV120023 both in vivo as well as in vitro. Inhibition of NHE3 activity by TRV120023 was associated with a decrease in NHE3 surface expression in OKP cells and with a redistribution from the body to the base of the microvilli in the rat proximal tubule. These findings indicate that biased signaling of the beta-arrestin pathway through the AT1 receptor inhibits NHE3 activity in the proximal tubule at least in part due to changes in NHE3 subcellular localization. PMID:26246427

  1. Arrestin Scaffolds NHERF1 to the P2Y12 Receptor to Regulate Receptor Internalization*

    PubMed Central

    Nisar, Shaista P.; Cunningham, Margaret; Saxena, Kunal; Pope, Robert J.; Kelly, Eamonn; Mundell, Stuart J.

    2012-01-01

    We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y12 receptor (P2Y12R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y12R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y12R internalization. In vitro and prior to agonist stimulation P2Y12R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization. PMID:22610101

  2. Arrestin scaffolds NHERF1 to the P2Y12 receptor to regulate receptor internalization.

    PubMed

    Nisar, Shaista P; Cunningham, Margaret; Saxena, Kunal; Pope, Robert J; Kelly, Eamonn; Mundell, Stuart J

    2012-07-13

    We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y(12) receptor (P2Y(12)R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y(12)R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y(12)R internalization. In vitro and prior to agonist stimulation P2Y(12)R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization. PMID:22610101

  3. Analysis of G Protein and β-Arrestin Activation in Chemokine Receptors Signaling.

    PubMed

    Vacchini, Alessandro; Busnelli, Marta; Chini, Bice; Locati, Massimo; Borroni, Elena Monica

    2016-01-01

    Chemokines are key regulators of leukocyte migration and play fundamental roles in immune responses. The chemokine system includes a set of over 40 ligands which engage in a promiscuous fashion a panel of over 25 receptors belonging to a distinct family of 7 transmembrane-domain receptors (7TM) widely expressed on a variety of cells. Although responses evoked by chemokine receptors have long been considered the result of balanced activation of the G protein- and β-arrestin-dependent signaling modules, evidence is accumulating showing that these receptors are capable, as other 7TMs, to activate different signaling modules in a ligand- and cell/tissue-specific manner. This biased signaling, or functional selectivity, confers a hitherto largely uncharacterized level of complexity to the chemokine system and challenges our present understanding of its redundancy. At the same time, it also provides new insights of relevance for chemokine receptors targeting drug development plans. Here, we provide current methods to study biased signaling of chemokine receptors by dissecting G proteins and β-arrestins activation upon chemokine stimulation. PMID:26921957

  4. c-Src regulates clathrin adapter protein 2 interaction with beta-arrestin and the angiotensin II type 1 receptor during clathrin- mediated internalization.

    PubMed

    Fessart, Delphine; Simaan, May; Laporte, Stéphane A

    2005-02-01

    Beta-arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to beta-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, beta-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between beta-arrestin2 and the beta-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both beta-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and beta-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and beta-arrestin during the clathrin-mediated internalization

  5. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

    PubMed Central

    Kang, Yanyong; Zhou, X. Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Ke, Jiyuan; Eileen Tan, M. H.; Zhang, Chenghai; Moeller, Arne; West, Graham M.; Pascal, Bruce; Van Eps, Ned; Caro, Lydia N.; Vishnivetskiy, Sergey A.; Lee, Regina J.; Suino-Powell, Kelly M.; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J.; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A.; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N.; Spence, John C. H.; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P.; Katritch, Vsevolod; Gurevich, Vsevolod V.; Griffin, Patrick R.; Hubbell, Wayne L.; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

    2015-01-01

    G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly, in which rhodopsin uses distinct structural elements, including TM7 and Helix 8 to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the N- and C- domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signaling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology. PMID:26200343

  6. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

    SciTech Connect

    Kang, Yanyong; Zhou, X. Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Ke, Jiyuan; Tan, M. H. Eileen; Zhang, Chenghai; Moeller, Arne; West, Graham M.; Pascal, Bruce D.; Van Eps, Ned; Caro, Lydia N.; Vishnivetskiy, Sergey A.; Lee, Regina J.; Suino-Powell, Kelly M.; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J.; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A.; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie E.; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S.; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N.; Spence, John C. H.; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P.; Katritch, Vsevolod; Gurevich, Vsevolod V.; Griffin, Patrick R.; Hubbell, Wayne L.; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

    2015-07-22

    G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. In conclusion, this structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

  7. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

    DOE PAGESBeta

    Kang, Yanyong; Zhou, X. Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; et al

    2015-07-22

    G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotationmore » between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. In conclusion, this structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.« less

  8. Formation and Decay of the Arrestin·Rhodopsin Complex in Native Disc Membranes*

    PubMed Central

    Beyrière, Florent; Sommer, Martha E.; Szczepek, Michal; Bartl, Franz J.; Hofmann, Klaus Peter; Heck, Martin; Ritter, Eglof

    2015-01-01

    In the G protein-coupled receptor rhodopsin, light-induced cis/trans isomerization of the retinal ligand triggers a series of distinct receptor states culminating in the active Metarhodopsin II (Meta II) state, which binds and activates the G protein transducin (Gt). Long before Meta II decays into the aporeceptor opsin and free all-trans-retinal, its signaling is quenched by receptor phosphorylation and binding of the protein arrestin-1, which blocks further access of Gt to Meta II. Although recent crystal structures of arrestin indicate how it might look in a precomplex with the phosphorylated receptor, the transition into the high affinity complex is not understood. Here we applied Fourier transform infrared spectroscopy to monitor the interaction of arrestin-1 and phosphorylated rhodopsin in native disc membranes. By isolating the unique infrared signature of arrestin binding, we directly observed the structural alterations in both reaction partners. In the high affinity complex, rhodopsin adopts a structure similar to Gt-bound Meta II. In arrestin, a modest loss of β-sheet structure indicates an increase in flexibility but is inconsistent with a large scale structural change. During Meta II decay, the arrestin-rhodopsin stoichiometry shifts from 1:1 to 1:2. Arrestin stabilizes half of the receptor population in a specific Meta II protein conformation, whereas the other half decays to inactive opsin. Altogether these results illustrate the distinct binding modes used by arrestin to interact with different functional forms of the receptor. PMID:25847250

  9. β-Arrestin-mediated Angiotensin II Signaling Controls the Activation of ARF6 Protein and Endocytosis in Migration of Vascular Smooth Muscle Cells.

    PubMed

    Charles, Ricardo; Namkung, Yoon; Cotton, Mathieu; Laporte, Stéphane A; Claing, Audrey

    2016-02-19

    Angiotensin II (Ang II) is a vasopressive hormone but is also a potent activator of cellular migration. We have previously shown that it can promote the activation of the GTPase ARF6 in a heterologous overexpressing system. The molecular mechanisms by which receptors control the activation of this small G protein remain, however, largely unknown. Furthermore, how ARF6 coordinates the activation of complex cellular responses needs to be further elucidated. In this study, we demonstrate that Ang II receptors engage β-arrestin, but not Gq, to mediate ARF6 activation in HEK 293 cells. To further confirm the key role of β-arrestin proteins, we overexpressed β-arrestin2-(1-320), a dominant negative mutant known to block receptor endocytosis. We show that expression of this truncated construct does not support the activation of the GTPase nor cell migration. Interestingly, β-arrestin2 can interact with the ARF guanine nucleotide exchange factor ARNO, although the C-terminally lacking mutant does not. We finally examined whether receptor endocytosis controlled ARF6 activation and cell migration. Although the clathrin inhibitor PitStop2 did not impact the ability of Ang II to activate ARF6, cell migration was markedly impaired. To further show that ARF activation regulates key signaling events leading to migration, we also examined MAPK activation. We demonstrate that this signaling axis is relevant in smooth muscle cells of the vasculature. Altogether, our findings show for the first time that Ang II receptor signaling to β-arrestin regulates ARF6 activation. These proteins together control receptor endocytosis and ultimately cell migration. PMID:26703465

  10. β-Arrestin-Dependent Dopaminergic Regulation of Calcium Channel Activity in the Axon Initial Segment.

    PubMed

    Yang, Sungchil; Ben-Shalom, Roy; Ahn, Misol; Liptak, Alayna T; van Rijn, Richard M; Whistler, Jennifer L; Bender, Kevin J

    2016-08-01

    G-protein-coupled receptors (GPCRs) initiate a variety of signaling cascades, depending on effector coupling. β-arrestins, which were initially characterized by their ability to "arrest" GPCR signaling by uncoupling receptor and G protein, have recently emerged as important signaling effectors for GPCRs. β-arrestins engage signaling pathways that are distinct from those mediated by G protein. As such, arrestin-dependent signaling can play a unique role in regulating cell function, but whether neuromodulatory GPCRs utilize β-arrestin-dependent signaling to regulate neuronal excitability remains unclear. Here, we find that D3 dopamine receptors (D3R) regulate axon initial segment (AIS) excitability through β-arrestin-dependent signaling, modifying CaV3 voltage dependence to suppress high-frequency action potential generation. This non-canonical D3R signaling thereby gates AIS excitability via pathways distinct from classical GPCR signaling pathways. PMID:27452469

  11. Visualization of arrestin recruitment by a G Protein-Coupled Receptor

    PubMed Central

    Reis, Rosana I.; Huang, Li-Yin; Tripathi-Shukla, Prachi; Qian, Jiang; Li, Sheng; Blanc, Adi; Oleskie, Austin N.; Dosey, Anne M.; Su, Min; Liang, Cui-Rong; Gu, Ling-Ling; Shan, Jin-Ming; Chen, Xin; Hanna, Rachel; Choi, Minjung; Yao, Xiao Jie; Klink, Bjoern U.; Kahsai, Alem W.; Sidhu, Sachdev S.; Koide, Shohei; Penczek, Pawel A.; Kossiakoff, Anthony A.; Jr, Virgil L. Woods; Kobilka, Brian K.; Skiniotis, Georgios; Lefkowitz, Robert J.

    2014-01-01

    G Protein Coupled Receptors (GPCRs) are critically regulated by β-arrestins (βarrs), which not only desensitize G protein signaling but also initiate a G protein independent wave of signaling1-5. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G protein complex, has provided novel insights into the structural basis of receptor activation6-11. Lacking however has been complementary information on recruitment of βarrs to activated GPCRs primarily due to challenges in obtaining stable receptor-βarr complexes for structural studies. Here, we devised a strategy for forming and purifying a functional β2AR-βarr1 complex that allowed us to visualize its architecture by single particle negative stain electron microscopy (EM) and to characterize the interactions between β2AR and βarr1 using hydrogen-deuterium exchange mass spectrometry (HDXMS) and chemical cross-linking. EM 2D averages and 3D reconstructions reveal bimodal binding of βarr1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy-terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of cross-linked residues suggest engagement of the finger loop of βarr1 with the seven-transmembrane core of the receptor. In contrast, focal areas of increased HDX indicate regions of increased dynamics in both N and C domains of βarr1 when coupled to the β2AR. A molecular model of the β2AR-βarr signaling complex was made by docking activated βarr1 and β2AR crystal structures into the EM map densities with constraints provided by HDXMS and cross-linking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented herein provides a framework for better understanding the basis of GPCR regulation by arrestins. PMID:25043026

  12. Functional map of arrestin binding to phosphorylated opsin, with and without agonist.

    PubMed

    Peterhans, Christian; Lally, Ciara C M; Ostermaier, Martin K; Sommer, Martha E; Standfuss, Jörg

    2016-01-01

    Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct. PMID:27350090

  13. Functional map of arrestin binding to phosphorylated opsin, with and without agonist

    PubMed Central

    Peterhans, Christian; Lally, Ciara C. M.; Ostermaier, Martin K.; Sommer, Martha E.; Standfuss, Jörg

    2016-01-01

    Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct. PMID:27350090

  14. β-arrestin-biased signaling through the β2-adrenergic receptor promotes cardiomyocyte contraction.

    PubMed

    Carr, Richard; Schilling, Justin; Song, Jianliang; Carter, Rhonda L; Du, Yang; Yoo, Sungsoo M; Traynham, Christopher J; Koch, Walter J; Cheung, Joseph Y; Tilley, Douglas G; Benovic, Jeffrey L

    2016-07-12

    β-adrenergic receptors (βARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of Gs-dependent β1AR and Gi-dependent β2AR pathways leads to enhanced cardiomyocyte death, reduced β1AR expression, and decreased inotropic reserve. β-blockers act to block excessive catecholamine stimulation of βARs to decrease cellular apoptotic signaling and normalize β1AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein-dependent signaling, β-arrestin-dependent signaling promotes cardiomyocyte survival. Given that β2AR expression is unaltered in CHF, a β-arrestin-biased agonist that operates through the β2AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective β-blocker, has been classified as a β-arrestin-biased agonist that can inhibit basal signaling from βARs and also stimulate cell survival signaling pathways. To understand the relative contribution of β-arrestin bias to the efficacy of select β-blockers, a specific β-arrestin-biased pepducin for the β2AR, intracellular loop (ICL)1-9, was used to decouple β-arrestin-biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1-9 was able to promote β2AR phosphorylation, β-arrestin recruitment, β2AR internalization, and β-arrestin-biased signaling. Interestingly, ICL1-9 was also able to induce β2AR- and β-arrestin-dependent and Ca(2+)-independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1-9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the β2AR and serves as a model for the next generation of cardiovascular drug development. PMID

  15. Wyoming Social Studies Content and Performance Standards.

    ERIC Educational Resources Information Center

    Wyoming State Dept. of Education, Cheyenne.

    The Wyoming Social Studies Content and Performance Standards were developed in the recognition that social studies is the integrated study of the social sciences and humanities to promote civic competence. The mission of social studies is to help young people develop the ability to make informed and reasoned decisions as citizens of a culturally…

  16. Visual Cone Arrestin 4 Contributes to Visual Function and Cone Health

    PubMed Central

    Deming, Janise D.; Pak, Joseph S.; Brown, Bruce M.; Kim, Moon K.; Aung, Moe H.; Eom, Yun Sung; Shin, Jung-a; Lee, Eun-Jin; Pardue, Machelle T.; Craft, Cheryl Mae

    2015-01-01

    Purpose Visual arrestins (ARR) play a critical role in shutoff of rod and cone phototransduction. When electrophysiological responses are measured for a single mouse cone photoreceptor, ARR1 expression can substitute for ARR4 in cone pigment desensitization; however, each arrestin may also contribute its own, unique role to modulate other cellular functions. Methods A combination of ERG, optokinetic tracking, immunohistochemistry, and immunoblot analysis was used to investigate the retinal phenotypes of Arr4 null mice (Arr4−/−) compared with age-matched control, wild-type mice. Results When 2-month-old Arr4−/− mice were compared with wild-type mice, they had diminished visual acuity and contrast sensitivity, yet enhanced ERG flicker response and higher photopic ERG b-wave amplitudes. In contrast, in older Arr4−/− mice, all ERG amplitudes were significantly reduced in magnitude compared with age-matched controls. Furthermore, in older Arr4−/− mice, the total cone numbers decreased and cone opsin protein immunoreactive expression levels were significantly reduced, while overall photoreceptor outer nuclear layer thickness was unchanged. Conclusions Our study demonstrates that Arr4−/− mice display distinct phenotypic differences when compared to controls, suggesting that ARR4 modulates essential functions in high acuity vision and downstream cellular signaling pathways that are not fulfilled or substituted by the coexpression of ARR1, despite its high expression levels in all mouse cones. Without normal ARR4 expression levels, cones slowly degenerate with increasing age, making this a new model to study age-related cone dystrophy. PMID:26284544

  17. β-Arrestin1 inhibits chemotherapy-induced intestinal stem cell apoptosis and mucositis

    PubMed Central

    Zhan, Y; Xu, C; Liu, Z; Yang, Y; Tan, S; Yang, Y; Jiang, J; Liu, H; Chen, J; Wu, B

    2016-01-01

    The mechanism of chemotherapy-induced gastrointestinal (GI) syndrome (CIGIS) is still controversial, and it is unclear whether chemotherapy induces intestinal stem cell (ISC) apoptosis. β-Arrestins are regulators and mediators of G protein-coupled receptor signaling in cell apoptosis, division and growth. In this study, we aimed to investigate whether chemotherapy induces ISC apoptosis to contribute to mucositis in CIGIS and whether β-arrestin1 (β-arr1) is involved in this apoptosis. Different chemotherapeutic agents were used to generate a CIGIS model. Lgr5-EGFP-IRES-creERT2+/− knock-in mice were used as a CIGIS model to investigate ISC apoptosis. β-arr1 knockout mice were used to determine whether β-arr1 is involved in the apoptosis in CIGIS. Intestinal histology was performed, the ISC apoptosis was analyzed and the mucosal barrier was examined. The effects of β-arr1 in apoptosis were investigated in the samples from humans and mice as well as in cell lines. Here, we demonstrate that chemotherapy induced intestinal mucositis by promoting crypt cell apoptosis, especially in Lgr5+ stem cells and Paneth cells but not in goblet cells, epithelial cells or vascular endothelial cells. Furthermore, β-arr1 deficiency exacerbated the Lgr5+ stem cell apoptosis, but not Paneth cell apoptosis, in CIGIS. In addition, the data showed that β-arr1 reduced the chemotherapy-induced Lgr5+ stem cell apoptosis by inhibiting endoplasmic reticulum stress-mediated mitochondrial apoptotic signaling. Our study indicates that β-arr1 inhibits chemotherapy-induced ISC apoptosis to alleviate intestinal mucositis in CIGIS. PMID:27195676

  18. β-Arrestin1 inhibits chemotherapy-induced intestinal stem cell apoptosis and mucositis.

    PubMed

    Zhan, Y; Xu, C; Liu, Z; Yang, Y; Tan, S; Yang, Y; Jiang, J; Liu, H; Chen, J; Wu, B

    2016-01-01

    The mechanism of chemotherapy-induced gastrointestinal (GI) syndrome (CIGIS) is still controversial, and it is unclear whether chemotherapy induces intestinal stem cell (ISC) apoptosis. β-Arrestins are regulators and mediators of G protein-coupled receptor signaling in cell apoptosis, division and growth. In this study, we aimed to investigate whether chemotherapy induces ISC apoptosis to contribute to mucositis in CIGIS and whether β-arrestin1 (β-arr1) is involved in this apoptosis. Different chemotherapeutic agents were used to generate a CIGIS model. Lgr5-EGFP-IRES-creERT2(+/-) knock-in mice were used as a CIGIS model to investigate ISC apoptosis. β-arr1 knockout mice were used to determine whether β-arr1 is involved in the apoptosis in CIGIS. Intestinal histology was performed, the ISC apoptosis was analyzed and the mucosal barrier was examined. The effects of β-arr1 in apoptosis were investigated in the samples from humans and mice as well as in cell lines. Here, we demonstrate that chemotherapy induced intestinal mucositis by promoting crypt cell apoptosis, especially in Lgr5+ stem cells and Paneth cells but not in goblet cells, epithelial cells or vascular endothelial cells. Furthermore, β-arr1 deficiency exacerbated the Lgr5+ stem cell apoptosis, but not Paneth cell apoptosis, in CIGIS. In addition, the data showed that β-arr1 reduced the chemotherapy-induced Lgr5+ stem cell apoptosis by inhibiting endoplasmic reticulum stress-mediated mitochondrial apoptotic signaling. Our study indicates that β-arr1 inhibits chemotherapy-induced ISC apoptosis to alleviate intestinal mucositis in CIGIS. PMID:27195676

  19. Identifying bias in CCR1 antagonists using radiolabelled binding, receptor internalization, β-arrestin translocation and chemotaxis assays

    PubMed Central

    Gilchrist, A; Gauntner, T D; Fazzini, A; Alley, K M; Pyen, D S; Ahn, J; Ha, S J; Willett, A; Sansom, S E; Yarfi, J L; Bachovchin, K A; Mazzoni, M R; Merritt, J R

    2014-01-01

    Background and Purpose Investigators have suggested that the chemokine receptor CCR1 plays a role in multiple myeloma. Studies using antisense and neutralizing antibodies to CCR1 showed that down-regulation of the receptor altered disease progression in a mouse model. More recently, experiments utilizing scid mice injected with human myeloma cells demonstrated that the CCR1 antagonist BX471 reduced osteolytic lesions, while the CCR1 antagonist MLN-3897 prevented myeloma cell adhesion to osteoclasts. However, information is limited regarding the pharmacology of CCR1 antagonists in myeloma cells. Experimental Approach We compared several well-studied CCR1 antagonists including AZD4818, BX471, CCX354, CP-481715, MLN-3897 and PS899877 for their ability to inhibit binding of [125I]-CCL3 in vitro using membranes prepared from RPMI 8226 cells, a human multiple myeloma cell line that endogenously expresses CCR1. In addition, antagonists were assessed for their ability to modulate CCL3-mediated internalization of CCR1 and CCL3-mediated cell migration using RPMI 8226 cells. As many GPCRs signal through β–arrestin-dependent pathways that are separate and distinct from those driven by G-proteins, we also evaluated the compounds for their ability to alter β-arrestin translocation. Key Results There were clear differences between the CCR1 antagonists in their ability to inhibit CCL3 binding to myeloma cells, as well as in their ability to inhibit G–protein-dependent and -independent functional responses. Conclusions and Implications Our studies demonstrate that tissue phenotype seems to be relevant with regards to CCR1. Moreover, it appears that for CCR1 antagonists, inhibition of β-arrestin translocation is not necessarily linked to chemotaxis or receptor internalization. PMID:24990525

  20. Regulatory role of β-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells.

    PubMed

    Manson, Mary E; Corey, Deborah A; Bederman, Ilya; Burgess, James D; Kelley, Thomas J

    2012-07-01

    Cystic fibrosis (CF) cells exhibit an increase in the protein expression of β-arrestin-2 (βarr2) coincident with perinuclear accumulation of free cholesterol. Arrestins are proteins that both serve as broad signaling regulators and contribute to G-protein coupled receptor internalization after agonist stimulation. The hypothesis of this study is that βarr2 is an important component in the mechanisms leading to cholesterol accumulation characteristic of CF cells. To test this hypothesis, epithelial cells stably expressing GFP-tagged βarr2 (βarr2-GFP) and respective GFP-expressing control cells (cont-GFP) were analyzed by filipin staining. The βarr2-GFP cells show a late endosomal/lysosomal cholesterol accumulation that is identical to that seen in CF cells. This βarr2-mediated accumulation is sensitive to Rp-cAMPS treatment, and depleting βarr2 expression in CF-model cells by shRNA alleviates cholesterol accumulation compared with controls. Cftr/βarr2 double knockout mice also exhibit wild-type (WT) levels of cholesterol synthesis, and WT profiles of signaling protein expression have previously been shown to be altered in CF due to cholesterol-related pathways. These data indicate a significant regulatory role for βarr2 in the development of CF-like cholesterol accumulation and give further insight into cholesterol processing mechanisms. An impact of βarr2 expression on Niemann-Pick type C-1 (NPC1)-containing organelle movement is proposed as the mechanism of βarr2-mediated alterations on cholesterol processing. It is concluded that βarr2 expression contributes to altered cholesterol trafficking observed in CF cells. PMID:22523395

  1. Inhibitory effects of omega-3 fatty acids on early brain injury after subarachnoid hemorrhage in rats: Possible involvement of G protein-coupled receptor 120/β-arrestin2/TGF-β activated kinase-1 binding protein-1 signaling pathway.

    PubMed

    Yin, Jia; Li, Haiying; Meng, Chengjie; Chen, Dongdong; Chen, Zhouqing; Wang, Yibin; Wang, Zhong; Chen, Gang

    2016-06-01

    Omega-3 fatty acids have been reported to improve neuron functions during aging and in patients affected by mild cognitive impairment, and mediate potent anti-inflammatory via G protein-coupled receptor 120 (GPR120) signal pathway. Neuron dysfunction and inflammatory response also contributed to the progression of subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). This study was to examine the effects of omega-3 fatty acids on SAH-induced EBI. Two weeks before SAH, 30% Omega-3 fatty acids was administered by oral gavage at 1g/kg body weight once every 24h. Specific siRNA for GPR120 was exploited. Terminal deoxynucleotidyl transferase dUTP nick end labeling, fluoro-Jade B staining, and neurobehavioral scores and brain water content test showed that omega-3 fatty acids effectively suppressed SAH-induced brain cell apoptosis and neuronal degradation, behavioral impairment, and brain edema. Western blot, immunoprecipitation, and electrophoretic mobility shift assays results showed that omega-3 fatty acids effectively suppressed SAH-induced elevation of inflammatory factors, including cyclooxygenase-2, monocyte chemoattractant protein-1, and inducible nitric oxide synthase. In addition, omega-3 fatty acids could inhibit phosphorylation of transforming growth factor β activated kinase-1 (TAK1), MEK4, c-Jun N-terminal kinase, and IkappaB kinase as well as activation of nuclear factor kappa B through regulating GPR120/β-arrestin2/TAK1 binding protein-1 pathway. Furthermore, siRNA-induced GPR120 silencing blocked the protective effects of omega-3 fatty acids. Here, we show that stimulation of GPR120 with omega-3 fatty acids pretreatment causes anti-apoptosis and anti-inflammatory effects via β-arrestin2/TAK1 binding protein-1/TAK1 pathway in the brains of SAH rats. Fish omega-3 fatty acids as part of a daily diet may reduce EBI in an experimental rat model of SAH. PMID:27000704

  2. Characterization of the interaction between the dopamine D4 receptor, KLHL12 and β-arrestins.

    PubMed

    Skieterska, Kamila; Shen, Ao; Clarisse, Dorien; Rondou, Pieter; Borroto-Escuela, Dasiel Oscar; Lintermans, Béatrice; Fuxe, Kjell; Xiang, Yang Kevin; Van Craenenbroeck, Kathleen

    2016-08-01

    Dopamine receptors are G protein-coupled receptors involved in regulation of cognition, learning, movement and endocrine signaling. The action of G protein-coupled receptors is highly regulated by multifunctional proteins, such as β-arrestins which can control receptor desensitization, ubiquitination and signaling. Previously, we have reported that β-arrestin 2 interacts with KLHL12, a BTB-Kelch protein which functions as an adaptor in a Cullin3-based E3 ligase complex and promotes ubiquitination of the dopamine D4 receptor. Here, we have investigated the molecular basis of the interaction between KLHL12 and β-arrestins and questioned its functional relevance. Our data demonstrate that β-arrestin 1 and β-arrestin 2 bind constitutively to the most common dopamine D4 receptor polymorphic variants and to KLHL12 and that all three proteins can interact within a single macromolecular complex. Surprisingly, stimulation of the receptor has no influence on the association between these proteins or their cellular distribution. We found that Cullin3 also interacts with both β-arrestins but has no influence on their ubiquitination. Knockout of one of the two β-arrestins hampers neither interaction between the dopamine D4 receptor and KLHL12, nor ubiquitination of the receptor. Finally, our results indicate that p44/42 MAPK phosphorylation, the signaling pathway which is often regulated by β-arrestins is not influenced by KLHL12, but seems to be exclusively mediated by Gαi protein upon dopamine D4 receptor stimulation. PMID:27155323

  3. Regulation of mitochondrial oxidative stress by β-arrestins in cultured human cardiac fibroblasts

    PubMed Central

    Philip, Jennifer L.; Razzaque, Md. Abdur; Han, Mei; Li, Jinju; Theccanat, Tiju; Xu, Xianyao; Akhter, Shahab A.

    2015-01-01

    ABSTRACT Oxidative stress in cardiac fibroblasts (CFs) promotes transformation to myofibroblasts and collagen synthesis leading to myocardial fibrosis, a precursor to heart failure (HF). NADPH oxidase 4 (Nox4) is a major source of cardiac reactive oxygen species (ROS); however, mechanisms of Nox4 regulation are unclear. β-arrestins are scaffold proteins that signal in G-protein-dependent and -independent pathways; for example, in ERK activation. We hypothesize that β-arrestins regulate oxidative stress in a Nox4-dependent manner and increase fibrosis in HF. CFs were isolated from normal and failing adult human left ventricles. Mitochondrial ROS/superoxide production was quantitated using MitoSox. β-arrestin and Nox4 expressions were manipulated using adenoviral overexpression or short interfering RNA (siRNA)-mediated knockdown. Mitochondrial oxidative stress and Nox4 expression in CFs were significantly increased in HF. Nox4 knockdown resulted in inhibition of mitochondrial superoxide production and decreased basal and TGF-β-stimulated collagen and α-SMA expression. CF β-arrestin expression was upregulated fourfold in HF. β-arrestin knockdown in failing CFs decreased ROS and Nox4 expression by 50%. β-arrestin overexpression in normal CFs increased mitochondrial superoxide production twofold. These effects were prevented by inhibition of either Nox or ERK. Upregulation of Nox4 seemed to be a primary mechanism for increased ROS production in failing CFs, which stimulates collagen deposition. β-arrestin expression was upregulated in HF and plays an important and newly identified role in regulating mitochondrial superoxide production via Nox4. The mechanism for this effect seems to be ERK-mediated. Targeted inhibition of β-arrestins in CFs might decrease oxidative stress as well as pathological cardiac fibrosis. PMID:26449263

  4. Conserved phosphoprotein interaction motif is functionally interchangeable between ataxin-7 and arrestins.

    PubMed

    Mushegian, A R; Vishnivetskiy, S A; Gurevich, V V

    2000-06-13

    Olivopontocerebellar atrophy with retinal degeneration is a hereditary neurodegenerative disorder that belongs to the subtype II of the autosomal dominant cerebellar ataxias and is characterized by early-onset cerebellar and macular degeneration preceded by diagnostically useful tritan colorblindness. The gene mutated in the disease (SCA7) has been mapped to chromosome 3p12-13.5, and positional cloning identified the cause of the disease as CAG repeat expansion in this gene. The SCA7 gene product, ataxin-7, is an 897 amino acid protein with an expandable polyglutamine tract close to its N-terminus. No clues to ataxin-7 function have been obtained from sequence database searches. Here we report that ataxin-7 has a motif of ca. 50 amino acids, related to the phosphate-binding site of arrestins. To test the relevance of this sequence similarity, we introduced the putative ataxin-7 phosphate-binding site into visual arrestin and beta-arrestin. Both chimeric arrestins retain receptor-binding affinity and show characteristic high selectivity for phosphorylated activated forms of rhodopsin and beta-adrenergic receptor, respectively. Although the insertion of a Gly residue (absent in arrestins but present in the putative phosphate-binding site of ataxin-7) disrupts the function of visual arrestin-ataxin-7 chimera, it enhances the function of beta-arrestin-ataxin-7 chimera. Taken together, our data suggest that the arrestin-like site in the ataxin-7 sequence is a functional phosphate-binding site. The presence of the phosphate-binding site in ataxin-7 suggests that this protein may be involved in phosphorylation-dependent binding to its protein partner(s) in the cell. PMID:10841760

  5. Development of an MRI biomarker sensitive to tetrameric visual arrestin 1 and its reduction via light-evoked translocation in vivo

    PubMed Central

    Berkowitz, Bruce A.; Gorgis, Jawan; Patel, Ankit; Baameur, Faiza; Gurevich, Vsevolod V.; Craft, Cheryl M.; Kefalov, Vladimir J.; Roberts, Robin

    2015-01-01

    Rod tetrameric arrestin 1 (tet-ARR1), stored in the outer nuclear layer/inner segments in the dark, modulates photoreceptor synaptic activity; light exposure stimulates a reduction via translocation to the outer segments for terminating G-protein coupled phototransduction signaling. Here, we test the hypothesis that intraretinal spin-lattice relaxation rate in the rotating frame (1/T1ρ), an endogenous MRI contrast mechanism, has high potential for evaluating rod tet-ARR1 and its reduction via translocation. Dark- and light-exposed mice (null for the ARR1 gene, overexpressing ARR1, diabetic, or wild type with or without treatment with Mn2+, a calcium channel probe) were studied using 1/T1ρ MRI. Immunohistochemistry and single-cell recordings of the retinas were also performed. In wild-type mice with or without treatment with Mn2+, 1/T1ρ of avascular outer retina (64% to 72% depth) was significantly (P < 0.05) greater in the dark than in the light; a significant (P < 0.05) but opposite pattern was noted in the inner retina (<50% depth). Light-evoked outer retina Δ1/T1ρ was absent in ARR1-null mice and supernormal in overexpressing mice. In diabetic mice, the outer retinal Δ1/T1ρ pattern suggested normal dark-to-light tet-ARR1 translocation and chromophore content, conclusions confirmed ex vivo. Light-stimulated Δ1/T1ρ in inner retina was linked to changes in blood volume. Our data support 1/T1ρ MRI for noninvasively assessing rod tet-ARR1 and its reduction via protein translocation, which can be combined with other metrics of retinal function in vivo.—Berkowitz, B. A., Gorgis, J., Patel, A., Baameur, F., Gurevich, V. V., Craft, C. M., Kefalov, V. J., Roberts, R. Development of an MRI biomarker sensitive to tetrameric visual arrestin 1 and its reduction via light-evoked translocation in vivo. PMID:25351983

  6. Arrestins and two receptor kinases are upregulated in Parkinson's disease with dementia.

    PubMed Central

    ER, Bychkov; VV, Gurevich; JN, Joyce; JL, Benovic; EV, Gurevich

    2008-01-01

    Arrestins and G proteins-coupled receptor kinases (GRKs) regulate signaling and trafficking of G protein-coupled receptors. We investigated changes in the expression of arrestins and GRKs in the striatum of patients with Parkinson's disease without (PD) or with dementia (PDD) at post mortem using Western blotting and ribonuclease protection assay. Both PD and PDD groups had similar degree of dopamine depletion in all striatal regions. Arrestin proteins and mRNAs were increased in the PDD group throughout striatum. Protein and mRNA of GRK5, the major subtype in the human striatum, and GRK3 were also upregulated, whereas GRK2 and 6 were mostly unchanged. The PD group had lower concentration of arrestins and GRKs than the PDD group. There was no statistical link between the load of Alzheimer's pathology and the expression of these signaling proteins. Upregulation of arrestins and GRK in PDD may confer resistance to the therapeutic effects of levodopa often observed in these patients. In addition, increased arrestin and GRK concentrations may lead to dementia via perturbation of multiple signaling mechanisms. PMID:17125886

  7. Evaluation of the arrestin gene in patients with retinitis pigmentosa or an allied disease

    SciTech Connect

    DeStefano, D.J.; Berson, E.L.; Dryja, T.P.

    1994-09-01

    Arrestin, also called 48K protein or S-antigen, plays a role in deactivating rhodopsin, the photosensitive, seven-helix, G-protein receptor found in rod photoreceptors. In Drosophila, null mutations in arrestin genes cause a light-dependent photoreceptor degeneration. It is possible that a comparable photoreceptor degeneration in humans is caused by defects in the rod arrestin gene. In order to evaluate this possibility, we are characterizing the human arrestin locus on chromosome 2q. We screened a genomic library (5 million plaques) using an arrestin cDNA clone. Sixty-eight hybridizing clones were identified; portions of 7 clones were sequenced to determine the intron sequence flanking the exons. We are using SSCP analysis and direct genomic sequencing to screen the entire coding region, splice donor and acceptor sites, and the promoter region of the arrestin gene in 188 patients with autosomal dominant and 104 patients with autosomal recessive retinitis pigmentosa. We have already obtained flanking intron sequences necessary for SSCP analysis for 13 of 16 exons. So far, we have identified 4 silent base changes at codons 67 (TGC-to-TGT), 107 (CTG-to-CTC), 163 (GCC-to-GCT), and 288 (CTG-to-TGT), all with allele frequencies at 1% or less. Several other variant bands detected by SSCP analysis are currently being sequenced.

  8. The structural basis of arrestin-mediated regulation of G-protein-coupled receptors

    PubMed Central

    Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2008-01-01

    The 4 mammalian arrestins serve as almost universal regulators of the largest known family of signaling proteins, G-protein-coupled receptors (GPCRs). Arrestins terminate receptor interactions with G proteins, redirect the signaling to a variety of alternative pathways, and orchestrate receptor internalization and subsequent intracellular trafficking. The elucidation of the structural basis and fine molecular mechanisms of the arrestin–receptor interaction paved the way to the targeted manipulation of this interaction from both sides to produce very stable or extremely transient complexes that helped to understand the regulation of many biologically important processes initiated by active GPCRs. The elucidation of the structural basis of arrestin interactions with numerous non-receptor-binding partners is long overdue. It will allow the construction of fully functional arrestins in which the ability to interact with individual partners is specifically disrupted or enhanced by targeted mutagenesis. These “custom-designed” arrestin mutants will be valuable tools in defining the role of various interactions in the intricate interplay of multiple signaling pathways in the living cell. The identification of arrestin-binding sites for various signaling molecules will also set the stage for designing molecular tools for therapeutic intervention that may prove useful in numerous disorders associated with congenital or acquired disregulation of GPCR signaling. PMID:16460808

  9. Few Residues within an Extensive Binding Interface Drive Receptor Interaction and Determine the Specificity of Arrestin Proteins*

    PubMed Central

    Vishnivetskiy, Sergey A.; Gimenez, Luis E.; Francis, Derek J.; Hanson, Susan M.; Hubbell, Wayne L.; Klug, Candice S.; Gurevich, Vsevolod V.

    2011-01-01

    Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements. PMID:21471193

  10. Phospho-selective mechanisms of arrestin conformations and functions revealed by unnatural amino acid incorporation and 19F-NMR

    PubMed Central

    Yang, Fan; Yu, Xiao; Liu, Chuan; Qu, Chang-Xiu; Gong, Zheng; Liu, Hong-Da; Li, Fa-Hui; Wang, Hong-Mei; He, Dong-Fang; Yi, Fan; Song, Chen; Tian, Chang-Lin; Xiao, Kun-Hong; Wang, Jiang-Yun; Sun, Jin-Peng

    2015-01-01

    Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific β-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, β-arrestin-1 ‘reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by 19F-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of β-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific β-arrestin-1 conformations. The identified receptor-phospho-selective mechanism for arrestin conformation and the spacing of the multiple phosphate-binding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors. PMID:26347956

  11. Nicotinic acetylcholine receptors induce c-Kit ligand/Stem Cell Factor and promote stemness in an ARRB1/ β-arrestin-1 dependent manner in NSCLC

    PubMed Central

    Perumal, Deepak; Pillai, Smitha; Nguyen, Jonathan; Schaal, Courtney; Coppola, Domenico; Chellappan, Srikumar P.

    2014-01-01

    Lung cancer remains the leading cause of cancer-related deaths worldwide. β-arrestin-1 (ARRB1), a scaffolding protein involved in the desensitization of signals arising from activated G-protein-coupled receptors (GPCRs), has been shown to play a role in invasion and proliferation of cancer cells, including nicotine-induced proliferation of human non–small cell lung cancers (NSCLCs). In this study, we identified genes that are differentially regulated by nicotine in an ARRB1/β-arrestin-1 dependent manner in NSCLC cells by microarray analysis. Among the identified genes, SCF (Stem cell factor) strongly differentiated smokers from non-smokers in the Director's Challenge Set expression data and its high expression correlated with poor prognosis. SCF, a major cytokine is the ligand for the c-Kit proto-oncogene and was found to be over expressed in human lung adenocarcinomas, but not squamous cell carcinomas. Data presented here show that transcription factor E2F1 can induce SCF expression at the transcriptional level and depletion of E2F1 or ARRB1/β-arrestin-1 could not promote self-renewal of SP cells. These studies suggest that nicotine might be promoting NSCLC growth and metastasis by inducing the secretion of SCF, and raise the possibility that targeting signalling cascades that activate E2F1 might be an effective way to combat NSCLC. PMID:25401222

  12. Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling

    PubMed Central

    Mundell, SJ; Matharu, A-L; Nisar, S; Palmer, TM; Benovic, JL; Kelly, E

    2010-01-01

    Background and purpose: We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A2B adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5′-(N-ethylcarboxamido)-adenosine. Experimental approach: The trafficking of the wild type A2B adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. Key results: The wild type A2B adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln325-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu330-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln325-stop, Ser326-stop and Phe328-stop receptors. Following internalization, the wild type A2B adenosine receptor recycled rapidly to the cell surface, whereas the Gln325-stop receptor did not recycle. Conclusions and implications: Deletion of the COOH-terminus of the A2B adenosine receptor beyond Leu330 switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A2B adenosine receptor following prolonged agonist addition. PMID:20128803

  13. Fanpage metrics analysis. "Study on content engagement"

    NASA Astrophysics Data System (ADS)

    Rahman, Zoha; Suberamanian, Kumaran; Zanuddin, Hasmah Binti; Moghavvemi, Sedigheh; Nasir, Mohd Hairul Nizam Bin Md

    2016-08-01

    Social Media is now determined as an excellent communicative tool to connect directly with consumers. One of the most significant ways to connect with the consumers through these Social Networking Sites (SNS) is to create a facebook fanpage with brand contents and to place different posts periodically on these fanpages. In measuring social networking sites' effectiveness, corporate houses are now analyzing metrics in terms of calculating engagement rate, number of comments/share and likings in fanpages. So now, it is very important for the marketers to know the effectiveness of different contents or posts of fanpages in order to increase the fan responsiveness and engagement rate in the fan pages. In the study the authors have analyzed total 1834 brand posts from 17 international brands of Electronics companies. Data of 9 months (From December 2014 to August 2015) have been collected for analyses, which were available online in the Brand' fan pages. An econometrics analysis is conducted using Eviews 9, to determine the impact of different contents on fanpage engagement. The study picked the four most frequently posted content to determine their impact on PTA (people Talking About) metrics and Fanpage engagement activities.

  14. The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

    PubMed Central

    de Munnik, Sabrina M.; Kooistra, Albert J.; van Offenbeek, Jody; Nijmeijer, Saskia; de Graaf, Chris; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Kaposi’s sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi’s sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74. PMID:25894435

  15. Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET.

    PubMed

    Namkung, Yoon; Le Gouill, Christian; Lukashova, Viktoria; Kobayashi, Hiroyuki; Hogue, Mireille; Khoury, Etienne; Song, Mideum; Bouvier, Michel; Laporte, Stéphane A

    2016-01-01

    Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes. PMID:27397672

  16. β-Arrestin-1 Drives Endothelin-1–Mediated Podocyte Activation and Sustains Renal Injury

    PubMed Central

    Buelli, Simona; Rosanò, Laura; Gagliardini, Elena; Corna, Daniela; Longaretti, Lorena; Pezzotta, Anna; Perico, Luca; Conti, Sara; Rizzo, Paola; Novelli, Rubina; Morigi, Marina; Zoja, Carlamaria; Remuzzi, Giuseppe; Bagnato, Anna

    2014-01-01

    Activation of endothelin-A receptor (ETAR) by endothelin-1 (ET-1) drives epithelial-to-mesenchymal transition in ovarian tumor cells through β-arrestin signaling. Here, we investigated whether this pathogenetic pathway could affect podocyte phenotype in proliferative glomerular disorders. In cultured mouse podocytes, ET-1 caused loss of the podocyte differentiation marker synaptopodin and acquisition of the mesenchymal marker α-smooth muscle actin. ET-1 promoted podocyte migration via ETAR activation and increased β-arrestin-1 expression. Activated ETAR recruited β-arrestin-1 to form a trimeric complex with Src leading to epithelial growth factor receptor (EGFR) transactivation and β-catenin phosphorylation, which promoted gene transcription of Snail. Increased Snail expression fostered ET-1–induced migration as confirmed by Snail knockdown experiments. Silencing of β-arrestin-1 prevented podocyte phenotypic changes and motility and inhibited ETAR-driven signaling. In vitro findings were confirmed in doxorubicin (Adriamycin)-induced nephropathy. Mice receiving Adriamycin developed renal injury with loss of podocytes and hyperplastic lesion formation; β-arrestin-1 expression increased in visceral podocytes and in podocytes entrapped in pseudo-crescents. Administration of the selective ETAR antagonist sitaxsentan prevented podocyte loss, formation of the hyperplastic lesions, and normalized expression of glomerular β-arrestin-1 and Snail. Increased β-arrestin-1 levels in podocytes retrieved from crescents of patients with proliferative glomerulopathies confirmed the translational relevance of these findings and suggest the therapeutic potential of ETAR antagonism for a group of diseases still needing a specific treatment. PMID:24371298

  17. Fenoterol inhibits LPS-induced AMPK activation and inflammatory cytokine production through β-arrestin-2 in THP-1 cell line

    SciTech Connect

    Wang, Wei; Zhang, Yuan; Xu, Ming; Zhang, You-Yi; He, Bei

    2015-06-26

    The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a β{sub 2}-adrenergic receptor (β{sub 2}-AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of β{sub 2}-AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and β-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1β (IL-1β) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of β-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1β (IL-1β) release, thus β-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1β release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the β{sub 2}-AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1β release via β-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases. - Highlights: • β{sub 2}-AR agonist fenoterol exerts its protective effect on LPS-treated THP-1 cells. • Fenoterol inhibits LPS-induced AMPK activation and IL-1β production. • β-arrestin2 mediates fenoterol-inhibited AMPK activation and IL-1β release. • AMPKα1 is involved in LPS-induced NF-κB activation and IL-1β production.

  18. β-arrestin Deficiency Protects Against Pulmonary Fibrosis in Mice and Prevents Fibroblast Invasion of Extracellular Matrix

    PubMed Central

    Lovgren, Alysia Kern; Kovacs, Jeffrey J.; Xie, Ting; Potts, Erin N.; Li, Yuejuan; Foster, W. Michael; Liang, Jiurong; Meltzer, Eric B.; Jiang, Dianhua; Lefkowitz, Robert J.; Noble, Paul W.

    2011-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive disease causing unremitting extracellular matrix deposition with resultant distortion of pulmonary architecture and impaired gas exchange. β-arrestins regulate G-protein-coupled receptors through receptor desensitization while acting as signaling scaffolds that facilitate numerous effector pathways. Here we examine the role of β-arrestin1 and β-arrestin2 in the pathobiology of pulmonary fibrosis. In the bleomycin-induced mouse lung fibrosis model, loss of eitherβ-arrestin1 or β-arrestin2 results in protection from mortality, inhibition of matrix deposition, and protected lung function. Fibrosis is prevented despite preserved recruitment of inflammatory cells and fibroblast chemotaxis. However, isolated lung fibroblasts from bleomycin-treated β-arrestin null mice fail to invade extracellular matrix while displaying altered expression of genes involved in matrix production and degradation. Furthermore, knockdown of β-arrestin2 in fibroblasts from IPF patients attenuated the invasive phenotype. These data implicate β-arrestins as mediators of fibroblast invasion and development of pulmonary fibrosis, thus representing a potential target for therapeutic intervention for patients with IPF. PMID:21411739

  19. Targeted Disruption of β-Arrestin 2-Mediated Signaling Pathways by Aptamer Chimeras Leads to Inhibition of Leukemic Cell Growth

    PubMed Central

    Li, Margie; Pratico, Elizabeth D.; Fereshteh, Mark P.; Ahrens, Douglas P.; Sullenger, Bruce A.; Kovacs, Jeffrey J.

    2014-01-01

    β-arrestins, ubiquitous cellular scaffolding proteins that act as signaling mediators of numerous critical cellular pathways, are attractive therapeutic targets because they promote tumorigenesis in several tumor models. However, targeting scaffolding proteins with traditional small molecule drugs has been challenging. Inhibition of β-arrestin 2 with a novel aptamer impedes multiple oncogenic signaling pathways simultaneously. Additionally, delivery of the β-arrestin 2-targeting aptamer into leukemia cells through coupling to a recently described cancer cell-specific delivery aptamer, inhibits multiple β-arrestin-mediated signaling pathways known to be required for chronic myelogenous leukemia (CML) disease progression, and impairs tumorigenic growth in CML patient samples. The ability to target scaffolding proteins such as β-arrestin 2 with RNA aptamers may prove beneficial as a therapeutic strategy. Highlights An RNA aptamer inhibits β-arrestin 2 activity. Inhibiting β-arrestin 2 impedes multiple tumorigenic pathways simultaneously. The therapeutic aptamer is delivered to cancer cells using a cell-specific DNA aptamer. Targeting β-arrestin 2 inhibits tumor progression in CML models and patient samples. PMID:24736311

  20. Delineation of a Conserved Arrestin-Biased Signaling Repertoire In Vivo

    PubMed Central

    Martin, Bronwen; Gesty-Palmer, Diane; Cheung, Huey; Johnson, Calvin; Patel, Shamit; Becker, Kevin G.; Wood, William H.; Zhang, Yongqing; Lehrmann, Elin; Luttrell, Louis M.

    2015-01-01

    Biased G protein–coupled receptor agonists engender a restricted repertoire of downstream events from their cognate receptors, permitting them to produce mixed agonist-antagonist effects in vivo. While this opens the possibility of novel therapeutics, it complicates rational drug design, since the in vivo response to a biased agonist cannot be reliably predicted from its in cellula efficacy. We have employed novel informatic approaches to characterize the in vivo transcriptomic signature of the arrestin pathway-selective parathyroid hormone analog [d-Trp12, Tyr34]bovine PTH(7-34) in six different murine tissues after chronic drug exposure. We find that [d-Trp12, Tyr34]bovine PTH(7-34) elicits a distinctive arrestin-signaling focused transcriptomic response that is more coherently regulated across tissues than that of the pluripotent agonist, human PTH(1-34). This arrestin-focused network is closely associated with transcriptional control of cell growth and development. Our demonstration of a conserved arrestin-dependent transcriptomic signature suggests a framework within which the in vivo outcomes of arrestin-biased signaling may be generalized. PMID:25637603

  1. Exploratory Activity in Drosophila Requires the kurtz Nonvisual Arrestin

    PubMed Central

    Liu, Lingzhi; Davis, Ronald L.; Roman, Gregg

    2007-01-01

    When Drosophila adults are placed into an open field arena, they initially exhibit an elevated level of activity followed by a reduced stable level of spontaneous activity. We have found that the initial elevated component arises from the fly's interaction with the novel arena since: (1) the increased activity is independent of handling prior to placement within the arena, (2) the fly's elevated activity is proportional to the size of the arena, and (3) the decay in activity to spontaneous levels requires both visual and olfactory input. These data indicate that active exploration is the major component of elevated initial activity. There is a specific requirement for the kurtz nonvisual arrestin in the nervous system for both the exploration stimulated by the novel arena and the mechanically stimulated activity. kurtz is not required for spontaneous activity; kurtz mutants display normal levels of spontaneous activity and average the same velocities as wild-type controls. Inhibition of dopamine signaling has no effect on the elevated initial activity phase in either wild-type or krz1 mutants. Therefore, the exploratory phase of open field activity requires kurtz in the nervous system, but is independent of dopamine's stimulation of activity. PMID:17151232

  2. X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex.

    PubMed

    Zhou, X Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Suino-Powell, Kelly M; Boutet, Sébastien; Williams, Garth J; Wang, Meitian; Li, Dianfan; Caffrey, Martin; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

    2016-01-01

    Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes. PMID:27070998

  3. X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex

    PubMed Central

    Zhou, X. Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Suino-Powell, Kelly M.; Boutet, Sébastien; Williams, Garth J.; Wang, Meitian; Li, Dianfan; Caffrey, Martin; Chapman, Henry N.; Spence, John C.H.; Fromme, Petra; Weierstall, Uwe; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

    2016-01-01

    Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes. PMID:27070998

  4. Protective Role of β-arrestin2 in Colitis Through Modulation of T-cell Activation.

    PubMed

    Sharma, Deepika; Malik, Ankit; Steury, Michael D; Lucas, Peter C; Parameswaran, Narayanan

    2015-12-01

    β-arrestin2 (β-arr2), identified as a scaffolding protein in G-protein-coupled receptor desensitization, is a negative regulator of inflammation in polymicrobial sepsis. In this study, we wanted to investigate the role of β-arr2 in intestinal inflammation, a site of persistent microbial stimulation. In the absence of β-arr2, mice exhibited greater extent of mucosal inflammation determined by cellular infiltration and expression of inflammatory mediators even under homeostatic conditions. Furthermore, β-arr2-deficient mice were more susceptible to dextran sulfate sodium-induced colitis as demonstrated by greater body weight loss, higher disease activity index, and shortened colon as compared with wild-type mice. We also show that T cells from β-arr2 knockout mice exhibit altered activation status under both basal and colitic conditions, implicating their involvement in disease induction. Further assessment of the role of β-arr2 in intrinsic T-cell differentiation confirmed its importance in T-cell polarization. Using the T-cell transfer model of colitis, we demonstrate that T-cell-specific β-arr2 is important in limiting colitic inflammation; however, it plays a paradoxical role in concurrent systemic wasting disease. Together, our study highlights a critical negative regulatory role of β-arr2 in intestinal inflammation and demonstrates a distinct role of T-cell-specific β-arr2 in systemic wasting disease. PMID:26296063

  5. Multiquantum EPR spectroscopy of spin-labeled arrestin K267C at 35 GHz.

    PubMed

    Klug, Candice S; Camenisch, Theodore G; Hubbell, Wayne L; Hyde, James S

    2005-05-01

    Three- and five-quantum absorption and dispersion multiquantum electron paramagnetic resonance spectra of a spin-labeled protein have been obtained for the first time at Q-band (35 GHz). Spectra of arrestin spin-labeled at site 267 were recorded at room temperature as a function of microwave power. The separation of irradiating microwave frequencies, Deltaf, was 10 kHz, and a newly-designed multiquantum Q-band electron paramagnetic resonance bridge was utilized, operating in a superheterodyne detection mode. The sample volume was 30 nL using a 3-loop-2-gap resonator. Most spectra were obtained at a 300 microM concentration in single, 2-min scans, but spectra were also successfully obtained at 30 microM, corresponding to one picomole of protein. Enhanced sensitivity to T(1) and T(2) was evident in the spectra, and linewidths varied considerably across the spectra. The pure absorption displays are beneficial relative to field modulation methods for spectral characterization. The presence of two states of the nitroxide spin-label with different relaxation times is evident, particularly in the dispersion spectra, which are expected to exhibit enhanced sensitivity to lineshape variation relative to absorption. Feasibility has been established for the use of this technique for site-directed spin-labeling studies of biologically relevant samples, particularly the study of protein structure and dynamics. PMID:15749769

  6. Content Validity Studies of Licensing Examinations.

    ERIC Educational Resources Information Center

    Smith, I. Leon; Hambleton, Ronald K.

    1990-01-01

    Implementing measurement specialists' ideas about content validity with licensure examinations and the problem of court litigation are discussed. Validity issues surfacing when sponsors of national licensure examinations conduct validity investigations are considered. Issues include local versus national focus on content validity, job analysis,…

  7. Beta-arrestin 1 is involved in the catabolic response stimulated by hyaluronan degradation in mouse chondrocytes.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-08-01

    Beta-arrestin-1 (β-arrestin-1) is an adaptor protein that functions in the termination of G-protein activation and seems to be involved in the mediation of the inflammatory response. Interleukin-1β (IL-1β) elicits the expression of inflammatory mediators through a mechanism involving hyaluronan (HA) degradation, thereby contributing to toll-like receptor 4 (TLR-4) and CD44 activation. Stimulation of both receptors induces nuclear factor kappaB (NF-kB) activation that, through transforming-growth-factor-activated-kinase-1 (TAK-1), in turn stimulates the inflammatory mediators of transcription. As β-arrestin-1 seems to play an inflammatory role in arthritis, we have investigated the involvement of β-arrestin-1 in a model of IL-1β-induced inflammatory response in mouse chondrocytes. IL-1β treatment significantly increases chondrocytes TLR-4, CD44, β-arrestin-1, TAK-1, and serine/threonine kinase (AKT) mRNA expression and related protein levels. NF-kB is also markedly activated with consequent tumor-necrosis-factor-alpha, interleukin-6, and inducible-nitric-oxide-synthase up-regulation. Treatment of IL-1β-stimulated chondrocytes with β-arrestin-1 and/or AKT and/or TAK-1-specific inhibitors significantly reduces all parameters, although the inhibitory effect exerted by TAK-1-mediated pathways is more effective than that of β-arrestin-1. β-Arrestin-1-induced NF-kB activation is mediated by the AKT pathway as shown by IL-1β-stimulated chondrocytes treated with AKT inhibitor. Finally, a specific HA-blocking peptide (Pep-1) has confirmed the inflammatory role of degraded HA as a mediator of the IL-1β-induced activation of β-arrestin-1. PMID:25673209

  8. Light-Dependent Phosphorylation of Bardet Biedl Syndrome 5 in Photoreceptor Cells Modulates its Interaction with Arrestin1

    PubMed Central

    Smith, Tyler S.; Spitzbarth, Benjamin; Li, Jian; Dugger, Donald R.; Stern-Schneider, Gabi; Sehn, Elisabeth; Bolch, Susan N.; McDowell, J. Hugh; Tipton, Jeremiah; Wolfrum, Uwe; Smith, W. Clay

    2013-01-01

    Arrestins are dynamic proteins which move between cell compartments triggered by stimulation of G-protein-coupled receptors. Even more dynamically in vertebrate photoreceptors, arrestin1 (Arr1) moves between the inner and outer segments according to the lighting conditions. Previous studies have shown that the light-driven translocation of Arr1 in rod photoreceptors is initiated by rhodopsin through a phospholipase C/protein kinase C (PKC) signaling cascade. The purpose of this study is to identify the PKC substrate that regulates the translocation of Arr1. Mass spectrometry was used to identify the primary phosphorylated proteins in extracts prepared from PKC-stimulated mouse eye cups, confirming the finding with in vitro phosphorylation assays. Our results show that BBS5 is the principal protein phosphorylated either by phorbol ester stimulation or by light stimulation of PKC. Via immunoprecipitation of BBS5 in rod outer segments, Arr1 was pulled down; phosphorylation of BBS5 reduced this co-precipitation of Arr1. Immunofluorescence and immunoelectron microscopy showed that BBS5 principally localizes along the axonemes of rods and cones, but also in photoreceptor inner segments, and synaptic regions. Our principal findings in this study are three-fold. First, we demonstrate that BBS5 is post-translationally regulated by phosphorylation via PKC, an event that is triggered by light in photoreceptor cells. Second, we find a direct interaction between BBS5 and Arr1, an interaction that is modulated by phosphorylation of BBS5. Finally, we show that BBS5 is distributed along the photoreceptor axoneme, co-localizing with Arr1 in the dark. These findings suggest a role for BBS5 in regulating light-dependent translocation of Arr1 and a model describing its role in Arr1 translocation is proposed. PMID:23817741

  9. Learning of Media Content: A Developmental Study

    ERIC Educational Resources Information Center

    Collins, W. Andrew

    1970-01-01

    Suggests an increase with age in children's ability to focus on essential content from a media presentation. Children in early adolescence seemed better able than younger ones to ignore nonessential information. (WY)

  10. CXCR7 Controls Competition for Recruitment of β-Arrestin 2 in Cells Expressing Both CXCR4 and CXCR7

    PubMed Central

    Coggins, Nathaniel L.; Trakimas, Danielle; Chang, S. Laura; Ehrlich, Anna; Ray, Paramita; Luker, Kathryn E.

    2014-01-01

    Chemokine CXCL12 promotes growth and metastasis of more than 20 different human cancers, as well as pathogenesis of other common diseases. CXCL12 binds two different receptors, CXCR4 and CXCR7, both of which recruit and signal through the cytosolic adapter protein β-arrestin 2. Differences in CXCL12-dependent recruitment of β-arrestin 2 in cells expressing one or both receptors remain poorly defined. To quantitatively investigate parameters controlling association of β-arrestin 2 with CXCR4 or CXCR7 in cells co-expressing both receptors, we used a systems biology approach combining real-time, multi-spectral luciferase complementation imaging with computational modeling. Cells expressing only CXCR4 maintain low basal association with β-arrestin 2, and CXCL12 induces a rapid, transient increase in this interaction. In contrast, cells expressing only CXCR7 have higher basal association with β-arrestin 2 and exhibit more gradual, prolonged recruitment of β-arrestin 2 in response to CXCL12. We developed and fit a data-driven computational model for association of either CXCR4 or CXCR7 with β-arrestin 2 in cells expressing only one type of receptor. We then experimentally validated model predictions that co-expression of CXCR4 and CXCR7 on the same cell substantially decreases both the magnitude and duration of CXCL12-regulated recruitment of β-arrestin 2 to CXCR4. Co-expression of both receptors on the same cell only minimally alters recruitment of β-arrestin 2 to CXCR7. In silico experiments also identified β-arrestin 2 as a limiting factor in cells expressing both receptors, establishing that CXCR7 wins the “competition” with CXCR4 for CXCL12 and recruitment of β-arrestin 2. These results reveal how competition for β-arrestin 2 controls integrated responses to CXCL12 in cells expressing both CXCR4 and CXCR7. These results advance understanding of normal and pathologic functions of CXCL12, which is critical for developing effective strategies to target

  11. Effects of the Dopamine D2 Allosteric Modulator, PAOPA, on the Expression of GRK2, Arrestin-3, ERK1/2, and on Receptor Internalization

    PubMed Central

    Basu, Dipannita; Tian, Yuxin; Bhandari, Jayant; Jiang, Jian Ru; Hui, Patricia; Johnson, Rodney L.; Mishra, Ram K.

    2013-01-01

    The activity of G protein-coupled receptors (GPCRs) is intricately regulated by a range of intracellular proteins, including G protein-coupled kinases (GRKs) and arrestins. Understanding the effects of ligands on these signaling pathways could provide insights into disease pathophysiologies and treatment. The dopamine D2 receptor is a GPCR strongly implicated in the pathophysiology of a range of neurological and neuropsychiatric disorders, particularly schizophrenia. Previous studies from our lab have shown the preclinical efficacy of a novel allosteric drug, 3(R)- [(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA), in attenuating schizophrenia-like behavioural abnormalities in rodent models of the disease. As an allosteric modulator, PAOPA binds to a site on the D2 receptor, which is distinct from the endogenous ligand-binding site, in order to modulate the binding of the D2 receptor ligand, dopamine. The exact signaling pathways affected by this allosteric modulator are currently unknown. The objectives of this study were to decipher the in vivo effects, in rats, of chronic PAOPA administration on D2 receptor regulatory and downstream molecules, including GRK2, arrestin-3 and extracellular receptor kinase (ERK) 1/2. Additionally, an in vitro cellular model was also used to study PAOPA’s effects on D2 receptor internalization. Results from western immunoblots showed that chronic PAOPA treatment increased the striatal expression of GRK2 by 41%, arrestin-3 by 34%, phospho-ERK1 by 51% and phospho-ERK2 by 36%. Results also showed that the addition of PAOPA to agonist treatment in cells increased D2 receptor internalization by 33%. This study provides the foundational evidence of putative signaling pathways, and changes in receptor localization, affected by treatment with PAOPA. It improves our understanding on the diverse mechanisms of action of allosteric modulators, while advancing PAOPA’s development into a novel drug for the improved

  12. A Functional Role for Anopheles gambiae Arrestin1 in Olfactory Signal Transduction

    PubMed Central

    Walker, William B.; Smith, Elaine M.; Jan, Taha; Zwiebel, L.J.

    2008-01-01

    Insect sensory arrestins act to desensitize visual and olfactory signal transduction pathways, as evidenced by the phenotypic effects of mutations in the genes encoding both Arr1 and Arr2 in Drosophila melanogaster. To assess whether such arrestins play similar roles in other, more medically relevant dipterans, we examined the ability of Anopheles gambiae sensory arrestin homologues AgArr1 and AgArr2 to rescue phenotypes associated with an olfactory deficit observed in D. melanogaster arrestin mutants. Of these, only AgArr1 facilitated significant phenotypic rescue of the corresponding Drosophila arr mutant olfactory phenotype, consistent with the view that functional orthology is shared by these Arr1 homologues. These results represent the first step in the functional characterization of AgArr1, which is highly expressed in olfactory appendages of An. gambiae in which it is likely to play an essential role in olfactory signal transduction. In addition to providing insight into the common elements of the peripheral olfactory system of dipterans, this work validates the importance of AgArr1 as a potential target for novel anti-malaria strategies that focus on olfactory-based behaviors in An. gambiae. PMID:18328499

  13. Development of an MRI biomarker sensitive to tetrameric visual arrestin 1 and its reduction via light-evoked translocation in vivo.

    PubMed

    Berkowitz, Bruce A; Gorgis, Jawan; Patel, Ankit; Baameur, Faiza; Gurevich, Vsevolod V; Craft, Cheryl M; Kefalov, Vladimir J; Roberts, Robin

    2015-02-01

    Rod tetrameric arrestin 1 (tet-ARR1), stored in the outer nuclear layer/inner segments in the dark, modulates photoreceptor synaptic activity; light exposure stimulates a reduction via translocation to the outer segments for terminating G-protein coupled phototransduction signaling. Here, we test the hypothesis that intraretinal spin-lattice relaxation rate in the rotating frame (1/T1ρ), an endogenous MRI contrast mechanism, has high potential for evaluating rod tet-ARR1 and its reduction via translocation. Dark- and light-exposed mice (null for the ARR1 gene, overexpressing ARR1, diabetic, or wild type with or without treatment with Mn2+, a calcium channel probe) were studied using 1/T1ρ MRI. Immunohistochemistry and single-cell recordings of the retinas were also performed. In wild-type mice with or without treatment with Mn2+, 1/T1ρ of avascular outer retina (64% to 72% depth) was significantly (P < 0.05) greater in the dark than in the light; a significant (P < 0.05) but opposite pattern was noted in the inner retina (<50% depth). Light-evoked outer retina Δ1/T1ρ was absent in ARR1-null mice and supernormal in overexpressing mice. In diabetic mice, the outer retinal Δ1/T1ρ pattern suggested normal dark-to-light tet-ARR1 translocation and chromophore content, conclusions confirmed ex vivo. Light-stimulated Δ1/T1ρ in inner retina was linked to changes in blood volume. Our data support 1/T1ρ MRI for noninvasively assessing rod tet-ARR1 and its reduction via protein translocation, which can be combined with other metrics of retinal function in vivo. PMID:25351983

  14. The role of beta-arrestin2 in the severity of antinociceptive tolerance and physical dependence induced by different opioid pain therapeutics

    PubMed Central

    Raehal, Kirsten M.; Bohn, Laura M.

    2010-01-01

    Ligands acting at the same receptor can differentially activate distinct signal transduction pathways, which in turn, can have diverse functional consequences. Further, receptors expressed in different tissues may utilize intracellular signaling proteins in response to a ligand differently as well. The mu opioid receptor (MOR), which mediates many of the pharmacological actions of opiate therapeutics, is also subject to differential signaling in response to diverse agonists. To study the effect of diverse agonists on MOR signaling, we examined the effects of chronic opiate treatment on two distinct physiological endpoints, antinociceptive tolerance and physical dependence, in mice lacking the intracellular regulatory molecule, βarrestin2. While βarrestin2 knockout (βarr2-KO) mice do not become tolerant to the antinociceptive effects of chronic morphine in a hot plate test, tolerance develops to the same degree in both wild type and βarr2-KO mice following chronic infusion with methadone, fentanyl, and oxycodone. Studies here also assess the severity of withdrawal signs precipitated by naloxone following chronic infusions at three different doses of each opiate agonist. While there are no differences in withdrawal responses between genotypes at the highest dose of morphine tested (48 mg/kg/day), the βarr2-KO mice display several less severe withdrawal responses when the infusion dose is lowered (12 or 24 mg/kg/day). Chronic infusion of methadone, fentanyl, and oxycodone all lead to equivalent naloxone-precipitated withdrawal responses in both genotypes at all doses tested. These results lend further evidence that distinct agonists can differentially impact on opioid-mediated responses in vivo in a βarrestin2-dependent manner. PMID:20713067

  15. Differential Phosphorylation Provides a Switch to Control How α-Arrestin Rod1 Down-regulates Mating Pheromone Response in Saccharomyces cerevisiae

    PubMed Central

    Alvaro, Christopher G.; Aindow, Ann; Thorner, Jeremy

    2016-01-01

    G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate stimulus-dependent activation of cognate heterotrimeric G-proteins, triggering ensuing downstream cellular responses. Tight regulation of GPCR-evoked pathways is required because prolonged stimulation can be detrimental to an organism. Ste2, a GPCR in Saccharomyces cerevisiae that mediates response of MATa haploids to the peptide mating pheromone α-factor, is down-regulated by both constitutive and agonist-induced endocytosis. Efficient agonist-stimulated internalization of Ste2 requires its association with an adaptor protein, the α-arrestin Rod1/Art4, which recruits the HECT-domain ubiquitin ligase Rsp5, allowing for ubiquitinylation of the C-terminal tail of the receptor and its engagement by the clathrin-dependent endocytic machinery. We previously showed that dephosphorylation of Rod1 by calcineurin (phosphoprotein phosphatase 2B) is required for optimal Rod1 function in Ste2 down-regulation. We show here that negative regulation of Rod1 by phosphorylation is mediated by two distinct stress-activated protein kinases, Snf1/AMPK and Ypk1/SGK1, and demonstrate both in vitro and in vivo that this phospho-regulation impedes the ability of Rod1 to promote mating pathway desensitization. These studies also revealed that, in the absence of its phosphorylation, Rod1 can promote adaptation independently of Rsp5-mediated receptor ubiquitinylation, consistent with recent evidence that α-arrestins can contribute to cargo recognition by both clathrin-dependent and clathrin-independent mechanisms. However, in cells lacking a component (formin Bni1) required for clathrin-independent entry, Rod1 derivatives that are largely unphosphorylated and unable to associate with Rsp5 still promote efficient adaptation, indicating a third mechanism by which this α-arrestin promotes desensitization of the pheromone-response pathway. PMID:26920760

  16. Differential Phosphorylation Provides a Switch to Control How α-Arrestin Rod1 Down-regulates Mating Pheromone Response in Saccharomyces cerevisiae.

    PubMed

    Alvaro, Christopher G; Aindow, Ann; Thorner, Jeremy

    2016-05-01

    G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate stimulus-dependent activation of cognate heterotrimeric G-proteins, triggering ensuing downstream cellular responses. Tight regulation of GPCR-evoked pathways is required because prolonged stimulation can be detrimental to an organism. Ste2, a GPCR in Saccharomyces cerevisiae that mediates response of MATa haploids to the peptide mating pheromone α-factor, is down-regulated by both constitutive and agonist-induced endocytosis. Efficient agonist-stimulated internalization of Ste2 requires its association with an adaptor protein, the α-arrestin Rod1/Art4, which recruits the HECT-domain ubiquitin ligase Rsp5, allowing for ubiquitinylation of the C-terminal tail of the receptor and its engagement by the clathrin-dependent endocytic machinery. We previously showed that dephosphorylation of Rod1 by calcineurin (phosphoprotein phosphatase 2B) is required for optimal Rod1 function in Ste2 down-regulation. We show here that negative regulation of Rod1 by phosphorylation is mediated by two distinct stress-activated protein kinases, Snf1/AMPK and Ypk1/SGK1, and demonstrate both in vitro and in vivo that this phospho-regulation impedes the ability of Rod1 to promote mating pathway desensitization. These studies also revealed that, in the absence of its phosphorylation, Rod1 can promote adaptation independently of Rsp5-mediated receptor ubiquitinylation, consistent with recent evidence that α-arrestins can contribute to cargo recognition by both clathrin-dependent and clathrin-independent mechanisms. However, in cells lacking a component (formin Bni1) required for clathrin-independent entry, Rod1 derivatives that are largely unphosphorylated and unable to associate with Rsp5 still promote efficient adaptation, indicating a third mechanism by which this α-arrestin promotes desensitization of the pheromone-response pathway. PMID:26920760

  17. Agonist-selective, Receptor-specific Interaction of Human P2Y Receptors with β-Arrestin-1 and -2*S⃞

    PubMed Central

    Hoffmann, Carsten; Ziegler, Nicole; Reiner, Susanne; Krasel, Cornelius; Lohse, Martin J.

    2008-01-01

    Interaction of G-protein-coupled receptors with β-arrestins is an important step in receptor desensitization and in triggering “alternative” signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with β-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with β-arrestin-1-GFP or β-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y1 receptor stimulated with ADP strongly translocated β-arrestin-2-YFP, whereas only a slight translocation was observed for β-arrestin-1-GFP. The P2Y4 receptor exhibited equally strong translocation for β-arrestin-1-GFP and β-arrestin-2-YFP when stimulated with UTP. The P2Y6, P2Y11, and P2Y12 receptor internalized only when GRK2 was additionally co-transfected, but β-arrestin translocation was only visible for the P2Y6 and P2Y11 receptor. The P2Y2 receptor showed a β-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated β-arrestin-1-GFP and β-arrestin-2-YFP equally well, whereas ATP translocated β-arrestin-1-GFP to a much lower extent than β-arrestin-2-YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y2 receptor and β-arrestins. Thus, the P2Y2 receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of β-arrestins by ATP and UTP stimulation of P2Y2 receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with β-arrestins. PMID:18703513

  18. Multifaceted roles of beta-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling.

    PubMed Central

    Shenoy, Sudha K; Lefkowitz, Robert J

    2003-01-01

    Beta-arrestins are cytosolic proteins that bind to activated and phosphorylated G-protein-coupled receptors [7MSRs (seven-membrane-spanning receptors)] and uncouple them from G-protein-mediated second messenger signalling pathways. The binding of beta-arrestins to 7MSRs also leads to new signals via activation of MAPKs (mitogen-activated protein kinases) such as JNK3 (c-Jun N-terminal kinase 3), ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 MAPKs. By binding to endocytic proteins [clathrin, AP2 (adapter protein 2), NSF (N -ethylmaleimide-sensitive fusion protein) and ARF6 (ADP-ribosylation factor 6)], beta-arrestins also serve as adapters to link the receptors to the cellular trafficking machinery. Agonist-promoted ubiquitination of beta-arrestins is a prerequisite for their role in receptor internalization, as well as a determinant of the differing trafficking patterns of distinct classes of receptors. Recently, beta-arrestins have also been implicated as playing novel roles in cellular chemotaxis and apoptosis. By virtue of their ability to bind, in a stimulus-dependent fashion, to 7MSRs as well as to different classes of cellular proteins, beta-arrestins serve as versatile adapter proteins that regulate the signalling and trafficking of the receptors. PMID:12959637

  19. ERK and β-arrestin interaction: a converging-point of signaling pathways for multiple types of cell-surface receptors

    PubMed Central

    Eishingdrelo, Haifeng; Sun, Wei; Li, Hua; Wang, Li; Eishingdrelo, Alex; Dai, Sheng; McKew, John C.; Zheng, Wei

    2016-01-01

    β-arrestin, a signal adaptor protein, mediates intracellular signal transductions through protein-protein interactions by bringing two or more proteins in proximity. Extracellular signal-regulated kinase (ERK), a protein kinase in the family of mitogen-activated protein kinases (MAPKs), is involved in various receptor signal pathways. Interaction of ERK with β-arrestin or formation of ERK/β-arrestin signal complex occurs in response to activation of a variety of cell-surface receptors. The ERK/β-arrestin signal complex may be a common transducer to converge a variety of extracellular stimuli to similar downstream intracellular signaling pathways. By using a cell based protein-protein interaction LinkLight assay technology, we demonstrate a direct interaction between ERK and β-arrestin in respond to extracellular stimuli, which can be sensitively and quantitatively monitored. Activations of G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and cytokine receptors promote formation of the ERK/β-arrestin signal complex. Our data indicate that the ERK/β-arrestin signal complex is a common transducer participated in a variety of receptor signaling pathways. Furthermore, we demonstrate that receptor antagonists or kinase inhibitors can block the agonist induced ERK and β-arrestin interaction. Thus, the ERK/β-arrestin interaction assay is useful for screening of new receptor modulators. PMID:25361946

  20. Binding of rhodopsin and rhodopsin analogues to transducin, rhodopsin kinase and arrestin-1

    PubMed Central

    Araujo, Nelson A; Sanz-Rodríguez, Carlos E; Bubis, José

    2014-01-01

    AIM: To investigate the interaction of reconstituted rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin with transducin, rhodopsin kinase and arrestin-1. METHODS: Rod outer segments (ROS) were isolated from bovine retinas. Following bleaching of ROS membranes with hydroxylamine, rhodopsin and rhodopsin analogues were generated with the different retinal isomers and the concentration of the reconstituted pigments was calculated from their UV/visible absorption spectra. Transducin and arrestin-1 were purified to homogeneity by column chromatography, and an enriched-fraction of rhodopsin kinase was obtained by extracting freshly prepared ROS in the dark. The guanine nucleotide binding activity of transducin was determined by Millipore filtration using β,γ-imido-(3H)-guanosine 5’-triphosphate. Recognition of the reconstituted pigments by rhodopsin kinase was determined by autoradiography following incubation of ROS membranes containing the various regenerated pigments with partially purified rhodopsin kinase in the presence of (γ-32P) ATP. Binding of arrestin-1 to the various pigments in ROS membranes was determined by a sedimentation assay analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. RESULTS: Reconstituted rhodopsin and rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal rendered an absorption spectrum showing a maximum peak at 498 nm, 486 nm and about 467 nm, respectively, in the dark; which was shifted to 380 nm, 404 nm and about 425 nm, respectively, after illumination. The percentage of reconstitution of rhodopsin and the rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal was estimated to be 88%, 81% and 24%, respectively. Although only residual activation of transducin was observed in the dark when reconstituted rhodopsin and 9-cis-retinal-rhodopsin was used, the rhodopsin analogue containing the 13-cis isomer of retinal was capable of activating transducin independently of light. Moreover

  1. Custom-designed proteins as novel therapeutic tools? The case of arrestins

    PubMed Central

    Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2010-01-01

    Multiple genetic disorders can be associated with excessive signalling by mutant G-protein-coupled receptors (GPCRs) that are either constitutively active or have lost sites where phosphorylation by GPCR kinases is necessary for desensitisation by cognate arrestins. Phosphorylation-independent arrestin1 can compensate for defects in phosphorylation of the GPCR rhodopsin in retinal rod cells, facilitating recovery, improving light responsiveness, and promoting photoreceptor survival. These proof-of-principle experiments show that, based on mechanistic understanding of the inner workings of a protein, one can modify its functional characteristics to generate custom-designed mutants that improve the balance of signalling in congenital and acquired disorders. Manipulations of arrestin elements responsible for scaffolding mitogen-activated protein kinase cascades and binding other signalling proteins involved in life-or-death decisions in the cell are likely to yield mutants that affect cell survival and proliferation in the desired direction. Although this approach is still in its infancy, targeted redesign of individual functions of many proteins offers a promise of a completely new therapeutic toolbox with huge potential. PMID:20412604

  2. Noncanonical Control of Vasopressin Receptor Type 2 Signaling by Retromer and Arrestin*

    PubMed Central

    Feinstein, Timothy N.; Yui, Naofumi; Webber, Matthew J.; Wehbi, Vanessa L.; Stevenson, Hilary P.; King, J. Darwin; Hallows, Kenneth R.; Brown, Dennis; Bouley, Richard; Vilardaga, Jean-Pierre

    2013-01-01

    The vasopressin type 2 receptor (V2R) is a critical G protein-coupled receptor (GPCR) for vertebrate physiology, including the balance of water and sodium ions. It is unclear how its two native hormones, vasopressin (VP) and oxytocin (OT), both stimulate the same cAMP/PKA pathway yet produce divergent antinatriuretic and antidiuretic effects that are either strong (VP) or weak (OT). Here, we present a new mechanism that differentiates the action of VP and OT on V2R signaling. We found that vasopressin, as opposed to OT, continued to generate cAMP and promote PKA activation for prolonged periods after ligand washout and receptor internalization in endosomes. Contrary to the classical model of arrestin-mediated GPCR desensitization, arrestins bind the VP-V2R complex yet extend rather than shorten the generation of cAMP. Signaling is instead turned off by the endosomal retromer complex. We propose that this mechanism explains how VP sustains water and Na+ transport in renal collecting duct cells. Together with recent work on the parathyroid hormone receptor, these data support the existence of a novel “noncanonical” regulatory pathway for GPCR activation and response termination, via the sequential action of β-arrestin and the retromer complex. PMID:23935101

  3. Autophagy-associated alpha-arrestin signaling is required for conidiogenous cell development in Magnaporthe oryzae.

    PubMed

    Dong, Bo; Xu, Xiaojin; Chen, Guoqing; Zhang, Dandan; Tang, Mingzhi; Xu, Fei; Liu, Xiaohong; Wang, Hua; Zhou, Bo

    2016-01-01

    Conidiation patterning is evolutionarily complex and mechanism concerning conidiogenous cell differentiation remains largely unknown. Magnaporthe oryzae conidiates in a sympodial way and uses its conidia to infect host and disseminate blast disease. Arrestins are multifunctional proteins that modulate receptor down-regulation and scaffold components of intracellular trafficking routes. We here report an alpha-arrestin that regulates patterns of conidiation and contributes to pathogenicity in M. oryzae. We show that disruption of ARRDC1 generates mutants which produce conidia in an acropetal array and ARRDC1 significantly affects expression profile of CCA1, a virulence-related transcription factor required for conidiogenous cell differentiation. Although germ tubes normally develop appressoria, penetration peg formation is dramatically impaired and Δarrdc1 mutants are mostly nonpathogenic. Fluorescent analysis indicates that EGFP-ARRDC1 puncta are well colocalized with DsRed2-Atg8, and this distribution profile could not be altered in Δatg9 mutants, suggesting ARRDC1 enters into autophagic flux before autophagosome maturation. We propose that M. oryzae employs ARRDC1 to regulate specific receptors in response to conidiation-related signals for conidiogenous cell differentiation and utilize autophagosomes for desensitization of conidiogenous receptor, which transmits extracellular signal to the downstream elements of transcription factors. Our investigation extends novel significance of autophagy-associated alpha-arrestin signaling to fungal parasites. PMID:27498554

  4. Autophagy-associated alpha-arrestin signaling is required for conidiogenous cell development in Magnaporthe oryzae

    PubMed Central

    Dong, Bo; Xu, Xiaojin; Chen, Guoqing; Zhang, Dandan; Tang, Mingzhi; Xu, Fei; Liu, Xiaohong; Wang, Hua; Zhou, Bo

    2016-01-01

    Conidiation patterning is evolutionarily complex and mechanism concerning conidiogenous cell differentiation remains largely unknown. Magnaporthe oryzae conidiates in a sympodial way and uses its conidia to infect host and disseminate blast disease. Arrestins are multifunctional proteins that modulate receptor down-regulation and scaffold components of intracellular trafficking routes. We here report an alpha-arrestin that regulates patterns of conidiation and contributes to pathogenicity in M. oryzae. We show that disruption of ARRDC1 generates mutants which produce conidia in an acropetal array and ARRDC1 significantly affects expression profile of CCA1, a virulence-related transcription factor required for conidiogenous cell differentiation. Although germ tubes normally develop appressoria, penetration peg formation is dramatically impaired and Δarrdc1 mutants are mostly nonpathogenic. Fluorescent analysis indicates that EGFP-ARRDC1 puncta are well colocalized with DsRed2-Atg8, and this distribution profile could not be altered in Δatg9 mutants, suggesting ARRDC1 enters into autophagic flux before autophagosome maturation. We propose that M. oryzae employs ARRDC1 to regulate specific receptors in response to conidiation-related signals for conidiogenous cell differentiation and utilize autophagosomes for desensitization of conidiogenous receptor, which transmits extracellular signal to the downstream elements of transcription factors. Our investigation extends novel significance of autophagy-associated alpha-arrestin signaling to fungal parasites. PMID:27498554

  5. X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex

    DOE PAGESBeta

    Zhou, X. Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; et al

    2016-04-12

    Here, serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solvedmore » with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.« less

  6. Desensitization of human CRF2(a) receptor signaling governed by agonist potency and βarrestin2 recruitment.

    PubMed

    Hauger, Richard L; Olivares-Reyes, J Alberto; Braun, Sandra; Hernandez-Aranda, Judith; Hudson, Christine C; Gutknecht, Eric; Dautzenberg, Frank M; Oakley, Robert H

    2013-09-10

    The primary goal was to determine agonist-specific regulation of CRF2(a) receptor function. Exposure of human retinoblastoma Y79 cells to selective (UCN2, UCN3 or stresscopins) and non-selective (UCN1 or sauvagine) agonists prominently desensitized CRF2(a) receptors in a rapid, concentration-dependent manner. A considerably slower rate and smaller magnitude of desensitization developed in response to the weak agonist CRF. CRF1 receptor desensitization stimulated by CRF, cortagine or stressin1-A had no effect on CRF2(a) receptor cyclic AMP signaling. Conversely, desensitization of CRF2(a) receptors by UCN2 or UCN3 did not cross-desensitize Gs-coupled CRF1 receptor signaling. In transfected HEK293 cells, activation of CRF2(a) receptors by UCN2, UCN3 or CRF resulted in receptor phosphorylation and internalization proportional to agonist potency. Neither protein kinase A nor casein kinases mediated CRF2(a) receptor phosphorylation or desensitization. Exposure of HEK293 or U2OS cells to UCN2 or UCN3 (100nM) produced strong βarrestin2 translocation and colocalization with membrane CRF2(a) receptors while CRF (1μM) generated only weak βarrestin2 recruitment. βarrestin2 did not internalize with the receptor, however, indicating that transient CRF2(a) receptor-arrestin complexes dissociate at or near the cell membrane. Since deletion of the βarrestin2 gene upregulated Gs-coupled CRF2(a) receptor signaling in MEF cells, a βarrestin2 mechanism restrains Gs-coupled CRF2(a) receptor signaling activated by urocortins. We further conclude that the rate and extent of homologous CRF2(a) receptor desensitization are governed by agonist-specific mechanisms affecting GRK phosphorylation, βarrestin2 recruitment, and internalization thereby producing unique signal transduction profiles that differentially affect the stress response. PMID:23820308

  7. Kappa Opioid Receptor Activation of p38 MAPK Is GRK3- and Arrestin-dependent in Neurons and Astrocytes*

    PubMed Central

    Bruchas, Michael R.; Macey, Tara A.; Lowe, Janet D.; Chavkin, Charles

    2007-01-01

    AtT-20 cells expressing the wild-type kappa opioid receptor (KOR) increased phospho-p38 MAPK following treatment with the kappa agonist U50,488. The increase was blocked by the kappa antagonist norbinaltorphimine and not evident in untransfected cells. In contrast, U50,488 treatment of AtT-20 cells expressing KOR having alanine substituted for serine-369 (KSA) did not increase phospho-p38. Phosphorylation of serine 369 in the KOR carboxyl terminus by G-protein receptor kinase 3 (GRK3) was previously shown to be required for receptor desensitization, and the results suggest that p38 MAPK activation by KOR may require arrestin recruitment. This hypothesis was tested by transfecting arrestin3-(R170E), a dominant positive form of arrestin that does not require receptor phosphorylation for activation. AtT-20 cells expressing both KSA and arrestin3-(R170E) responded to U50,488 treatment with an increase in phospho-p38 consistent with the hypothesis. Primary cultured astrocytes (glial fibrillary acidic protein-positive) and neurons (γ-aminobutyric acid-positive) isolated from mouse striata also responded to U50,488 by increasing phospho-p38 immunolabeling. p38 activation was not evident in either striatal astrocytes or neurons isolated from KOR knock-out mice or GRK3 knock-out mice. Astrocytes pretreated with small interfering RNA for arrestin3 were also unable to activate p38 in response to U50,488 treatment. Furthermore, in striatal neurons, the kappa-mediated phospho-p38 labeling was colocalized with arrestin3. These findings suggest that KOR may activate p38 MAPK in brain by a GRK3 and arrestin-dependent mechanism. PMID:16648139

  8. Arrestins 2 and 3 differentially regulate ETA and P2Y2 receptor-mediated cell signaling and migration in arterial smooth muscle

    PubMed Central

    Morris, Gavin E.; Nelson, Carl P.; Brighton, Paul J.; Standen, Nicholas B.; Challiss, R. A. John

    2012-01-01

    Overstimulation of endothelin type A (ETA) and nucleotide (P2Y) Gαq-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied Gαq-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca2+ levels were assessed in isolated ASMCs loaded with Ca2+-sensitive dyes, P2Y2 and ETA receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y2 receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ETA receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ETA and P2Y2 receptor-stimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease. PMID:22159081

  9. Arrestins 2 and 3 differentially regulate ETA and P2Y2 receptor-mediated cell signaling and migration in arterial smooth muscle.

    PubMed

    Morris, Gavin E; Nelson, Carl P; Brighton, Paul J; Standen, Nicholas B; Challiss, R A John; Willets, Jonathon M

    2012-03-01

    Overstimulation of endothelin type A (ET(A)) and nucleotide (P2Y) Gα(q)-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied Gα(q)-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca(2+) levels were assessed in isolated ASMCs loaded with Ca(2+)-sensitive dyes, P2Y(2) and ET(A) receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y(2) receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ET(A) receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ET(A) and P2Y(2) receptor-stimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease

  10. A Study in Content Language Acquisition

    ERIC Educational Resources Information Center

    Broer, Kathleen

    2003-01-01

    This study examines how young second language learners acquire academic language. Among the main language groups represented were Punjabi, Hindi, Tamil, Estonian, Serbian, Arabic as well as 23 other language groups. I monitored over 75 students, in grades 1, 2 and 4. I was interested in exploring what strategies best promoted coherence in…

  11. β-Arrestin-Selective G Protein-Coupled Receptor Agonists Engender Unique Biological Efficacy in Vivo

    PubMed Central

    Gesty-Palmer, Diane; Yuan, Ling; Martin, Bronwen; Wood, William H.; Lee, Mi-Hye; Janech, Michael G.; Tsoi, Lam C.; Zheng, W. Jim; Maudsley, Stuart

    2013-01-01

    Biased G protein-coupled receptor agonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors. In vitro, d-Trp12,Tyr34-bPTH(7–34) [bPTH(7–34)], a biased agonist for the type 1 PTH receptor, antagonizes receptor-G protein coupling but activates arrestin-dependent signaling. In vivo, both bPTH(7–34) and the conventional agonist hPTH(1–34) stimulate anabolic bone formation. To understand how two PTH receptor ligands with markedly different in vitro efficacy could elicit similar in vivo responses, we analyzed transcriptional profiles from calvarial bone of mice treated for 8 wk with vehicle, bPTH(7–34) or hPTH(1–34). Treatment of wild-type mice with bPTH(7–34) primarily affected pathways that promote expansion of the osteoblast pool, notably cell cycle regulation, cell survival, and migration. These responses were absent in β-arrestin2-null mice, identifying them as downstream targets of β-arrestin2-mediated signaling. In contrast, hPTH(1–34) primarily affected pathways classically associated with enhanced bone formation, including collagen synthesis and matrix mineralization. hPTH(1–34) actions were less dependent on β-arrestin2, as might be expected of a ligand capable of G protein activation. In vitro, bPTH(7–34) slowed the rate of preosteoblast proliferation, enhanced osteoblast survival when exposed to an apoptotic stimulus, and stimulated cell migration in wild-type, but not β-arrestin2-null, calvarial osteoblasts. These results suggest that bPTH(7–34) and hPTH(1–34) affect bone mass in vivo through predominantly separate genomic mechanisms created by largely distinct receptor-signaling networks and demonstrate that functional selectivity can be exploited to change the quality of G protein-coupled receptor efficacy. PMID:23315939

  12. Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling

    PubMed Central

    Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi; Asbury, Charles L.; Hille, Bertil

    2016-01-01

    Activated Gq protein–coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein–tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein–coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to

  13. Regulation of amygdalar PKA by β-arrestin-2/phosphodiesterase-4 complex is critical for fear conditioning

    PubMed Central

    Li, Yuting; Li, Haohong; Liu, Xing; Bao, Guobin; Tao, Yezheng; Wu, Ziyan; Xia, Peng; Wu, Chunfu; Li, Baoming; Ma, Lan

    2009-01-01

    β-arrestins, key regulators of receptor signaling, are highly expressed in the central nervous system, but their roles in brain physiology are largely unknown. Here we show that β-arrestin-2 is critically involved in the formation of associative fear memory and amygdalar synaptic plasticity. In response to fear conditioning, β-arrestin-2 translocates to amygdalar membrane where it interacts with PDE-4, a cAMP-degrading enzyme, to inhibit PKA activation. Arrb2−/− mice exhibit impaired conditioned fear memory and long-term potentiation at the lateral amygdalar synapses. Moreover, expression of the β-arrestin-2 in the lateral amygdala of Arrb2−/− mice, but not its mutant form that is incapable of binding PDE-4, restores basal PKA activity and rescues conditioned fear memory. Taken together, our data demonstrate that the feedback regulation of amygdalar PKA activation by β-arrestin-2 and PDE-4 complex is critical for the formation of conditioned fear memory. PMID:19955404

  14. Insulin-Like Growth Factor-1 Contributes to Mucosal Repair by β-Arrestin2-Mediated Extracellular Signal-Related Kinase Signaling in Experimental Colitis.

    PubMed

    Chen, Tingting; Zheng, Fengping; Tao, Jin; Tan, Siwei; Zeng, Lixian; Peng, Xiaojie; Wu, Bin

    2015-09-01

    Insulin-like growth factor-1 (IGF-1) possesses the ability to attenuate intestinal damage and promote mucosal repair of colitis. β-Arrestins, as the scaffolding proteins of G protein-coupled receptors or non-G protein-coupled receptors signaling, can be involved in IGF-1-mediated signaling pathways. However, the interaction of IGF-1 and β-arrestin2 in the mucosal repair of experimental colitis remains unexplored. Ulcerative colitis was induced in β-arrestin2 wild-type mice and β-arrestin2 knockout littermates by using 3% dextran sulfate sodium for 5 days, followed by regular water consumption for 1, 2, 3, and 4 weeks to analyze the mucosal repair from experimental colitis. Disease activity index and histologic score analyses were performed. Apoptosis and proliferation were assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and Ki-67 staining, respectively. The expressions of β-arrestin2, phospho (p)-IGF-1R, and p-extracellular signal-regulated kinase (ERK)1/2 were examined. Furthermore, β-arrestin2 was overexpressed or altered in HCT116 cells by transfection before IGF-1 treatment in vitro. IGF-1 and β-arrestin2 expression was up-regulated in the repairing phase of experimental colitis. Targeted deletion of β-arrestin2 delayed the repair of colitis by inhibiting cell proliferation without affecting the levels of IGF-1 and p-IGF-1R. The β-arrestin2/ERK signaling pathway was involved in IGF-1-mediated mucosal repair through promoting epithelial cell and goblet cell regeneration from experimental colitis. These results indicate that IGF-1 contributes to the mucosal repair by β-arrestin2-mediated ERK signaling in experimental colitis. PMID:26362717

  15. The calcium-sensing receptor changes cell shape via a beta-arrestin-1 ARNO ARF6 ELMO protein network.

    PubMed

    Bouschet, Tristan; Martin, Stéphane; Kanamarlapudi, Venkateswarlu; Mundell, Stuart; Henley, Jeremy M

    2007-08-01

    G-protein-coupled receptors (GPCRs) transduce the binding of extracellular stimuli into intracellular signalling cascades that can lead to morphological changes. Here, we demonstrate that stimulation of the calcium-sensing receptor (CaSR), a GPCR that promotes chemotaxis by detecting increases in extracellular calcium, triggers plasma membrane (PM) ruffling via a pathway that involves beta-arrestin 1, Arf nucleotide binding site opener (ARNO), ADP-ribosylating factor 6 (ARF6) and engulfment and cell motility protein (ELMO). Expression of dominant negative beta-arrestin 1 or its knockdown with siRNA impaired the CaSR-induced PM ruffling response. Expression of a catalytically inactive ARNO also reduced CaSR-induced PM ruffling. Furthermore, beta-arrestin 1 co-immunoprecipitated with the CaSR and ARNO under resting conditions. Agonist treatment did not markedly alter beta-arrestin 1 binding to the CaSR or to ARNO but it did elicit the translocation and colocalisation of the CaSR, beta-arrestin 1 and ARNO to membrane protrusions. Furthermore, ARF6 and ELMO, two proteins known to couple ARNO to the cytoskeleton, were required for CaSR-dependent morphological changes and translocated to the PM ruffles. These data suggest that cells ruffle upon CaSR stimulation via a mechanism that involves translocation of beta-arrestin 1 pre-assembled with the CaSR or ARNO, and that ELMO plays an essential role in this CaSR-signalling-induced cytoskeletal reorganisation. PMID:17623778

  16. Dissociating ghrelin-dependent G protein from β-arrestin-2 signaling in transgenic rats.

    PubMed

    Smith, Roy G

    2016-01-01

    The gut-derived hormone ghrelin regulates growth hormone release, appetite, metabolism, and immune function through its receptor, the growth hormone secretagogue receptor 1a (GHSR1a). In this issue ofScience Signaling, Chebaniet al decode GHSR1a signaling by using transgenic rats with a mutation in GHSR1a that prevents its interaction with β-arrestin. These mutant rats are more responsive to endogenous ghrelin, resulting in increased fat deposition and insulin resistance without affecting food intake. PMID:27095592

  17. Suppression of adrenal βarrestin1-dependent aldosterone production by ARBs: head-to-head comparison

    PubMed Central

    Dabul, Samalia; Bathgate-Siryk, Ashley; Valero, Thairy Reyes; Jafferjee, Malika; Sturchler, Emmanuel; McDonald, Patricia; Koch, Walter J.; Lymperopoulos, Anastasios

    2015-01-01

    The known angiotensin II (AngII) physiological effect of aldosterone synthesis and secretion is mediated by either Gq/11 proteins or βarrestin1 (βarr1), both of which can couple to its type 1 receptors (AT1Rs), present in adrenocortical zona glomerulosa (AZG) cell membranes. In the present study, we examined the relative potencies of all the currently used in the clinic AT1R antagonist drugs (angiotensin receptor blockers, ARBs, or sartans) at preventing activation of these two signaling mediators (G proteins and βarrs) at the AngII-bound AT1R and, consequently, at suppression of aldosterone in vitro. All ARBs were found to be potent inhibitors of G protein activation at the AT1R. However, candesartan and valsartan were the most potent at blocking AngII-induced βarr activation at this receptor, among the tetrazolo-biphenyl-methyl derivatives, translating into excellent efficacies at aldosterone suppression in H295R cells. Conversely, irbesartan and losartan were largely G protein-selective inhibitors at the AT1R, with very low potency towards βarr inhibition. As a result, they were very weak suppressors of βarr1-dependent aldosterone production in H295R cells. These findings provide important pharmacological insights into the drug class of ARBs and medicinal chemistry insights for future drug development in the field of AngII antagonism. PMID:25631300

  18. Distinct phosphorylation sites on the ghrelin receptor, GHSR1a, establish a code that determines the functions of ß-arrestins.

    PubMed

    Bouzo-Lorenzo, Monica; Santo-Zas, Icía; Lodeiro, Maria; Nogueiras, Rubén; Casanueva, Felipe F; Castro, Marian; Pazos, Yolanda; Tobin, Andrew B; Butcher, Adrian J; Camiña, Jesús P

    2016-01-01

    The growth hormone secretagogue receptor, GHSR1a, mediates the biological activities of ghrelin, which includes the secretion of growth hormone, as well as the stimulation of appetite, food intake and maintenance of energy homeostasis. Mapping phosphorylation sites on GHSR1a and knowledge of how these sites control specific functional consequences unlocks new strategies for the development of therapeutic agents targeting individual functions. Herein, we have identified the phosphorylation of different sets of sites within GHSR1a which engender distinct functionality of ß-arrestins. More specifically, the Ser(362), Ser(363) and Thr(366) residues at the carboxyl-terminal tail were primarily responsible for ß-arrestin 1 and 2 binding, internalization and ß-arrestin-mediated proliferation and adipogenesis. The Thr(350) and Ser(349) are not necessary for ß-arrestin recruitment, but are involved in the stabilization of the GHSR1a-ß-arrestin complex in a manner that determines the ultimate cellular consequences of ß-arrestin signaling. We further demonstrated that the mitogenic and adipogenic effect of ghrelin were mainly dependent on the ß-arrestin bound to the phosphorylated GHSR1a. In contrast, the ghrelin function on GH secretion was entirely mediated by G protein signaling. Our data is consistent with the hypothesis that the phosphorylation pattern on the C terminus of GHSR1a determines the signaling and physiological output. PMID:26935831

  19. Distinct phosphorylation sites on the ghrelin receptor, GHSR1a, establish a code that determines the functions of ß-arrestins

    PubMed Central

    Bouzo-Lorenzo, Monica; Santo-Zas, Icía; Lodeiro, Maria; Nogueiras, Rubén; Casanueva, Felipe F.; Castro, Marian; Pazos, Yolanda; Tobin, Andrew B; Butcher, Adrian J.; Camiña, Jesús P.

    2016-01-01

    The growth hormone secretagogue receptor, GHSR1a, mediates the biological activities of ghrelin, which includes the secretion of growth hormone, as well as the stimulation of appetite, food intake and maintenance of energy homeostasis. Mapping phosphorylation sites on GHSR1a and knowledge of how these sites control specific functional consequences unlocks new strategies for the development of therapeutic agents targeting individual functions. Herein, we have identified the phosphorylation of different sets of sites within GHSR1a which engender distinct functionality of ß-arrestins. More specifically, the Ser362, Ser363 and Thr366 residues at the carboxyl-terminal tail were primarily responsible for ß-arrestin 1 and 2 binding, internalization and ß-arrestin-mediated proliferation and adipogenesis. The Thr350 and Ser349 are not necessary for ß-arrestin recruitment, but are involved in the stabilization of the GHSR1a-ß-arrestin complex in a manner that determines the ultimate cellular consequences of ß-arrestin signaling. We further demonstrated that the mitogenic and adipogenic effect of ghrelin were mainly dependent on the ß-arrestin bound to the phosphorylated GHSR1a. In contrast, the ghrelin function on GH secretion was entirely mediated by G protein signaling. Our data is consistent with the hypothesis that the phosphorylation pattern on the C terminus of GHSR1a determines the signaling and physiological output. PMID:26935831

  20. Social Studies and Grade Level Content Expectations in Michigan

    ERIC Educational Resources Information Center

    DeChano-Cook, Lisa M.

    2012-01-01

    In 2007 the Michigan Department of Education (MDOE) unveiled Social Studies Grade Level Content Expectations (GLCEs) that went into implementation during the 2008-2009 academic year. The purpose of this research was to examine social studies teaching in grades K-8 and what effects, if any, the GLCEs had on the curriculum in these grades.…

  1. Pedagogical Content Knowledge: A Case Study of ESL Teacher Educator

    ERIC Educational Resources Information Center

    Liu, Siping

    2013-01-01

    This single-case study focuses on the pedagogical content knowledge (PCK) of a university faculty member teaching Second Language Acquisition to elementary teacher candidates. The research questions address the pattern and development of PCK for ESL teaching. Based on data from classroom observation, interviews and document review, the study finds…

  2. General Religious Studies Content in the Psychology of Religion.

    ERIC Educational Resources Information Center

    Goldsmith, W. Mack

    Results of a survey to determine student demand for religious studies content in introductory psychology and sociology of religion courses are discussed. The survey was administered to 382 members of the American Psychological Association and the Society for the Scientific Study of Religion. Respondents reported that approximately one-fourth of…

  3. Internalization of gonadotropin-releasing hormone receptors (GnRHRs): does arrestin binding to the C-terminal tail target GnRHRs for dynamin-dependent internalization?

    PubMed

    Hislop, James N; Caunt, Christopher J; Sedgley, Kathleen R; Kelly, Eammon; Mundell, Stuart; Green, Lisa D; McArdle, Craig A

    2005-08-01

    Activation of seven-transmembrane receptors is typically followed by desensitization and arrestin-dependent internalization via vesicles that are pinched off by a dynamin collar. Arrestins also scaffold Src, which mediates dynamin-dependent internalization of beta2-adrenergic receptors. Type I mammalian gonadotropin-releasing hormone receptors (GnRHRs) do not rapidly desensitize or internalize (characteristics attributed to their unique lack of C-terminal tails) whereas non-mammalian GnRHRs (that have C-terminal tails) are rapidly internalized and desensitized. Moreover, internalization of Xenopus (X) GnRHRs is dynamin-dependent whereas that of human (h) GnRHRs is not, raising the possibility that binding of arrestin to the C-terminal tails of GnRHRs targets them to the dynamin-dependent internalization pathway. To test this we have compared wild-type GnRHRs with chimeric receptors (XGnRHR C-terminal tail added to the hGnRHR alone (h.XtGnRHR) or with exchange of the third intracellular loops (h.Xl.XtGnRHR)). We show that adding the XGnRHR C-terminal tail facilitates arrestin- and dynamin-dependent internalization as well as arrestin/green fluorescent protein translocation, but Src (or mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase) inhibition does not slow internalization, and h.XtGnRHR internalization is slower than that of the hGnRHR. Moreover, arrestin expression increased XGnRHR internalization even when dynamin was inhibited and h.Xl.XtGnRHR underwent rapid arrestin-dependent internalization without signaling to G(q/11). Thus, although the C-terminal tail can direct GnRHRs for arrestin- and dynamin-dependent internalization, this effect is not dependent on Src activation and arrestin can also facilitate dynamin-independent internalization. PMID:16087731

  4. β-Arrestin 2 is required for B1 receptor-dependent post-translational activation of inducible nitric oxide synthase

    PubMed Central

    Kuhr, Frank K.; Zhang, Yongkang; Brovkovych, Viktor; Skidgel, Randal A.

    2010-01-01

    A major source of “high-output” NO in inflammation is inducible nitric oxide synthase (iNOS). iNOS is primarily transcriptionally regulated and is thought to function as an uncontrolled generator of high NO. We found that iNOS in cytokine-stimulated human lung microvascular endothelial cells (HLMVECs) is highly regulated post-translationally via activation of the B1 kinin G protein-coupled receptor (B1R). We report here that B1R-mediated iNOS activation was significantly inhibited by knockdown of β-arrestin 2 with siRNA in cytokine-treated HLMVECs or HEK293 cells transfected with iNOS and B1R. In contrast, β-arrestin 1 siRNA had no effect. The prolonged phase of B1R-dependent ERK activation was also inhibited by β-arrestin 2 knockdown. Furthermore, robust ERK activation by the epidermal growth factor receptor (a β-arrestin 2 independent pathway) had no effect on iNOS-derived NO production. β-arrestin 2 and iNOS coimmunoprecipitated, and there was significant fluorescence resonance energy transfer between CFP-iNOS and β-arrestin 2-YFP (but not β-arrestin 1-YFP) that increased 3-fold after B1R stimulation. These data show that β-arrestin 2 mediates B1R-dependent high-output NO by scaffolding iNOS and ERK to allow post-translational activation of iNOS. This could play a critical role in mediating endothelial function in inflammation.—Kuhr, F. K., Zhang, Y., Brovkovych, V., Skidgel, R. A. β-Arrestin 2 is required for B1 receptor-dependent post-translational activation of inducible nitric oxide synthase. PMID:20228252

  5. Competing G protein-coupled receptor kinases balance G protein and β-arrestin signaling

    PubMed Central

    Heitzler, Domitille; Durand, Guillaume; Gallay, Nathalie; Rizk, Aurélien; Ahn, Seungkirl; Kim, Jihee; Violin, Jonathan D; Dupuy, Laurence; Gauthier, Christophe; Piketty, Vincent; Crépieux, Pascale; Poupon, Anne; Clément, Frédérique; Fages, François; Lefkowitz, Robert J; Reiter, Eric

    2012-01-01

    Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT1AR) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on β-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT1AR, and HEK293 cells expressing other 7TMRs. PMID:22735336

  6. Tango assay for ligand-induced GPCR-β-arrestin2 interaction: Application in drug discovery.

    PubMed

    Dogra, Shalini; Sona, Chandan; Kumar, Ajeet; Yadav, Prem N

    2016-01-01

    G protein-coupled receptors (GPCRs) are widely known to modulate almost all physiological functions and have been demonstrated over the time as therapeutic targets for wide gamut of diseases. The design and implementation of high-throughput GPCR-based assays that permit the efficient screening of large compound libraries to discover novel drug candidates are essential for a successful drug discovery endeavor. Usually, GPCR-based functional assays depend primarily on the measurement of G protein-mediated second messenger generation. However, with advent of advanced molecular biology tools and increased understanding of GPCR signal transduction, many G protein-independent pathways such as β-arrestin translocation are being utilized to detect the activity of GPCRs. These assays provide additional information on functional selectivity (also known as biased agonism) of compounds that could be harnessed to develop pathway-selective drug candidates to reduce the adverse effects associated with given GPCR target. In this chapter, we describe the basic principle, detailed methodologies and assay setup, result analysis and data interpretations of the β-arrestin2 Tango assay, and its comparison with cell-based G protein-dependent GPCR assays, which could be employed in a simple academic setup to facilitate GPCR-based drug discovery. PMID:26928547

  7. Mathematics Education Research in Turkey: A Content Analysis Study

    ERIC Educational Resources Information Center

    Ciltas, Alper; Guler, Gursel; Sozbilir, Mustafa

    2012-01-01

    In this study, a content analysis of research is aimed in the field of mathematics education of Turkish researchers. To this aim, the investigation of 359 article were made which were accessed from web in full text between 1987 and 2009 years and which were published in the field of mathematics education from 32 different journals. 27 of these…

  8. Children's Content Interest--A Factor Analytic Study.

    ERIC Educational Resources Information Center

    Feeley, Joan T.

    Recognizing that interest is essential to motivation, this study was designed both to identify and describe the content interest patterns and media preferences (print and television) of middle-grade children and to determine any relationship between these interests and sex, race, or socioeconomic status (SES). An inventory was administered to 250…

  9. Integrating Language and Content Learning in the Social Studies Classroom.

    ERIC Educational Resources Information Center

    Kang, Hee-Won; LeSourd, Sandra J.

    This paper focuses on helping social studies teachers discover ways to help second language students comprehend, use, and learn language as well as content in the classroom. Activities conducive to this purpose include: providing contextual support such as pictures, globes, videotapes, diagrams, body and facial gestures, pantomime and role…

  10. Disrupted-in-schizophrenia-1 Gln31Leu polymorphism results in social anhedonia associated with monoaminergic imbalance and reduction of CREB and β-arrestin-1,2 in the nucleus accumbens in a mouse model of depression.

    PubMed

    Lipina, Tatiana V; Fletcher, Paul J; Lee, Frankie H; Wong, Albert H C; Roder, John C

    2013-02-01

    Disrupted-in-schizophrenia-1 (DISC1) is associated with mental disorders, including major depression. We previously showed that DISC1-Q31L mutant mice have depression-like behaviors and can therefore be used to study neurobiological mechanisms of depression and antidepressant (AD) medication action. First, we found reduced levels of dopamine, serotonin and norepinephrine in the nucleus accumbens (NAC) of DISC1-Q31L mutants. Next, we assessed social-conditioned place preference as a reward-dependent task and the capacity of distinct ADs to correct impaired social behavior in DISC1-Q31L mice. Bupropion, but not fluoxetine or desipramine, was able to correct deficient social facilitation, social reward, and social novelty in DISC1-Q31L mutants, whereas all three ADs were able to improve social motivation and behavioral despair in DISC1-Q31L mutants. Furthermore, we sought to correlate social anhedonia with molecular and cellular features including dendritic spine density, β-arrestin-1,2, and cAMP-response-element-binding protein (CREB) in the NAC as biomarkers related to depression and the DISC1 pathway. DISC1-Q31L mutants showed reduced levels of β-arrestin-1,2, CREB, and spine density in the NAC, further supporting the construct validity of the genetic model. Bupropion induced the greatest effect on CREB in DISC1-Q31L mutants, whereas all studied ADs corrected the reduced levels of β-arrestin-1,2 and modestly ameliorated deficient spine density in this brain region. Overall, we find neurobiological changes accompanying social anhedonia in the NAC of DISC1-Q31L mutant mice, consistent with a role for DISC1 in regulating social reward as an endophenotype of depression. PMID:23011268

  11. RalA employs GRK2 and β-arrestins for the filamin A-mediated regulation of trafficking and signaling of dopamine D2 and D3 receptor.

    PubMed

    Zheng, Mei; Zhang, Xiaohan; Sun, NingNing; Min, Chengchun; Zhang, Xiaowei; Kim, Kyeong-Man

    2016-08-01

    Filamin A (FLNA) is known to act as platform for the signaling and intracellular trafficking of various GPCRs including dopamine D2 and D3 receptors (D2R, D3R). To understand molecular mechanisms involved in the FLNA-mediated regulation of D2R and D3R, comparative studies were conducted on the signaling and intracellular trafficking of the D2R and D3R in FLNA-knockdown cells, with a specific focus on the roles of the proteins that interact with FLNA and the D2R and D3R. Lowering the level of cellular FLNA caused an elevation in RalA activity and resulted in selective interference with the normal intracellular trafficking and signaling of the D2R and D3R, through GRK2 and β-arrestins, respectively. Knockdown of FLNA or coexpression of active RalA interfered with the recycling of the internalized D2R and resulted in the development of receptor tolerance. Active RalA was found to interact with GRK2 to sequester it from D2R. Knockdown of FLNA or coexpression of active RalA prevented D3R from coupling with G protein. The selective involvement of GRK2- and β-arrestins in the RalA-mediated cellular processes of the D2R and D3R was achieved via their different modes of interactions with the receptor and their distinct functional roles in receptor regulation. Our results show that FLNA is a multi-functional protein that acts as a platform on which D2R and D3R can interact with various proteins, through which selective regulation of these receptors occurs in combination with GRK2 and β-arrestins. PMID:27188791

  12. Genetic association between G protein-coupled receptor kinase 6/β-arrestin 2 and dopamine supersensitivity psychosis in schizophrenia

    PubMed Central

    Oda, Yasunori; Kanahara, Nobuhisa; Kimura, Hiroshi; Watanabe, Hiroyuki; Hashimoto, Kenji; Iyo, Masaomi

    2015-01-01

    Background/aim Dopamine supersensitivity psychosis (DSP), clinically characterized by unstable and severe psychosis or tardive dyskinesia and often categorized as treatment-resistant schizophrenia, is promoted by long-term antipsychotic treatment. An upregulation of the dopamine D2 receptor caused by antipsychotic(s) is involved in the development of DSP. The present study explored the potential roles of G protein-coupled receptor kinase 6 (GRK6) and β-arrestin 2 (ARRB2) that are involved in the trafficking of DRD2 in patients with DSP. Methods We conducted a genetic association study of GRK6/ARRB2 between the patients with DSP episodes [DSP(+) group: N=108] and the patients without DSP(−) episodes [DSP(−) group: N=169] from the total group of patients (N=333). Based on the patients’ treatment history, a DSP episode was defined as withdrawal psychosis, developed tolerance to antipsychotic effect, and tardive dyskinesia (the remaining 56 patients were excluded due to insufficient information). Results The results revealed that none of the allelic or genotyping distributions of five single nucleotide polymorphisms (SNPs) of GRK6 and three SNPs of ARRB2 showed any significant difference between the DSP(+) and DSP(−) groups. Conclusion The results suggest that the SNP analyses of these two molecules fail to classify patients into the potential clinical subtype of DSP(+) or DSP(−) group. However, since GRK6 and ARRB2 are surely involved in dopamine D2 receptor metabolism, further studies based on prospective observations of the onset of DSP under specific antipsychotic treatments are needed. PMID:26251601

  13. Regulatory arrestin cycle secures the fidelity and maintenance of the fly photoreceptor cell.

    PubMed Central

    Byk, T; Bar-Yaacov, M; Doza, Y N; Minke, B; Selinger, Z

    1993-01-01

    Excitation of fly photoreceptor cells is initiated by photoisomerization of rhodopsin to the active form of metarhodopsin. Fly metarhodopsin is thermostable, does not bleach, and does not regenerate spontaneously to rhodopsin. For this reason, the activity of metarhodopsin must be stopped by an effective termination reaction. On the other hand, there is also a need to restore the inactivated photopigment to an excitable state in order to keep a sufficient number of photopigment molecules available for excitation. The following findings reveal how these demands are met. The photopigment undergoes rapid phosphorylation upon photoconversion of rhodopsin to metarhodopsin and an efficient Ca2+ dependent dephosphorylation upon regeneration of metarhodopsin to rhodopsin. Phosphorylation decreases the ability of metarhodopsin to activate the guanine nucleotide-binding protein. Binding of 49-kDa arrestin further quenches the activity of metarhodopsin and protects it from dephosphorylation. Light-dependent binding and release of 49-kDa arrestin from metarhodopsin- and rhodopsin-containing membranes, respectively, directs the dephosphorylation reaction toward rhodopsin. This ensures the return of phosphorylated metarhodopsin to the rhodopsin pool without initiating transduction in the dark. Assays of rhodopsin dephosphorylation in the Drosophila retinal degeneration C (rdgC) mutant, a mutant in a gene previously cloned and predicted to encode a serine/threonine protein phosphatase, reveal that phosphorylated rhodopsin is a major substrate for the rdgC phosphatase. We propose that mutations resulting in either a decrease or an improper regulation of rhodopsin phosphatase activity bring about degeneration of the fly photoreceptor cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8446607

  14. Role of rhodopsin and arrestin phosphorylation in retinal degeneration of Drosophila.

    PubMed

    Kristaponyte, Inga; Hong, Yuan; Lu, Haiqin; Shieh, Bih-Hwa

    2012-08-01

    Arrestins belong to a family of multifunctional adaptor proteins that regulate internalization of diverse receptors including G-protein-coupled receptors (GPCRs). Defects associated with endocytosis of GPCRs have been linked to human diseases. We used enhanced green fluorescent protein-tagged arrestin 2 (Arr2) to monitor the turnover of the major rhodopsin (Rh1) in live Drosophila. We demonstrate that during degeneration of norpA(P24) photoreceptors the loss of Rh1 is parallel to the disappearance of rhabdomeres, the specialized visual organelle that houses Rh1. The cause of degeneration in norpA(P24) is the failure to activate CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) and retinal degeneration C (RDGC) because of a loss of light-dependent Ca(2+) entry. A lack of activation in CaMKII, which phosphorylates Arr2, leads to hypophosphorylated Arr2, while a lack of activation of RDGC, which dephosphorylates Rh1, results in hyperphosphorylated Rh1. We investigated how reversible phosphorylation of Rh1 and Arr2 contributes to photoreceptor degeneration. To uncover the consequence underlying a lack of CaMKII activation, we characterized ala(1) flies in which CaMKII was suppressed by an inhibitory peptide, and showed that morphology of rhabdomeres was not affected. In contrast, we found that expression of phosphorylation-deficient Rh1s, which either lack the C terminus or contain Ala substitution in the phosphorylation sites, was able to prevent degeneration of norpA(P24) photoreceptors. This suppression is not due to a loss of Arr2 interaction. Importantly, co-expression of these modified Rh1s offered protective effects, which greatly delayed photoreceptor degeneration. Together, we conclude that phosphorylation of Rh1 is the major determinant that orchestrates its internalization leading to retinal degeneration. PMID:22855823

  15. Non-Hematopoietic β-Arrestin1 Confers Protection Against Experimental Colitis.

    PubMed

    Lee, Taehyung; Lee, Eunhee; Arrollo, David; Lucas, Peter C; Parameswaran, Narayanan

    2016-05-01

    β-Arrestins are multifunctional scaffolding proteins that modulate G protein-coupled receptor (GPCR)-dependent and -independent cell signaling pathways in various types of cells. We recently demonstrated that β-arrestin1 (β-arr1) deficiency strikingly attenuates dextran sodium sulfate (DSS)-induced colitis in mice. Since DSS-induced colitis is in part dependent on gut epithelial injury, we examined the role of β-arr1 in intestinal epithelial cells (IECs) using a colon epithelial cell line, SW480 cells. Surprisingly, we found that knockdown of β-arr1 in SW480 cells enhanced epithelial cell death via a caspase-3-dependent process. To understand the in vivo relevance and potential cell type-specific role of β-arr1 in colitis development, we generated bone marrow chimeras with β-arr1 deficiency in either the hematopoietic or non-hematopoietic compartment. Reconstituted chimeric mice were then subjected to DSS-induced colitis. Similar to our previous findings, β-arr1 deficiency in the hematopoietic compartment protected mice from DSS-induced colitis. However, consistent with the role of β-arr1 in epithelial apoptosis in vitro, non-hematopoietic β-arr1 deficiency led to an exacerbated colitis phenotype. To further understand signaling mechanisms, we examined the effect of β-arr1 on TNF-α-mediated NFκB and MAPK pathways. Our results demonstrate that β-arr1 has a critical role in modulating ERK, JNK and p38 MAPK pathways mediated by TNF-α in IECs. Together, our results show that β-arr1-dependent signaling in hematopoietic and non-hematopoietic cells differentially regulates colitis pathogenesis and further demonstrates that β-arr1 in epithelial cells inhibits TNF-α-induced cell death pathways. PMID:26479868

  16. Lipid G Protein-coupled Receptor Ligand Identification Using β-Arrestin PathHunter™ Assay

    PubMed Central

    Yin, Hong; Chu, Alan; Li, Wei; Wang, Bin; Shelton, Fabiola; Otero, Francella; Nguyen, Deborah G.; Caldwell, Jeremy S.; Chen, Yu Alice

    2009-01-01

    A growing number of orphan G-protein-coupled receptors (GPCRs) have been reported to be activated by lipid ligands, such as lysophosphatidic acid, sphingosine 1-phosphate (S1P), and cannabinoids, for which there are already well established receptors. These new ligand claims are controversial due to either lack of independent confirmations or conflicting reports. We used the β-arrestin PathHunter™ assay system, a newly developed, generic GPCR assay format that measures β-arrestin binding to GPCRs, to evaluate lipid receptor and ligand pairing. This assay eliminates interference from endogenous receptors on the parental cells because it measures a signal that is specifically generated by the tagged receptor and is immediately downstream of receptor activation. We screened a large number of newly “deorphaned” receptors (GPR23, GPR92, GPR55, G2A, GPR18, GPR3, GPR6, GPR12, and GPR63) and control receptors against a collection of ∼400 lipid molecules to try to identify the receptor ligand in an unbiased fashion. GPR92 was confirmed to be a lysophosphatidic acid receptor with weaker responses to farnesyl pyrophosphate and geranylgeranyl diphosphate. The putative cannabinoid receptor GPR55 responded strongly to AM251, rimonabant, and lysophosphatidylinositol but only very weakly to endocannabinoids. G2A receptor was confirmed to be an oxidized free fatty acid receptor. In addition, we discovered that 3,3′-diindolylmethane, a dietary molecule from cruciferous vegetables, which has known anti-cancer properties, to be a CB2 receptor partial agonist, with binding affinity around 1 μm. The anti-inflammatory effect of 3,3′-diindolylmethane in RAW264.7 cells was shown to be partially mediated by CB2. PMID:19286662

  17. Rhodopsin kinase and arrestin binding control the decay of photoactivated rhodopsin and dark adaptation of mouse rods.

    PubMed

    Frederiksen, Rikard; Nymark, Soile; Kolesnikov, Alexander V; Berry, Justin D; Adler, Leopold; Koutalos, Yiannis; Kefalov, Vladimir J; Cornwall, M Carter

    2016-07-01

    Photoactivation of vertebrate rhodopsin converts it to the physiologically active Meta II (R*) state, which triggers the rod light response. Meta II is rapidly inactivated by the phosphorylation of C-terminal serine and threonine residues by G-protein receptor kinase (Grk1) and subsequent binding of arrestin 1 (Arr1). Meta II exists in equilibrium with the more stable inactive form of rhodopsin, Meta III. Dark adaptation of rods requires the complete thermal decay of Meta II/Meta III into opsin and all-trans retinal and the subsequent regeneration of rhodopsin with 11-cis retinal chromophore. In this study, we examine the regulation of Meta III decay by Grk1 and Arr1 in intact mouse rods and their effect on rod dark adaptation. We measure the rates of Meta III decay in isolated retinas of wild-type (WT), Grk1-deficient (Grk1(-/-)), Arr1-deficient (Arr1(-/-)), and Arr1-overexpressing (Arr1(ox)) mice. We find that in WT mouse rods, Meta III peaks ∼6 min after rhodopsin activation and decays with a time constant (τ) of 17 min. Meta III decay slows in Arr1(-/-) rods (τ of ∼27 min), whereas it accelerates in Arr1(ox) rods (τ of ∼8 min) and Grk1(-/-) rods (τ of ∼13 min). In all cases, regeneration of rhodopsin with exogenous 11-cis retinal is rate limited by the decay of Meta III. Notably, the kinetics of rod dark adaptation in vivo is also modulated by the levels of Arr1 and Grk1. We conclude that, in addition to their well-established roles in Meta II inactivation, Grk1 and Arr1 can modulate the kinetics of Meta III decay and rod dark adaptation in vivo. PMID:27353443

  18. Morphine-induced μ-Opioid Receptor Rapid Desensitization is independent of Receptor Phosphorylation and β-Arrestins

    PubMed Central

    Chu, Ji; Zheng, Hui; Loh, Horace H.; Law, Ping-Yee

    2008-01-01

    Receptor desensitization involving receptor phosphorylation and subsequent βArrestin (βArr) recruitment has been implicated in the tolerance development mediated by μ-opioid receptor (OPRM1). However, the roles of receptor phosphorylation and βArr on morphine-induced OPRM1 desensitization remain to be demonstrated. Using OPRM1-induced intracellular Ca2+ ([Ca2+]i )release to monitor receptor activation, as predicted, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), induced OPRM1 desensitization in a receptor phosphorylation- and βArr-dependent manner. The DAMGO-induced OPRM1 desensitization was attenuated significantly when phosphorylation deficient OPRM1 mutants or Mouse Embryonic Fibroblast (MEF) cells from βArr1 and 2 knockout mice were used in the studies. Specifically, DAMGO-induced desensitization was blunted in HEK293 cells expressing the OPRM1S375A mutant and was eliminated in MEF cells isolated from βArr2 knockout mice expressing the wild type OPRM1. However, although morphine also could induce a rapid desensitization on [Ca2+]i release to a greater extent than that of DAMGO and could induce the phosphorylation of Ser375 residue, morphine-induced desensitization was not influenced by mutating the phosphorylation sites or in MEF cells lacking βArr1 and 2. Hence, morphine could induce OPRM1 desensitization via pathway independent of βArr, thus suggesting the in vivo tolerance development to morphine can occur in the absence of βArr. PMID:18558479

  19. Substance P enhances tissue factor release from granulocyte-macrophage colony-stimulating factor-dependent macrophages via the p22phox/β-arrestin 2/Rho A signaling pathway.

    PubMed

    Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

    2016-03-01

    Granulocyte-macrophage colony stimulating factor (GM-CSF) induces procoagulant activity of macrophages. Tissue factor (TF) is a membrane-bound glycoprotein and substance P (SP) is a pro-inflammatory neuropeptide involved in the formation of membrane blebs. This study investigated the role of SP in TF release by GM-CSF-dependent macrophages. SP significantly decreased TF levels in whole-cell lysates of GM-CSF-dependent macrophages. TF was detected in the culture supernatant by enzyme-linked immunosorbent assay after stimulation of macrophages by SP. Aprepitant (an SP/neurokinin 1 receptor antagonist) reduced TF release from macrophages stimulated with SP. Pretreatment of macrophages with a radical scavenger(pyrrolidinedithiocarbamate) also limited the decrease of TF in whole-cell lysates after stimulation with SP. A protein kinase C inhibitor (rottlerin) partially blocked this macrophage response to SP, while it was significantly inhibited by a ROCK inhibitor (Y-27632) or a dynamin inhibitor (dinasore). An Akt inhibitor (perifosine) also partially blocked this response. Furthermore, siRNA targeting p22phox, β-arrestin 2, or Rho A, blunted the release of TF from macrophages stimulated with SP. In other experiments, visceral adipocytes derived from cryopreserved preadipocytes were found to produce SP. In conclusion, SP enhances the release of TF from macrophages via the p22phox/β-arrestin 2/Rho A signaling pathway. PMID:26852662

  20. The α-arrestin ARRDC3 mediates ALIX ubiquitination and G protein–coupled receptor lysosomal sorting

    PubMed Central

    Dores, Michael R.; Lin, Huilan; J. Grimsey, Neil; Mendez, Francisco; Trejo, JoAnn

    2015-01-01

    The sorting of G protein–coupled receptors (GPCRs) to lysosomes is critical for proper signaling and cellular responses. We previously showed that the adaptor protein ALIX regulates lysosomal degradation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, independent of ubiquitin-binding ESCRTs and receptor ubiquitination. However, the mechanisms that regulate ALIX function during PAR1 lysosomal sorting are not known. Here we show that the mammalian α-arrestin arrestin domain–containing protein-3 (ARRDC3) regulates ALIX function in GPCR sorting via ubiquitination. ARRDC3 colocalizes with ALIX and is required for PAR1 sorting at late endosomes and degradation. Depletion of ARRDC3 by small interfering RNA disrupts ALIX interaction with activated PAR1 and the CHMP4B ESCRT-III subunit, suggesting that ARRDC3 regulates ALIX activity. We found that ARRDC3 is required for ALIX ubiquitination induced by activation of PAR1. A screen of nine mammalian NEDD4-family E3 ubiquitin ligases revealed a critical role for WWP2. WWP2 interacts with ARRDC3 and not ALIX. Depletion of WWP2 inhibited ALIX ubiquitination and blocked ALIX interaction with activated PAR1 and CHMP4B. These findings demonstrate a new role for the α-arrestin ARRDC3 and the E3 ubiquitin ligase WWP2 in regulation of ALIX ubiquitination and lysosomal sorting of GPCRs. PMID:26490116

  1. Casein kinase 1 controls the activation threshold of an α-arrestin by multisite phosphorylation of the interdomain hinge

    PubMed Central

    Herrador, Antonio; Livas, Daniela; Soletto, Lucía; Becuwe, Michel; Léon, Sébastien; Vincent, Olivier

    2015-01-01

    α-Arrestins play a key role as trafficking adaptors in both yeast and mammals. The yeast Rim8/Art9 α-arrestin mediates the recruitment of endosomal sorting complex required for transport (ESCRT) to the seven-transmembrane protein Rim21 in the ambient pH signaling RIM pathway. ESCRT is believed to function as a signaling platform that enables the proteolytic activation of the Rim101 transcription factor upon external alkalization. Here we provide evidence that the pH signal promotes the stable association of Rim8 with Rim21 at the plasma membrane. We show that Rim8 is phosphorylated in a pH-independent but Rim21-dependent manner by the plasma membrane–associated casein kinase 1 (CK1). We further show that this process involves a cascade of phosphorylation events within the hinge region connecting the arrestin domains. Strikingly, loss of casein kinase 1 activity causes constitutive activation of the RIM pathway, and, accordingly, pH signaling is activated in a phosphodeficient Rim8 mutant and impaired in the corresponding phosphomimetic mutant. Our results indicate that Rim8 phosphorylation prevents its accumulation at the plasma membrane at acidic pH and thereby inhibits RIM signaling. These findings support a model in which CK1-mediated phosphorylation of Rim8 contributes to setting a signaling threshold required to inhibit the RIM pathway at acidic pH. PMID:25851600

  2. The α-Arrestin ARRDC3 Regulates the Endosomal Residence Time and Intracellular Signaling of the β2-Adrenergic Receptor.

    PubMed

    Tian, Xufan; Irannejad, Roshanak; Bowman, Shanna L; Du, Yang; Puthenveedu, Manojkumar A; von Zastrow, Mark; Benovic, Jeffrey L

    2016-07-01

    Arrestin domain-containing protein 3 (ARRDC3) is a member of the mammalian α-arrestin family, which is predicted to share similar tertiary structure with visual-/β-arrestins and also contains C-terminal PPXY motifs that mediate interaction with E3 ubiquitin ligases. Recently, ARRDC3 has been proposed to play a role in regulating the trafficking of G protein-coupled receptors, although mechanistic insight into this process is lacking. Here, we focused on characterizing the role of ARRDC3 in regulating the trafficking of the β2-adrenergic receptor (β2AR). We find that ARRDC3 primarily localizes to EEA1-positive early endosomes and directly interacts with the β2AR in a ligand-independent manner. Although ARRDC3 has no effect on β2AR endocytosis or degradation, it negatively regulates β2AR entry into SNX27-occupied endosomal tubules. This results in delayed recycling of the receptor and a concomitant increase in β2AR-dependent endosomal signaling. Thus, ARRDC3 functions as a switch to modulate the endosomal residence time and subsequent intracellular signaling of the β2AR. PMID:27226565

  3. ARRB1/β-arrestin-1 mediates neuroprotection through coordination of BECN1-dependent autophagy in cerebral ischemia

    PubMed Central

    Wang, Pei; Xu, Tian-Ying; Wei, Kai; Guan, Yun-Feng; Wang, Xia; Xu, Hui; Su, Ding-Feng; Pei, Gang; Miao, Chao-Yu

    2014-01-01

    Autophagy, a highly conserved process conferring cytoprotection against stress, contributes to the progression of cerebral ischemia. β-arrestins are multifunctional proteins that mediate receptor desensitization and serve as important signaling scaffolds involved in numerous physiopathological processes. Here, we show that both ARRB1 (arrestin, β 1) and ARRB2 (arrestin, β 2) were upregulated by cerebral ischemic stress. Knockout of Arrb1, but not Arrb2, aggravated the mortality, brain infarction, and neurological deficit in a mouse model of cerebral ischemia. Accordingly, Arrb1-deficient neurons exhibited enhanced cell injury upon oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Deletion of Arrb1 did not affect the cerebral ischemia-induced inflammation, oxidative stress, and nicotinamide phosphoribosyltransferase upregulation, but markedly suppressed autophagy and induced neuronal apoptosis/necrosis in vivo and in vitro. Additionally, we found that ARRB1 interacted with BECN1/Beclin 1 and PIK3C3/Vps34, 2 major components of the BECN1 autophagic core complex, under the OGD condition but not normal conditions in neurons. Finally, deletion of Arrb1 impaired the interaction between BECN1 and PIK3C3, which is a critical event for autophagosome formation upon ischemic stress, and markedly reduced the kinase activity of PIK3C3. These findings reveal a neuroprotective role for ARRB1, in the context of cerebral ischemia, centered on the regulation of BECN1-dependent autophagosome formation. PMID:24988431

  4. Casein kinase 1 controls the activation threshold of an α-arrestin by multisite phosphorylation of the interdomain hinge.

    PubMed

    Herrador, Antonio; Livas, Daniela; Soletto, Lucía; Becuwe, Michel; Léon, Sébastien; Vincent, Olivier

    2015-06-01

    α-Arrestins play a key role as trafficking adaptors in both yeast and mammals. The yeast Rim8/Art9 α-arrestin mediates the recruitment of endosomal sorting complex required for transport (ESCRT) to the seven-transmembrane protein Rim21 in the ambient pH signaling RIM pathway. ESCRT is believed to function as a signaling platform that enables the proteolytic activation of the Rim101 transcription factor upon external alkalization. Here we provide evidence that the pH signal promotes the stable association of Rim8 with Rim21 at the plasma membrane. We show that Rim8 is phosphorylated in a pH-independent but Rim21-dependent manner by the plasma membrane-associated casein kinase 1 (CK1). We further show that this process involves a cascade of phosphorylation events within the hinge region connecting the arrestin domains. Strikingly, loss of casein kinase 1 activity causes constitutive activation of the RIM pathway, and, accordingly, pH signaling is activated in a phosphodeficient Rim8 mutant and impaired in the corresponding phosphomimetic mutant. Our results indicate that Rim8 phosphorylation prevents its accumulation at the plasma membrane at acidic pH and thereby inhibits RIM signaling. These findings support a model in which CK1-mediated phosphorylation of Rim8 contributes to setting a signaling threshold required to inhibit the RIM pathway at acidic pH. PMID:25851600

  5. Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding*

    PubMed Central

    Kook, Seunghyi; Zhan, Xuanzhi; Kaoud, Tamer S.; Dalby, Kevin N.; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2013-01-01

    Non-visual arrestins scaffold mitogen-activated protein kinase (MAPK) cascades. The c-Jun N-terminal kinases (JNKs) are members of MAPK family. Arrestin-3 has been shown to enhance the activation of JNK3, which is expressed mainly in neurons, heart, and testes, in contrast to ubiquitous JNK1 and JNK2. Although all JNKs are activated by MKK4 and MKK7, both of which bind arrestin-3, the ability of arrestin-3 to facilitate the activation of JNK1 and JNK2 has never been reported. Using purified proteins we found that arrestin-3 directly binds JNK1α1 and JNK2α2, interacting with the latter comparably to JNK3α2. Phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 or MKK7 is increased by arrestin-3. Endogenous arrestin-3 interacted with endogenous JNK1/2 in different cell types. Arrestin-3 also enhanced phosphorylation of endogenous JNK1/2 in intact cells upon expression of upstream kinases ASK1, MKK4, or MKK7. We observed a biphasic effect of arrestin-3 concentrations on phosphorylation of JNK1α1 and JNK2α2 both in vitro and in vivo. Thus, arrestin-3 acts as a scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation by MKK4 and MKK7 via bringing JNKs and their activators together. The data suggest that arrestin-3 modulates the activity of ubiquitous JNK1 and JNK2 in non-neuronal cells, impacting the signaling pathway that regulates their proliferation and survival. PMID:24257757

  6. Content and Workflow Management for Library Websites: Case Studies

    ERIC Educational Resources Information Center

    Yu, Holly, Ed.

    2005-01-01

    Using database-driven web pages or web content management (WCM) systems to manage increasingly diverse web content and to streamline workflows is a commonly practiced solution recognized in libraries today. However, limited library web content management models and funding constraints prevent many libraries from purchasing commercially available…

  7. Working experiences of Iranian retired nurses: a content analysis study.

    PubMed

    Nobahar, Monir; Ahmadi, Fazlollah; Alhani, Fatemah; Fallahi Khoshknab, Masood

    2013-10-01

    Understanding the experiences of retired nurses can be useful in increasing self-confidence, motivation to work and work enthusiasm among nurses. The purpose of this study was to explore the work experiences of Iranian retired nurses. A qualitative design was conducted using a content analysis approach. Purposive sampling was used to choose the study participants. Semi-structured interviews were held to collect the perspectives of 20 retired nurses (10 female and 10 male). Two main themes emerged in the data analysis: 'work problems and unpleasant experiences in a sense' with subthemes 'exhausting work', 'insufficient salary', 'inappropriate relation' and 'unsuitable social position'; and 'job satisfaction and pleasant experiences in a sense' with subthemes 'divine satisfaction and religious belief', 'satisfaction of patients and their companions' and 'love of nursing profession and relaxation experience'. The findings indicate the challenges that nurses face after retirement. These experiences will help nurse managers to adopt appropriate measures to support nurses after retirement. PMID:24093736

  8. Study of the detail content of Apollo orbital photography

    NASA Technical Reports Server (NTRS)

    Kinzly, R. E.

    1972-01-01

    The results achieved during a study of the Detail Content of Apollo Orbital Photography are reported. The effect of residual motion smear or image reproduction processes upon the detail content of lunar surface imagery obtained from the orbiting command module are assessed. Data and conclusions obtained from the Apollo 8, 12, 14 and 15 missions are included. For the Apollo 8, 12 and 14 missions, the bracket-mounted Hasselblad camera had no mechanism internal to the camera for motion compensation. If the motion of the command module were left totally uncompensated, these photographs would exhibit a ground smear varying from 12 to 27 meters depending upon the focal length of the lens and the exposure time. During the photographic sequences motion compensation was attempted by firing the attitude control system of the spacecraft at a rate to compensate for the motion relative to the lunar surface. The residual smear occurring in selected frames of imagery was assessed using edge analyses methods to obtain and achieved modulation transfer function (MTF) which was compared to a baseline MTF.

  9. Studying of tritium content in snowpack of Degelen mountain range.

    PubMed

    Turchenko, D V; Lukashenko, S N; Aidarkhanov, A O; Lyakhova, O N

    2014-06-01

    The paper presents the results of investigation of tritium content in the layers of snow located in the streambeds of the "Degelen" massif contaminated with tritium. The objects of investigation were selected watercourses Karabulak, Uzynbulak, Aktybai located beyond the "Degelen" site. We studied the spatial distribution of tritium relative to the streambed of watercourses and defined the borders of the snow cover contamination. In the centre of the creek watercourses the snow contamination in the surface layer is as high as 40 000 Bq/L. The values of the background levels of tritium in areas not related to the streambed, which range from 40 to 50 Bq/L. The results of snow cover measurements in different seasonal periods were compared. The main mechanisms causing tritium transfer in snow were examined and identified. The most important mechanism of tritium transfer in the streams is tritium emanation from ice or soil surface. PMID:24657814

  10. β-Arrestin-2 modulates radiation-induced intestinal crypt progenitor/stem cell injury.

    PubMed

    Liu, Z; Tian, H; Jiang, J; Yang, Y; Tan, S; Lin, X; Liu, H; Wu, B

    2016-09-01

    Intestinal crypt progenitor/stem (ICPS) cell apoptosis and vascular endothelial cell apoptosis are responsible for the initiation and development of ionizing radiation (IR)-evoked gastrointestinal syndrome. The signaling mechanisms underlying IR-induced ICPS cell apoptosis remain largely unclear. Our findings provide evidence that β-arrestin-2 (βarr2)-mediated ICPS cell apoptosis is crucial for IR-stimulated intestinal injury. βArr2-deficient mice exhibited decreased ICPS cell and intestinal Lgr5(+) (leucine-rich repeat-containing G-protein-coupled receptor 5-positive) stem cell apoptosis, promoted crypt proliferation and reproduction, and protracted survival following lethal doses of radiation. Radioprotection in the ICPS cells isolated from βarr2-deficient mice depended on prolonged nuclear factor-κB (NF-κB) activation via direct interaction of βarr2 with IκBα and subsequent inhibition of p53-upregulated modulator of apoptosis (PUMA)-mediated mitochondrial dysfunction. Unexpectedly, βarr2 deficiency had little effect on IR-induced intestinal vascular endothelial cell apoptosis in mice. Consistently, βarr2 knockdown also provided significant radioresistance by manipulating NF-κB/PUMA signaling in Lgr5(+) cells in vitro. Collectively, these observations show that targeting the βarr2/NF-κB/PUMA novel pathway is a potential radiomitigator for limiting the damaging effect of radiotherapy on the gastrointestinal system. Significance statement: acute injury to the intestinal mucosa is a major dose-limiting complication of abdominal radiotherapy. The issue of whether the critical factor for the initiation of radiation-induced intestinal injury is intestinal stem cell apoptosis or endothelial cell apoptosis remains unresolved. βArrs have recently been found to be multifunctional adaptor of apoptosis. Here, we found that β-arrestin-2 (βarr2) deficiency was associated with decreased radiation-induced ICPS cell apoptosis, which prolonged survival in

  11. G-Protein/β-Arrestin-Linked Fluctuating Network of G-Protein-Coupled Receptors for Predicting Drug Efficacy and Bias Using Short-Term Molecular Dynamics Simulation

    PubMed Central

    Ichikawa, Osamu; Fujimoto, Kazushi; Yamada, Atsushi; Okazaki, Susumu; Yamazaki, Kazuto

    2016-01-01

    The efficacy and bias of signal transduction induced by a drug at a target protein are closely associated with the benefits and side effects of the drug. In particular, partial agonist activity and G-protein/β-arrestin-biased agonist activity for the G-protein-coupled receptor (GPCR) family, the family with the most target proteins of launched drugs, are key issues in drug discovery. However, designing GPCR drugs with appropriate efficacy and bias is challenging because the dynamic mechanism of signal transduction induced by ligand—receptor interactions is complicated. Here, we identified the G-protein/β-arrestin-linked fluctuating network, which initiates large-scale conformational changes, using sub-microsecond molecular dynamics (MD) simulations of the β2-adrenergic receptor (β2AR) with a diverse collection of ligands and correlation analysis of their G protein/β-arrestin efficacy. The G-protein-linked fluctuating network extends from the ligand-binding site to the G-protein-binding site through the connector region, and the β-arrestin-linked fluctuating network consists of the NPxxY motif and adjacent regions. We confirmed that the averaged values of fluctuation in the fluctuating network detected are good quantitative indexes for explaining G protein/β-arrestin efficacy. These results indicate that short-term MD simulation is a practical method to predict the efficacy and bias of any compound for GPCRs. PMID:27187591

  12. G-Protein/β-Arrestin-Linked Fluctuating Network of G-Protein-Coupled Receptors for Predicting Drug Efficacy and Bias Using Short-Term Molecular Dynamics Simulation.

    PubMed

    Ichikawa, Osamu; Fujimoto, Kazushi; Yamada, Atsushi; Okazaki, Susumu; Yamazaki, Kazuto

    2016-01-01

    The efficacy and bias of signal transduction induced by a drug at a target protein are closely associated with the benefits and side effects of the drug. In particular, partial agonist activity and G-protein/β-arrestin-biased agonist activity for the G-protein-coupled receptor (GPCR) family, the family with the most target proteins of launched drugs, are key issues in drug discovery. However, designing GPCR drugs with appropriate efficacy and bias is challenging because the dynamic mechanism of signal transduction induced by ligand-receptor interactions is complicated. Here, we identified the G-protein/β-arrestin-linked fluctuating network, which initiates large-scale conformational changes, using sub-microsecond molecular dynamics (MD) simulations of the β2-adrenergic receptor (β2AR) with a diverse collection of ligands and correlation analysis of their G protein/β-arrestin efficacy. The G-protein-linked fluctuating network extends from the ligand-binding site to the G-protein-binding site through the connector region, and the β-arrestin-linked fluctuating network consists of the NPxxY motif and adjacent regions. We confirmed that the averaged values of fluctuation in the fluctuating network detected are good quantitative indexes for explaining G protein/β-arrestin efficacy. These results indicate that short-term MD simulation is a practical method to predict the efficacy and bias of any compound for GPCRs. PMID:27187591

  13. Studies on the energy content of pigeon feeds I. Determination of digestibility and metabolizable energy content.

    PubMed

    Hullar, I; Meleg, I; Fekete, S; Romvari, R

    1999-12-01

    The digestibility coefficient and metabolizable energy (ME) content of the most important pigeon feeds (corn, wheat, barley, red and white millet, sorghum, canary seed, peas, lentils, sunflower, and hemp) were determined. The experiment was carried out using 10 adult male homing pigeons. All feeds were fed alone, in a whole-grain form, ad libitum. Drinking water and grit were offered to the birds on a continuous basis. Each feedstuff was fed to five pigeons in 1-wk cycles. There was no significant difference between the values determined in pigeons and those reported in the literature for chickens among the digestibilities of the CP of the various feeds. For pigeons, the digestibility of carbohydrates (N-free extracts, NFE) was lower (e.g., 62.37 vs 83.00% for barley and 63.45 vs 77.00% for peas), whereas the ether extract (EE) was higher (e.g., 75.58 vs 61.00% for barley and 82.59 vs 80.00% for peas) in pigeons compared with chickens. As a result, the AMEn values determined in pigeons did not differ significantly from those reported for chickens but tended to be slightly higher. For feeds of high-oil content, that difference may be somewhat larger. The correlation between the CP, EE, crude fiber (CF), and NFE contents of the feeds and the ME values determined in this experiment were calculated by multivariate linear regression. It was concluded that it was more accurate to determine and tabulate the ME contents of other potential pigeon feeds directly by experimental methods rather than using an equation. PMID:10626652

  14. Adjunct Courses: Integrating Study Skills into Content Courses.

    ERIC Educational Resources Information Center

    Harding, Ida Beal

    College level remediation programs are generally unsatisfactory because students tend to give these noncredit courses low priority and do not seem to transfer the skills learned to their content courses. In addition, because students are simultaneously enrolled in regular content courses requiring skills they have not yet developed, they often…

  15. Teaching Reading and Study Strategies: The Content Areas.

    ERIC Educational Resources Information Center

    Robinson, H. Alan

    This guide to teaching reading in the content areas is designed for both experienced and inexperienced teachers and emphasizes the specific teaching and learning of significant reading strategies which a student should apply to the patterns of writing used in various content areas. The book is divided into three parts, all dealing with reading as…

  16. β2-Adrenoceptor involved in smoking-induced airway mucus hypersecretion through β-arrestin-dependent signaling.

    PubMed

    Zhou, Yujiao; Zhang, Yuan; Guo, Yang; Zhang, Youyi; Xu, Ming; He, Bei

    2014-01-01

    Progression of chronic obstructive pulmonary disease is associated with small airway obstruction by accumulation of inflammatory mucous exudates. However, the mechanism of mucin hypersecretion after exposure to cigarette smoke (CS) is still not clear. In this study, we explored the contribution of β2-adrenoceptor (β2-AR) signaling to CS extract (CSE)-induced mucus hypersecretion in vitro and examined the effect of a β-blocker on airway mucin hypersecretion in vivo. NCI-H292 epithelial cell line was used to determine the contribution of β2-AR signaling to CSE-induced MUC5AC production by treatment with β2-AR antagonists propranolol and ICI118551 and β2-AR-targeted small interfering RNA. The effect of propranolol on airway mucus hypersecretion was examined in a rat model exposed to CS. MUC5AC expression was assayed by real-time PCR, immunohistochemistry and ELISA. β2-AR and its downstream signaling were detected by western blot analysis. We found that pretreating NCI-H292 cells with propranolol, ICI118551 for 30 min or β2AR-targeted siRNA for 48 h reduced MUC5AC mRNA and protein levels stimulated by CSE. However,inhibiting the classical β2AR-cAMP-PKA pathway didn't attenuate CSE-induced MUC5AC production, while silencing β-arretin2 expression significantly decreased ERK and p38MAPK phosphorylation, thus reduced the CSE-stimulated MUC5AC production. In vivo, we found that administration of propranolol (25 mg kg(-1) d(-1)) for 28 days significantly attenuated the airway goblet cell metaplasia, mucus hypersecretion and MUC5AC expression of rats exposed to CS. From the study, β2-AR-β-arrestin2-ERK1/2 signaling was required for CS-induced airway MUC5AC expression. Chronic propranolol administration ameliorated airway mucus hypersecretion and MUC5AC expression in smoking rats. The exploration of these mechanisms may contribute to the optimization of β2-AR target therapy in chronic obstructive pulmonary disease. PMID:24905583

  17. Screening of the arrestin gene in dogs afflicted with generalized progressive retinal atrophy

    PubMed Central

    Dekomien, Gabriele; Epplen, Jörg Thomas

    2002-01-01

    Background Intronic DNA sequences of the canine arrestin (SAG) gene was screened to identify potential disease causing mutations in dogs with generalized progressive retinal atrophy (gPRA). The intronic sequences flanking each of the 16 exons were obtained from clones of a canine genomic library. Results Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequence analyses we screened affected and unaffected dogs of 23 breeds with presumed autosomal recessively (ar) transmitted gPRA. In the coding region of the SAG gene 12 nucleotide exchanges were identified, 5 of which lead to amino acid substitutions (H14C; A111V; A113T; D259T; A379E). 7 other exonic substitutions represent silent polymorphisms (C132C; Q199Q; H225H; V247V; P264P; T288T and L293L). 16 additional sequence variations were observed in intronic regions of different dog breeds. Conclusions In several breeds, these polymorphisms were found in homozygous state in unaffected and in heterozygous state in affected animals. Consequently these informative substitutions provide evidence to exclude mutations in the SAG gene as causing retinal degeneration in 14 of the 23 dog breeds with presumed ar transmitted gPRA. PMID:12123530

  18. Studying the HI content of the NGC 4930 group

    NASA Astrophysics Data System (ADS)

    Wolfinger, Kathrin; Kilborn, Virginia; Koribalski, Baerbel

    2011-10-01

    We propose to observe the neutral hydrogen (HI) content of the spiral-rich NGC 4930 group using the ATCA. This notable group lies 2.5° east of the Centaurus cluster core and is probably infalling for the first time. Our primary goal is to trace the evolutionary changes of spirals in different environments and to map the first signs of interaction and transformation. Our aims of the ATCA observations are (i) to study the HI properties of the group, (ii) to determine if there is an HI deficiency in the members, (iii) to look for any signs of ram pressure stripping that would indicate an interaction with a hot intra-group medium and (iv) to conduct a ‘blind’ survey for new group members, such as dwarf companions within the survey volume. We will further test the latest galaxy finding routines such as Duchamp, which are vital for the success of the upcoming ASKAP HI surveys. The NGC 4930 group is covered in the HI Parkes All Sky Survey but only two out of the nine group members are detected in HI. We propose to make mosaic observations and we expect to detect all of the known galaxies in this group.

  19. The calcium-sensing receptor changes cell shape via a β-arrestin-1–ARNO–ARF6–ELMO protein network

    PubMed Central

    Bouschet, Tristan; Martin, Stéphane; Kanamarlapudi, Venkateswarlu; Mundell, Stuart; Henley, Jeremy M.

    2012-01-01

    Summary G-protein-coupled receptors (GPCRs) transduce the binding of extracellular stimuli into intracellular signalling cascades that can lead to morphological changes. Here, we demonstrate that stimulation of the calcium-sensing receptor (CaSR), a GPCR that promotes chemotaxis by detecting increases in extracellular calcium, triggers plasma membrane (PM) ruffling via a pathway that involves β-arrestin 1, Arf nucleotide binding site opener (ARNO), ADP-ribosylating factor 6 (ARF6) and engulfment and cell motility protein (ELMO). Expression of dominant negative β-arrestin 1 or its knockdown with siRNA impaired the CaSR-induced PM ruffling response. Expression of a catalytically inactive ARNO also reduced CaSR-induced PM ruffling. Furthermore, β-arrestin 1 co-immunoprecipitated with the CaSR and ARNO under resting conditions. Agonist treatment did not markedly alter β-arrestin 1 binding to the CaSR or to ARNO but it did elicit the translocation and colocalisation of the CaSR, β-arrestin 1 and ARNO to membrane protrusions. Furthermore, ARF6 and ELMO, two proteins known to couple ARNO to the cytoskeleton, were required for CaSR-dependent morphological changes and translocated to the PM ruffles. These data suggest that cells ruffle upon CaSR stimulation via a mechanism that involves translocation of β-arrestin 1 pre-assembled with the CaSR or ARNO, and that ELMO plays an essential role in this CaSR-signalling-induced cytoskeletal reorganisation. PMID:17623778

  20. Study of the detail content of Apollo orbital photography

    NASA Technical Reports Server (NTRS)

    Kinzly, R. E.

    1974-01-01

    The development and application of image evaluation methods for assessing the detail content of Apollo orbital photography was demonstrated. Edge analysis using shadow to sunlight edges interior to craters was successfully used to evaluate the fine detail content of Apollo 15, 16, and 17 imagery. A method for evaluating tone quality was developed using a gain factor as a function of object contrast and average exposure level that can be related to object detectability.

  1. β-Arrestin recruitment and G protein signaling by the atypical human chemokine decoy receptor CCX-CKR.

    PubMed

    Watts, Anne O; Verkaar, Folkert; van der Lee, Miranda M C; Timmerman, Claudia A W; Kuijer, Martien; van Offenbeek, Jody; van Lith, Lambertus H C J; Smit, Martine J; Leurs, Rob; Zaman, Guido J R; Vischer, Henry F

    2013-03-01

    Chemokine receptors form a large subfamily of G protein-coupled receptors that predominantly activate heterotrimeric Gi proteins and are involved in immune cell migration. CCX-CKR is an atypical chemokine receptor with high affinity for CCL19, CCL21, and CCL25 chemokines, but is not known to activate intracellular signaling pathways. However, CCX-CKR acts as decoy receptor and efficiently internalizes these chemokines, thereby preventing their interaction with other chemokine receptors, like CCR7 and CCR9. Internalization of fluorescently labeled CCL19 correlated with β-arrestin2-GFP translocation. Moreover, recruitment of β-arrestins to CCX-CKR in response to CCL19, CCL21, and CCL25 was demonstrated using enzyme-fragment complementation and bioluminescence resonance energy transfer methods. To unravel why CCX-CKR is unable to activate Gi signaling, CCX-CKR chimeras were constructed by substituting its intracellular loops with the corresponding CCR7 or CCR9 domains. The signaling properties of chimeric CCX-CKR receptors were characterized using a cAMP-responsive element (CRE)-driven reporter gene assay. Unexpectedly, wild type CCX-CKR and a subset of the chimeras induced an increase in CRE activity in response to CCL19, CCL21, and CCL25 in the presence of the Gi inhibitor pertussis toxin. CCX-CKR signaling to CRE required an intact DRY motif. These data suggest that inactive Gi proteins impair CCX-CKR signaling most likely by hindering the interaction of this receptor with pertussis toxin-insensitive G proteins that transduce signaling to CRE. On the other hand, recruitment of the putative signaling scaffold β-arrestin to CCX-CKR in response to chemokines might allow activation of yet to be identified signal transduction pathways. PMID:23341447

  2. Dependence of seismoelectric amplitudes on water content - a field study

    NASA Astrophysics Data System (ADS)

    Strahser, M. H. P.; Matthey, P.-D.; Jouniaux, L.; Sailhac, P.

    2009-04-01

    In porous saturated media, seismic compressional waves can cause seismoelectric and seismoelectromagnetic signals through electrokinetic coupling. It has been observed that these measureable signals also occur in partially saturated media, but the theory is largely unknown for these circumstances. Seismoelectromagnetic tomography is expected to combine the sensitivity of electrical properties to water-content and permeability, to the high spatial resolution of seismic surveys. A better understanding of the physical processes and a reliable quantification of the conversion between seismic and electric energy are necessary and need to take into account the effect of water-content, especially for shallow subsurface investigations. In order to quantify seismoelectric signals with changing water content, we repeated seismoelectric and seismic measurements on the same profile in the Vosges Mountains during several months. The electrical resistivity was also monitored to take into account the water-content variations. We show that an exponential relation can be established between the seismoelectric amplitudes normalized with the seismic amplitudes and the resistivity which in turn is related to the saturation: Increasing resistivity (decreasing water content) leads to decreasing normalized seismoelectric amplitudes. These results imply that the electrokinetic coefficient should increase with water-saturation, as measured in laboratory, but not predicted by theory. This work was funded by CNRS and Université Louis Pasteur de Strasbourg.

  3. Carbetocin is a Functional Selective Gq Agonist That Does Not Promote Oxytocin Receptor Recycling After Inducing β-Arrestin-Independent Internalisation.

    PubMed

    Passoni, I; Leonzino, M; Gigliucci, V; Chini, B; Busnelli, M

    2016-04-01

    Carbetocin, a long-acting oxytocin analogue, has been reported to elicit interesting and peculiar behavioural effects. The present study investigated the molecular pharmacology of carbetocin, aiming to better understand the molecular basis of its action in the brain. Using bioluminescence resonance energy transfer biosensors, we characterised the effects of carbetocin on the three human oxytocin/vasopressin receptors expressed in the nervous system: the oxytocin receptor (OXTR) and the vasopressin V1a (V1aR) and V1b (V1bR) receptors. Our results indicate that (i) carbetocin activates the OXTR but not the V1aR and V1bR at which it may act as an antagonist; (ii) carbetocin selectively activates only the OXTR/Gq pathway displaying a strong functional selectivity; (iii) carbetocin is a partial agonist at the OXTR/Gq coupling; (iv) carbetocin promotes OXTR internalisation via a previously unreported β-arrestin-independent pathway; and (v) carbetocin does not induce OXTR recycling to the plasma membrane. Altogether, these molecular pharmacology features identify carbetocin as a substantially different analogue compared to the endogenous oxytocin and, consequently, carbetocin is not expected to mimic oxytocin in the brain. Whether these unique features of carbetocin could be exploited therapeutically remains to be established. PMID:26751410

  4. Searching for Educational Content in the For-Profit Internet: Case Study and Analysis.

    ERIC Educational Resources Information Center

    Fabos, Bettina

    This case study investigates the commercialized nature of Internet content and the ways educators and students negotiate and talk about such content. In factoring in the economic and historical context of educational Internet content, this case study also addresses educators' evolving attitudes towards commercialism in the classroom. A survey was…

  5. Central HIV-1 Tat exposure elevates anxiety and fear conditioned responses of male mice concurrent with altered mu-opioid receptor-mediated G-protein activation and β-arrestin 2 activity in the forebrain.

    PubMed

    Hahn, Yun K; Paris, Jason J; Lichtman, Aron H; Hauser, Kurt F; Sim-Selley, Laura J; Selley, Dana E; Knapp, Pamela E

    2016-08-01

    Co-exposure to opiates and HIV/HIV proteins results in enhanced CNS morphological and behavioral deficits in HIV(+) individuals and in animal models. Opiates with abuse liability, such as heroin and morphine, bind preferentially to and have pharmacological actions through μ-opioid-receptors (MORs). The mechanisms underlying opiate-HIV interactions are not understood. Exposure to the HIV-1 transactivator of transcription (Tat) protein causes neurodegenerative outcomes that parallel many aspects of the human disease. We have also observed that in vivo exposure to Tat results in apparent changes in morphine efficacy, and thus have hypothesized that HIV proteins might alter MOR activation. To test our hypothesis, MOR-mediated G-protein activation was determined in neuroAIDS-relevant forebrain regions of transgenic mice with inducible CNS expression of HIV-1 Tat. G-protein activation was assessed by MOR agonist-stimulated [(35)S]guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPγS) autoradiography in brain sections, and in concentration-effect curves of MOR agonist-stimulated [(35)S]GTPγS binding in membranes isolated from specific brain regions. Comparative studies were done using the MOR-selective agonist DAMGO ([D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin) and a more clinically relevant agonist, morphine. Tat exposure reduced MOR-mediated G-protein activation in an agonist, time, and regionally dependent manner. Levels of the GPCR regulatory protein β-arrestin-2, which is involved in MOR desensitization, were found to be elevated in only one affected brain region, the amygdala; amygdalar β-arrestin-2 also showed a significantly increased association with MOR by co-immunoprecipitation, suggesting decreased availability of MOR. Interestingly, this correlated with changes in anxiety and fear-conditioned extinction, behaviors that have substantial amygdalar input. We propose that HIV-1 Tat alters the intrinsic capacity of MOR to signal in response to agonist binding

  6. Optical sensing of vegetation water content: A synthesis study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vegetation Water Content (VWC) plays an important role in parameterizing the vegetation influence on microwave soil moisture retrieval. During the past decade, researchers have developed relationships between VWC and vegetation indices available from satellite optical sensors in order to create larg...

  7. MOISTURE CONTENT AND POROSITY OF CONCRETE RUBBLE STUDY.

    SciTech Connect

    Phifer, M

    2005-10-07

    Tritium contaminated concrete rubble from the 232-F Tritium Facility was disposed in the Slit 1 Trenches 1 and 2 in 1997. A Special Analysis (SA) has been performed to evaluate any impact this disposal may have on the groundwater. The SA assumed that the disposed concrete rubble was fully saturated at the time of disposal, however if the concrete was less than fully saturated, migration of tritium out of the concrete would be slower than under fully saturated conditions. Therefore if the concrete at disposal was less than fully saturated, the PA assumption of full saturation would be a conservative assumption. In order to evaluate whether the PA assumption resulted in a conservative analysis from the standpoint of the concrete saturation, concrete rubble samples were collected from various facilities being demolished at the Savannah River Site (SRS) and evaluated for in-field moisture content, absorbable moisture, and water exchangeable porosity. The purpose of this task was to collect concrete rubble samples from various demolished SRS facilities for the purpose of determining in-field moisture content, absorbable moisture, and water exchangeable porosity. Since moisture content testing for concrete rubble is not typical, existing ASTM Standards were reviewed for method and procedure development.

  8. A unique PDZ domain and arrestin-like fold interaction reveals mechanistic details of endocytic recycling by SNX27-retromer.

    PubMed

    Gallon, Matthew; Clairfeuille, Thomas; Steinberg, Florian; Mas, Caroline; Ghai, Rajesh; Sessions, Richard B; Teasdale, Rohan D; Collins, Brett M; Cullen, Peter J

    2014-09-01

    The sorting nexin 27 (SNX27)-retromer complex is a major regulator of endosome-to-plasma membrane recycling of transmembrane cargos that contain a PSD95, Dlg1, zo-1 (PDZ)-binding motif. Here we describe the core interaction in SNX27-retromer assembly and its functional relevance for cargo sorting. Crystal structures and NMR experiments reveal that an exposed β-hairpin in the SNX27 PDZ domain engages a groove in the arrestin-like structure of the vacuolar protein sorting 26A (VPS26A) retromer subunit. The structure establishes how the SNX27 PDZ domain simultaneously binds PDZ-binding motifs and retromer-associated VPS26. Importantly, VPS26A binding increases the affinity of the SNX27 PDZ domain for PDZ- binding motifs by an order of magnitude, revealing cooperativity in cargo selection. With disruption of SNX27 and retromer function linked to synaptic dysfunction and neurodegenerative disease, our work provides the first step, to our knowledge, in the molecular description of this important sorting complex, and more broadly describes a unique interaction between a PDZ domain and an arrestin-like fold. PMID:25136126

  9. Activity dependent internalization of the glutamate transporter GLT-1 mediated by β-arrestin 1 and ubiquitination.

    PubMed

    Ibáñez, Ignacio; Díez-Guerra, F Javier; Giménez, Cecilio; Zafra, Francisco

    2016-08-01

    GLT-1 is the main glutamate transporter in the brain and undergoes trafficking processes that control its concentration on the cell surface thereby shaping glutamatergic neurotransmission. We have investigated how the traffic of GLT-1 is regulated by transporter activity. We report that internalization of GLT-1 from the cell surface is accelerated by transportable substrates like glutamate or aspartate, as well as by the transportable inhibitor L-trans-2,4-PDC, but not by the non-substrate inhibitor WAY 213613 in primary mixed cultures and in transiently transfected HEK293 cells. Analysis of the mechanism of endocytosis in HEK293 cells revealed that glutamate promoted the association with the transporter of the adaptor protein β-arrestin and the ubiquitin ligase Nedd4-2. The addition of glutamate is accompanied by an increase in the transporter ubiquitination, and the internalization is suppressed by an ubiquitination inhibitor (PYR41), and in a mutant defective in C-terminal lysines. The glutamate triggered endocytosis was also suppressed by siRNA for β-arrestin. This regulatory mechanism might be relevant in controlling the amount of transporter on the cell surface in conditions such as ischemia or traumatic brain injury, where extracellular concentrations of glutamate are persistently elevated. PMID:27044663

  10. Smoothened determines β-arrestin-mediated removal of the G protein-coupled receptor Gpr161 from the primary cilium.

    PubMed

    Pal, Kasturi; Hwang, Sun-Hee; Somatilaka, Bandarigoda; Badgandi, Hemant; Jackson, Peter K; DeFea, Kathryn; Mukhopadhyay, Saikat

    2016-03-28

    Dynamic changes in membrane protein composition of the primary cilium are central to development and homeostasis, but we know little about mechanisms regulating membrane protein flux. Stimulation of the sonic hedgehog (Shh) pathway in vertebrates results in accumulation and activation of the effector Smoothened within cilia and concomitant disappearance of a negative regulator, the orphan G protein-coupled receptor (GPCR), Gpr161. Here, we describe a two-step process determining removal of Gpr161 from cilia. The first step involves β-arrestin recruitment by the signaling competent receptor, which is facilitated by the GPCR kinase Grk2. An essential factor here is the ciliary trafficking and activation of Smoothened, which by increasing Gpr161-β-arrestin binding promotes Gpr161 removal, both during resting conditions and upon Shh pathway activation. The second step involves clathrin-mediated endocytosis, which functions outside of the ciliary compartment in coordinating Gpr161 removal. Mechanisms determining dynamic compartmentalization of Gpr161 in cilia define a new paradigm for down-regulation of GPCRs during developmental signaling from a specialized subcellular compartment. PMID:27002170

  11. A unique PDZ domain and arrestin-like fold interaction reveals mechanistic details of endocytic recycling by SNX27-retromer

    PubMed Central

    Gallon, Matthew; Clairfeuille, Thomas; Steinberg, Florian; Mas, Caroline; Ghai, Rajesh; Sessions, Richard B.; Teasdale, Rohan D.; Collins, Brett M.; Cullen, Peter J.

    2014-01-01

    The sorting nexin 27 (SNX27)-retromer complex is a major regulator of endosome-to-plasma membrane recycling of transmembrane cargos that contain a PSD95, Dlg1, zo-1 (PDZ)-binding motif. Here we describe the core interaction in SNX27-retromer assembly and its functional relevance for cargo sorting. Crystal structures and NMR experiments reveal that an exposed β-hairpin in the SNX27 PDZ domain engages a groove in the arrestin-like structure of the vacuolar protein sorting 26A (VPS26A) retromer subunit. The structure establishes how the SNX27 PDZ domain simultaneously binds PDZ-binding motifs and retromer-associated VPS26. Importantly, VPS26A binding increases the affinity of the SNX27 PDZ domain for PDZ- binding motifs by an order of magnitude, revealing cooperativity in cargo selection. With disruption of SNX27 and retromer function linked to synaptic dysfunction and neurodegenerative disease, our work provides the first step, to our knowledge, in the molecular description of this important sorting complex, and more broadly describes a unique interaction between a PDZ domain and an arrestin-like fold. PMID:25136126

  12. KISS1R signaling promotes invadopodia formation in human breast cancer cell via β-arrestin2/ERK.

    PubMed

    Goertzen, Cameron G; Dragan, Magdalena; Turley, Eva; Babwah, Andy V; Bhattacharya, Moshmi

    2016-03-01

    Kisspeptins (KPs), peptide products of the KISS1 gene are endogenous ligands for the kisspeptin receptor (KISS1R), a G protein-coupled receptor. In numerous cancers, KISS1R signaling plays anti-metastatic roles. However, we have previously shown that in breast cancer cells lacking the estrogen receptor (ERα), kisspeptin-10 stimulates cell migration and invasion by cross-talking with the epidermal growth factor receptor (EGFR), via a β-arrestin-2-dependent mechanism. To further define the mechanisms by which KISS1R stimulates invasion, we determined the effect of down-regulating KISS1R expression in triple negative breast cancer cells. We found that depletion of KISS1R reduced their mesenchymal phenotype and invasiveness. We show for the first time that KISS1R signaling induces invadopodia formation and activation of key invadopodia proteins, cortactin, cofilin and membrane type I matrix metalloproteases (MT1-MMP). Moreover, KISS1R stimulated invadopodia formation occurs via a new pathway involving a β-arrestin2 and ERK1/2-dependent mechanism, independent of Src. Taken together, our findings suggest that targeting the KISS1R signaling axis might be a promising strategy to inhibit invasiveness and metastasis. PMID:26721186

  13. Multi-Core Processor Memory Contention Benchmark Analysis Case Study

    NASA Technical Reports Server (NTRS)

    Simon, Tyler; McGalliard, James

    2009-01-01

    Multi-core processors dominate current mainframe, server, and high performance computing (HPC) systems. This paper provides synthetic kernel and natural benchmark results from an HPC system at the NASA Goddard Space Flight Center that illustrate the performance impacts of multi-core (dual- and quad-core) vs. single core processor systems. Analysis of processor design, application source code, and synthetic and natural test results all indicate that multi-core processors can suffer from significant memory subsystem contention compared to similar single-core processors.

  14. Content Metadata Standards for Marine Science: A Case Study

    USGS Publications Warehouse

    Riall, Rebecca L.; Marincioni, Fausto; Lightsom, Frances L.

    2004-01-01

    The U.S. Geological Survey developed a content metadata standard to meet the demands of organizing electronic resources in the marine sciences for a broad, heterogeneous audience. These metadata standards are used by the Marine Realms Information Bank project, a Web-based public distributed library of marine science from academic institutions and government agencies. The development and deployment of this metadata standard serve as a model, complete with lessons about mistakes, for the creation of similarly specialized metadata standards for digital libraries.

  15. 42 CFR 456.243 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 4 2012-10-01 2012-10-01 false Content of medical care evaluation studies. 456.243... Ur Plan: Medical Care Evaluation Studies § 456.243 Content of medical care evaluation studies. Each medical care evaluation study must— (a) Identify and analyze medical or administrative factors related...

  16. 42 CFR 456.243 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 4 2013-10-01 2013-10-01 false Content of medical care evaluation studies. 456.243... Ur Plan: Medical Care Evaluation Studies § 456.243 Content of medical care evaluation studies. Each medical care evaluation study must— (a) Identify and analyze medical or administrative factors related...

  17. 42 CFR 456.243 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 4 2011-10-01 2011-10-01 false Content of medical care evaluation studies. 456.243... Ur Plan: Medical Care Evaluation Studies § 456.243 Content of medical care evaluation studies. Each medical care evaluation study must— (a) Identify and analyze medical or administrative factors related...

  18. 42 CFR 456.243 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 4 2010-10-01 2010-10-01 false Content of medical care evaluation studies. 456.243... Ur Plan: Medical Care Evaluation Studies § 456.243 Content of medical care evaluation studies. Each medical care evaluation study must— (a) Identify and analyze medical or administrative factors related...

  19. 42 CFR 456.243 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 4 2014-10-01 2014-10-01 false Content of medical care evaluation studies. 456.243... Ur Plan: Medical Care Evaluation Studies § 456.243 Content of medical care evaluation studies. Each medical care evaluation study must— (a) Identify and analyze medical or administrative factors related...

  20. 42 CFR 456.143 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 4 2010-10-01 2010-10-01 false Content of medical care evaluation studies. 456.143...: Medical Care Evaluation Studies § 456.143 Content of medical care evaluation studies. Each medical care evaluation study must— (a) Identify and analyze medical or administrative factors related to the...

  1. 7 CFR 4280.176 - Feasibility study grant applications-content.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 15 2013-01-01 2013-01-01 false Feasibility study grant applications-content. 4280... Energy for America Program General Renewable Energy System Feasibility Study Grants § 4280.176 Feasibility study grant applications—content. Applications for feasibility study grants must include a...

  2. 7 CFR 4280.176 - Feasibility study grant applications-content.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 15 2012-01-01 2012-01-01 false Feasibility study grant applications-content. 4280... Energy for America Program General Renewable Energy System Feasibility Study Grants § 4280.176 Feasibility study grant applications—content. Applications for feasibility study grants must include a...

  3. 42 CFR 456.143 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 4 2012-10-01 2012-10-01 false Content of medical care evaluation studies. 456.143... SERVICES (CONTINUED) MEDICAL ASSISTANCE PROGRAMS UTILIZATION CONTROL Utilization Control: Hospitals Ur Plan: Medical Care Evaluation Studies § 456.143 Content of medical care evaluation studies. Each medical...

  4. 42 CFR 456.143 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 4 2013-10-01 2013-10-01 false Content of medical care evaluation studies. 456.143... SERVICES (CONTINUED) MEDICAL ASSISTANCE PROGRAMS UTILIZATION CONTROL Utilization Control: Hospitals Ur Plan: Medical Care Evaluation Studies § 456.143 Content of medical care evaluation studies. Each medical...

  5. 42 CFR 456.143 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 4 2014-10-01 2014-10-01 false Content of medical care evaluation studies. 456.143... SERVICES (CONTINUED) MEDICAL ASSISTANCE PROGRAMS UTILIZATION CONTROL Utilization Control: Hospitals Ur Plan: Medical Care Evaluation Studies § 456.143 Content of medical care evaluation studies. Each medical...

  6. 42 CFR 456.143 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 4 2011-10-01 2011-10-01 false Content of medical care evaluation studies. 456.143... SERVICES (CONTINUED) MEDICAL ASSISTANCE PROGRAMS UTILIZATION CONTROL Utilization Control: Hospitals Ur Plan: Medical Care Evaluation Studies § 456.143 Content of medical care evaluation studies. Each medical...

  7. Strategic and Organisational Considerations in Planning Content and Language Integrated Learning: A Study on the Coordination between Content and Language Teachers

    ERIC Educational Resources Information Center

    Pavón Vázquez, Víctor; Ávila López, Javier; Gallego Segador, Arturo; Espejo Mohedano, Roberto

    2015-01-01

    Content and language integrated learning (CLIL) is generally recognised as a fruitful example of bilingual education. However, success in CLIL may not be straightforward and may require the establishment of coordination between content and language teachers. The aim of this study is to investigate if content and language teachers are able to plan…

  8. Moral content and assignment marking: an exploratory study.

    PubMed

    Lipscomb, Martin; Snelling, Paul C

    2006-08-01

    This small scale exploratory survey investigates the relation between the moral content of student assignments and potential marking behaviour by lecturers. A questionnaire containing a number of deliberately discriminatory statements was sent to a sample of nurse academics, and participants were asked to indicate whether the inclusion of these statements might affect the mark awarded. The sample was too small to undertake tests of statistical significance. However, the results tentatively suggest that a majority of participants would penalise assignments that contain morally questionable statements, and many would cite the code of professional conduct in justification. There appeared to be little difference in the response to statements that offered opinions (normative) or described actions (descriptive). However, differences between responses to statements discriminating against different groups were apparent, with racist statements being penalised more often and with greater severity than other categories. Qualitative data support the quantitative data. Several areas for discussion are identified, including the academic status of nursing, the use of the code of professional conduct, the moral claims made for nurses, a potential 'hierarchy of wrongness', student self censorship, and inconsistency in marking. Further research is justified. PMID:16458999

  9. I, Pronoun: A Study of Formality in Online Content

    ERIC Educational Resources Information Center

    Thayer, Alexander; Evans, Mary B.; McBride, Alicia A.; Queen, Matt; Spyridakis, Jan H.

    2010-01-01

    This article presents the results of a study that investigated readers' perceptions of tone formality in online text passages. The study found that readers perceived text passages to be less formal when they contained personal pronouns, active voice verbs, informal punctuation, or verb contractions. The study reveals that professional…

  10. A Comparative Study of Six Decades of General Science Textbooks: Evaluating the Evolution of Science Content

    ERIC Educational Resources Information Center

    Lewis, Anna

    2008-01-01

    This study examined science textbooks over time to better understand the "science content" expectations that the U.S. educational system deems appropriate for 8th and 9th grade science students. The study attempted to answer the questions: (1) What specific science content has been presented via the textbook from 1952 to 2008? (2) Within which…

  11. Preliminary assestment of lint cotton water content in gin-drying temperature studies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prior studies to measure total water (free and bound) in lint cotton by Karl Fischer Titration showed the method is more accurate and precise than moisture content by standard oven drying. The objective of the current study was to compare the moisture and total water contents from five cultivars de...

  12. Teaching University Freshmen to Employ, Regulate, and Transfer Study Strategies to the Content Areas.

    ERIC Educational Resources Information Center

    Simpson, Michele L.

    1986-01-01

    To determine whether students have study strategies that they can transfer to future learning tasks, a research study used a content-based model--the Supportive Seminar--a voluntary content review session held by a leader enrolled in a targeted college course for freshmen. The leader acts as a role model for the students by attending all lectures,…

  13. A National Study of Training Content and Activities for Faculty Development for Online Teaching

    ERIC Educational Resources Information Center

    Meyer, Katrina A.; Murrell, Vicki S.

    2014-01-01

    This article presents the results of a national study of 39 higher education institutions that collected information about their practices for faculty development for online teaching and particularly the content and training activities used during 2011-2012. This study found that the most frequently offered training content (97% of the…

  14. Connecting Content, Context, and Communication in a Sixth-Grade Social Studies Class through Political Cartoons

    ERIC Educational Resources Information Center

    Gallavan, Nancy P.; Webster-Smith, Angela; Dean, Sheila S.

    2012-01-01

    Sixth-grade students are challenged in understanding social studies content relevant to particular contexts, then connecting the content and context to their contemporary lives while communicating new knowledge to peers and teachers. Using political cartoons published after September 11, 2001, one sixth-grade social studies teacher designed…

  15. Content Analysis of Turkish Studies about the Multiple Intelligences Theory

    ERIC Educational Resources Information Center

    Saban, Ahmet

    2009-01-01

    Recently, there has been a significant increase in the number of multiple intelligences (MI) studies in Turkey. Consequently, a systematic analysis of these studies is crucial in order to be able to see the present situation and future trends in the field of education. By this way, it is also hoped that the current analysis will offer an avenue…

  16. ICT Student Teachers' Pedagogical Content Knowledge: A Case Study

    ERIC Educational Resources Information Center

    Yilmaz, Nuray Parlak

    2016-01-01

    This study aimed to identify the difficulties that information technology student teachers have in teaching concepts. This qualitative study was carried out with 12 student teachers. The student teachers were fourth-year students enrolled in the Special Teaching Methods II course in the spring semester of the 2010-2011 academic year. The research…

  17. β-Arrestin1 and distinct CXCR4 structures are required for stromal derived factor-1 to downregulate CXCR4 cell-surface levels in neuroblastoma.

    PubMed

    Clift, Ian C; Bamidele, Adebowale O; Rodriguez-Ramirez, Christie; Kremer, Kimberly N; Hedin, Karen E

    2014-04-01

    CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor (GPCR) located on the cell surface that signals upon binding the chemokine stromal derived factor-1 (SDF-1; also called CXCL 12). CXCR4 promotes neuroblastoma proliferation and chemotaxis. CXCR4 expression negatively correlates with prognosis and drives neuroblastoma growth and metastasis in mouse models. All functions of CXCR4 require its expression on the cell surface, yet the molecular mechanisms that regulate CXCR4 cell-surface levels in neuroblastoma are poorly understood. We characterized CXCR4 cell-surface regulation in the related SH-SY5Y and SK-N-SH human neuroblastoma cell lines. SDF-1 treatment caused rapid down-modulation of CXCR4 in SH-SY5Y cells. Pharmacologic activation of protein kinase C similarly reduced CXCR4, but via a distinct mechanism. Analysis of CXCR4 mutants delineated two CXCR4 regions required for SDF-1 treatment to decrease cell-surface CXCR4 in neuroblastoma cells: the isoleucine-leucine motif at residues 328 and 329 and residues 343-352. In contrast, and unlike CXCR4 regulation in other cell types, serines 324, 325, 338, and 339 were not required. Arrestin proteins can bind and regulate GPCR cell-surface expression, often functioning together with kinases such as G protein-coupled receptor kinase 2 (GRK2). Using SK-N-SH cells which are naturally deficient in β-arrestin1, we showed that β-arrestin1 is required for the CXCR4 343-352 region to modulate CXCR4 cell-surface expression following treatment with SDF-1. Moreover, GRK2 overexpression enhanced CXCR4 internalization, via a mechanism requiring both β-arrestin1 expression and the 343-352 region. Together, these results characterize CXCR4 structural domains and β-arrestin1 as critical regulators of CXCR4 cell-surface expression in neuroblastoma. β-Arrestin1 levels may therefore influence the CXCR4-driven metastasis of neuroblastoma as well as prognosis. PMID:24452472

  18. The Stem Cell-Expressed Receptor Lgr5 Possesses Canonical and Functionally Active Molecular Determinants Critical to β-arrestin-2 Recruitment

    PubMed Central

    Snyder, Joshua C.; Rochelle, Lauren K.; Barak, Larry S.; Caron, Marc G.

    2013-01-01

    Lgr5 is a membrane protein related to G protein-coupled receptors (GPCR)s whose expression identifies stem cells in multiple tissues and is strongly correlated with cancer. Despite the recent identification of endogenous ligands for Lgr5, its mode of signaling remains enigmatic. The ability to couple to G proteins and βarrestins are classical molecular behaviors of GPCRs that have yet to be observed for Lgr5. Therefore, the goal of this study was to determine if Lgr5 can engage a classical GPCR behavior and elucidate the molecular determinants of this process. Structural analysis of Lgr5 revealed several motifs consistent with its ability to recruit βarr2. Among them, a “SSS” serine cluster located at amino acid position 873-875 within the C-terminal tail (C-tail), is in a region consistent with other GPCRs that bind βarr2 with high-affinity. To test its functionality, a ligand-independent βarr2 translocation assay was implemented. We show that Lgr5 recruits βarr2 and that the “SSS” amino acids (873-875) are absolutely critical to this process. We also demonstrate that for full efficacy, this cluster requires other Lgr5 C-tail serines that were previously shown to be important for constitutive and βarr2 independent internalization of Lgr5. These data are proof of principle that a classical GPCR behavior can be manifested by Lgr5. The existence of alternative ligands or missing effectors of Lgr5 that scaffold this classical GPCR behavior and the downstream signaling pathways engaged should be considered. Characterizing Lgr5 signaling will be invaluable for assessing its role in tissue maintenance, repair, and disease. PMID:24386388

  19. Studying the information content of TMDs using Monte Carlo generators

    SciTech Connect

    Avakian, H.; Matevosyan, H.; Pasquini, B.; Schweitzer, P.

    2015-02-05

    Theoretical advances in studies of the nucleon structure have been spurred by recent measurements of spin and/or azimuthal asymmetries worldwide. One of the main challenges still remaining is the extraction of the parton distribution functions, generalized to describe transverse momentum and spatial distributions of partons from these observables with no or minimal model dependence. In this topical review we present the latest developments in the field with emphasis on requirements for Monte Carlo event generators, indispensable for studies of the complex 3D nucleon structure, and discuss examples of possible applications.

  20. Will More Diversified Staffs Diversify Newspaper Content? A Pilot Study.

    ERIC Educational Resources Information Center

    Fedler, Fred; and Others

    A pilot study asked 94 students enrolled in introductory newswriting classes at three separate universities to evaluate 18 news stories. About half the stories concerned topics that proponents of multiculturalism have suggested would receive more emphasis if newspapers employed more women and minorities: topics such as breast cancer, divorce,…

  1. Integration of Basic Skills into Social Studies Content.

    ERIC Educational Resources Information Center

    Lunstrum, John P.; Irvin, Judith L.

    1981-01-01

    A basic skills model is presented which stresses the skills of writing, reading, study, and research for elementary school pupils. The model focuses on lesson background, the purpose of the reading, independent reading, follow-up discussion, developing related skills, and extending and applying ideas. A lesson about the 1910 British expedition to…

  2. Secondary Social Studies: Arkansas Public School Course Content Guide.

    ERIC Educational Resources Information Center

    Arkansas State Dept. of Education, Little Rock.

    This guide is offered as a framework on which a comprehensive curriculum can be built. Within each subject area and grade level, skills have been identified at three instructional levels: basic, developmental, and extension. The study of political and economic systems, citizenship rights and responsibilities, and the foundations of the U.S.…

  3. 23 CFR 650.117 - Content of design studies.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... reasons, when applicable, why FEMA criteria (44 CFR 60.3, formerly 24 CFR 1910.3) are demonstrably... BRIDGES, STRUCTURES, AND HYDRAULICS Location and Hydraulic Design of Encroachments on Flood Plains § 650...) Studies by highway agencies shall contain: (1) The hydrologic and hydraulic data and design...

  4. A Study of the Contents of First Graders' Journals.

    ERIC Educational Resources Information Center

    Manning, Maryann; And Others

    What first graders chose to put in their journals when given no direct suggestions for topics was studied during the 1985-86 school year in a suburban Birmingham, Alabama, classroom. Journal writing was scheduled for 30 minutes daily throughout the school year, but not all children chose to write every day. At the end of the year, all of the…

  5. Virtual Worlds in Distance Education: A Content Analysis Study

    ERIC Educational Resources Information Center

    Wang, Feihong; Lockee, Barbara B.

    2010-01-01

    The three-dimensional (3D) virtual world is one of the current innovations that has been discussed extensively as the potential medium for online distance education. Despite the heated discussion on the possible applications of 3D virtual worlds for distance education, empirical studies were hard to locate, and little has been written about how…

  6. Evaluation of the Use of Team Teaching for Delivering Sensitive Content: A Pilot Study

    ERIC Educational Resources Information Center

    Kerridge, Joanna; Kyle, Gaye; Marks-Maran, Diane

    2009-01-01

    Many programmes in further and higher education contain sensitive areas of content, such as diversity, racism, power and privilege, breaking bad news, counselling, sex education and ethical decision making. Team teaching may be a useful method for delivering sensitive areas of course content. This article presents a pilot study that was undertaken…

  7. Creative Writing in the Social Studies Classroom: Promoting Literacy and Content Learning

    ERIC Educational Resources Information Center

    Firek, Hilve

    2006-01-01

    This article discusses creative writing which promotes literacy and content learning in a social studies classroom. In this article, the author states, that educators must encourage students' creative energies and enable them to engage with content in new and stimulating ways. One way to help students really learn about the concepts inherent in…

  8. Content-Based Instruction in Primary and Secondary School Settings. Case Studies in TESOL Practice Series

    ERIC Educational Resources Information Center

    Kaufman, Dorit, Ed.; Crandall, JoAnn, Ed.

    2005-01-01

    Content-based instruction (CBI) challenges English language educators to teach English using materials that learners encounter in their regular subject-area classes. This volume helps ESL and EFL teachers meet that challenge by providing them with creative ways to integrate English language learning with the content that students study at primary…

  9. New Histories for a New State: A Study of History Textbook Content in Northern Ireland

    ERIC Educational Resources Information Center

    Terra, Luke

    2014-01-01

    This study examined the changing content of history textbooks in Northern Ireland, drawing on a sample of 15 textbooks published from 1968 to 2010. Findings from the content and narrative analysis indicated that following the introduction of the Northern Ireland Curriculum in 1991, history textbooks shifted from a narrative to source-driven…

  10. A Trend Study of Advertising Content Used by Banks Before, During and After a Banking Collapse.

    ERIC Educational Resources Information Center

    De Riemer, Cynthia; Baxter, Richard L.

    To examine the content of newspaper advertisements used by banks before, during, and after a major bank collapse, issues of the Knoxville (Tennessee) "News Sentinel" from 1982, 1983, and 1984 were analyzed for bank sponsored product and nonproduct advertisements. These advertisements were studied for type, size, and content relating to categories…

  11. Every Day Successes: Powerful Integration of Social Studies Content and English-Language Arts

    ERIC Educational Resources Information Center

    Field, Sherry L.; Bauml, Michelle; Ledbetter, Mary

    2011-01-01

    Instructional time for social studies has been reduced nationwide, particularly in the elementary grades. Yet states continue to promote content standards in history, economics, civics, and geography for young learners. Purposeful integrative content that is appropriate for grades K-6, while sometimes difficult to find, seems to many elementary…

  12. Studies of the content and process of environmental education

    NASA Astrophysics Data System (ADS)

    Wolfe, Vickie Lynn

    2002-01-01

    In order to examine the extent to which four-year institutions in the U.S. provide for the environmental education of students in nonenvironmental majors, and to identify various approaches to increasing environmental literacy at the college level, chief academic officers at these institutions were surveyed electronically. Of the 496 responding institutions (representing a 42.3 percent response rate), 11.6 percent indicated that an "environmental literacy" course was required of all students, and 55.0 percent reported that such a course was available and countable toward the institution's general education requirements. At least one "environmental" minor (e.g. Environmental Science, Environmental Studies) was offered at 33.7 percent of the institutions. Thirty-nine percent reported the existence of an "environmental" academic program that offered a course appropriate for non-majors. Various approaches to achieving environmental literacy at the college level are discussed, as are statistical differences in survey responses (1) among Carnegie classifications, from Research to Baccalaureate; (2) between public and private institutions; and (3) among geographical regions. Generally, proportions of positive responses were greater among Research institutions than among the other Carnegie categories, and among public than private institutions. Also explored are ways in which higher education institutions can enhance environmental education at the K--12 level, as well as some of the obstacles to such efforts. Brief overviews are provided of several higher education institutions' activities in the area of K--12 environmental education, either in the form of direct outreach to K--12 students, teacher training (pre-service or in-service), or both. Methods used by some of the programs to assess their success are discussed briefly, as are impediments to higher education institutions' enhancement of K--12 environmental education. Prominent among these impediments are funding and

  13. Design and Implementation Content Validity Study: Development of an instrument for measuring Patient-Centered Communication

    PubMed Central

    Zamanzadeh, Vahid; Ghahramanian, Akram; Rassouli, Maryam; Abbaszadeh, Abbas; Alavi-Majd, Hamid; Nikanfar, Ali-Reza

    2015-01-01

    Introduction: The importance of content validity in the instrument psychometric and its relevance with reliability, have made it an essential step in the instrument development. This article attempts to give an overview of the content validity process and to explain the complexity of this process by introducing an example. Methods: We carried out a methodological study conducted to examine the content validity of the patient-centered communication instrument through a two-step process (development and judgment). At the first step, domain determination, sampling (item generation) and instrument formation and at the second step, content validity ratio, content validity index and modified kappa statistic was performed. Suggestions of expert panel and item impact scores are used to examine the instrument face validity. Results: From a set of 188 items, content validity process identified seven dimensions includes trust building (eight items), informational support (seven items), emotional support (five items), problem solving (seven items), patient activation (10 items), intimacy/friendship (six items) and spirituality strengthening (14 items). Content validity study revealed that this instrument enjoys an appropriate level of content validity. The overall content validity index of the instrument using universal agreement approach was low; however, it can be advocated with respect to the high number of content experts that makes consensus difficult and high value of the S-CVI with the average approach, which was equal to 0.93. Conclusion: This article illustrates acceptable quantities indices for content validity a new instrument and outlines them during design and psychometrics of patient-centered communication measuring instrument. PMID:26161370

  14. Teachers' Professional Development: A Content Analysis about the Tendencies in Studies

    ERIC Educational Resources Information Center

    Yurtseven, Nihal; Bademcioglu, Mehtap

    2016-01-01

    The purpose of this study is to carry out a content analysis about the studies on teachers' professional development and to determine the tendencies in these studies. Within this scope, 60 studies that were registered to Turkish National Thesis Centre and ProQuest database between the years 2005-2015 were examined. Of the 60 studies, 37 of them…

  15. Studying protein-reconstituted proteoliposome fusion with content indicators in vitro

    PubMed Central

    Diao, Jiajie; Zhao, Minglei; Zhang, Yunxiang; Kyoung, Minjoung; Brunger, Axel T.

    2015-01-01

    In vitro reconstitution assays are commonly used to study biological membrane fusion. However, to date, most ensemble and single-vesicle experiments involving SNARE proteins have been performed only with lipid-mixing, but not content-mixing indicators. Through simultaneous detection of lipid and small content-mixing indicators, we found that lipid mixing often occurs seconds prior to content mixing, or without any content mixing at all, during a 50-sec observation period, for Ca2+-triggered fusion with SNAREs, full-length synaptotagmin-1, and complexin. Our results illustrate the caveats of commonly used bulk lipid-mixing fusion experiments. We recommend that proteoliposome fusion experiments should always employ content-mixing indicators in addition to, or in place of, lipid-mixing indicators. PMID:23625805

  16. 78 FR 42085 - Draft Guidance for Industry on Pediatric Study Plans: Content of and Process for Submitting...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-15

    ...: Content of and Process for Submitting Initial Pediatric Study Plans and Amended Pediatric Study Plans... Plans: Content of and Process for Submitting Initial Pediatric Study Plans and Amended Pediatric Study... draft guidance for industry entitled ``Pediatric Study Plans: Content of and Process for...

  17. A study of the metal content of municipal solid waste. Final report

    SciTech Connect

    Churney, K.L.; Domalski, E.S.

    1998-01-01

    Knowledge of the content of toxic components, so called pollutant precursors, in the municipal solid waste (MSW) stream is essential to development of the strategies for source reduction and reuse, recycling, composting and disposal. Data are scarce; trends in composition for any locality even more so. In a previous study the total and water soluble chlorine content of the components of municipal solid waste were determined from sampling studies at two sites, Baltimore County, MD, and Brooklyn, NY, each for a five day period. The total sulfur content of the combined combustible components was also determined. Because of the scarcity of data and synergistic effects, it seemed appropriate to determine the heavy metal content of the preceding material prior to its disposal. The metals chosen were the so-called priority pollutant metals (PPM): antimony, arsenic, beryllium, cadmium, chromium, copper, lead, mercury, nickel, selenium, silver, thallium, and zinc.

  18. The Relationship Between Sexual Content on Mass Media and Social Media: A Longitudinal Study.

    PubMed

    Vandenbosch, Laura; van Oosten, Johanna M F; Peter, Jochen

    2015-12-01

    The goal of this study was to investigate whether exposure to sexual reality television content and Internet pornography (IP) is related to sexual self-presentation on social media. Based on a two-wave panel survey among 1,765 adolescents aged 13-17 years, we found that watching sexual reality television content stimulated adolescents to produce and distribute sexual images of themselves on social media. In turn, sexual self-presentation on social media led adolescents to watch sexual reality television content more frequently. These relationships were similar among boys and girls. No reciprocal relationship between exposure to IP and boys' and girls' sexual self-presentation on social media was found. The results suggest that sexual content in mainstream mass media may predict adolescents' sexually oriented behavior on social media and vice versa. Moreover, adolescents seem to differentiate between types of sexual content (i.e., mainstream versus more explicit sexual content) when incorporating sexual media content in their sexual behavior online. PMID:26588715

  19. Making Connections among Student Learning, Content, and Teaching: Teacher Talk Paths in Elementary Mathematics Lesson Study

    ERIC Educational Resources Information Center

    Murata, Aki; Bofferding, Laura; Pothen, Bindu E.; Taylor, Megan W.; Wischnia, Sarah

    2012-01-01

    This study investigated how elementary teachers in a mathematics lesson study made sense of student learning, teaching, and content, as related to using representations in teaching multidigit subtraction, and how changes occurred over time in their talk and practice. The lesson-study process paved a group talk path along which teacher talk shifted…

  20. The Strategic Content Learning Approach to Promoting Self-Regulated Learning: A Report of Three Studies.

    ERIC Educational Resources Information Center

    Butler, Deborah L.

    1998-01-01

    Reports findings from three studies investigating the efficacy of an instructional model designed to promote self-regulation, the Strategic Content Learning (SCL) approach. Each study comprised multiple in-depth case studies involving postsecondary students with learning disabilities who ranged in age from 19 to 48 years. Implications for theory,…

  1. [The Study of the Spectral Model for Estimating Pigment Contents of Tobacco Leaves in Field].

    PubMed

    Ren, Xiao; Lao, Cai-lian; Xu, Zhao-li; Jin, Yan; Guo, Yan; Li, Jun-hui; Yang, Yu-hong

    2015-06-01

    Fast and non-destructive measurements of tobacco leaf pigment contents by spectroscopy in situ in the field has great significance in production guidance for nutrient diagnosis and growth monitoring of tobacco in vegetative growth stage, and it is also very important for the quality evaluation of tobacco leaves in mature stage. The purpose of this study is to estimate the chlorophyll and carotenoid contents of tobacco leaves using tobacco leaf spectrum collected in the field. Reflectance spectrum of tobacco leaves in vegetative growth stage and mature stage were collected in situ in the field and the pigment contents of tobacco leaf samples were measured in this study, taking the tobacco leaf samples collected in each and both stages as modeling sets respectively, and using the methods of support vector machine (SVM) and spectral indice to establish the pigment content estimation models, and then compare the prediction performance of the models built by different methods. The study results indicated that the difference of estimation performance by each stage or mixed stages is not significant. For chlorophyll content, SVM and spectral indice modeling methods can both have a well estimation performance, while for carotenoid content, SVM modeling method has a better estimation performance than spectral indice. The coefficient of determination and the root mean square error of SVM model for estimating tobacco leaf chlorophyll content by each stage were 0.867 6 and 0.014 7, while the coefficient of determination and the root mean square error of SVM model for estimating tobacco leaf chlorophyll content by mixed stages were 0.898 6 and 0.012 3; The coefficient of determination and the root mean square error for estimating tobacco leaf carotenoid content by each stage were 0.861 4 and 0.002 5, while the coefficient of determination and the root mean square error of SVM model for estimating tobacco leaf carotenoid content by mixed stages were 0.839 9 and 0.002 5. The

  2. Beta-arrestin1 and 2 differently modulate metabotropic glutamate receptor 7 signaling in rat developmental sevoflurane-induced neuronal apoptosis.

    PubMed

    Wang, W-Y; Wu, X-M; Jia, L-J; Zhang, H-H; Cai, F; Mao, H; Xu, W-C; Chen, L; Zhang, J; Hu, S-F

    2016-01-28

    Beta-arrestins (β-arrs) are initially known as negative regulators of G protein-coupled receptors (GPCRs). Recently, there is increasing evidence suggesting that β-arrs also serve as scaffolds and adapters that mediate distinct intracellular signal transduction initiated by GPCR activation. In the previous study, we have shown that metabotropic glutamate receptor 7 (mGluR7) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling may be involved in the developmental sevoflurane neurotoxicity. In the present study, we showed that activation of mGluR7 with a group III mGluRs orthosteric agonist LAP4 or an atypical mGluR7 allosteric agonist N,N'-bis(diphenylmethyl)-1,2-ethanediamine dihydrochloride (AMN082) significantly attenuated sevoflurane-induced neuronal apoptosis. Interestingly, this neuroprotective role of LAP4 could be partially reduced by β-arr1 small interfering RNA (siRNA) or β-arr2 siRNA transfection. In contrast, β-arr2 siRNA transfection alone abolished the effects of AMN082 on sevoflurane neurotoxicity. In addition, administration of LAP4 or AMN082 significantly enhanced Phospho-ERK1/2 in sevoflurane neurotoxicity, which could be abrogated by β-arr2 siRNA transfection, but not by β-arr1 siRNA transfection. Increased β-arr2-dependent Phospho-ERK1/2 signaling alleviated sevoflurane neurotoxicity by inhibiting bad phosphorylation. We also found that the neuroprotective role of AMN082 was completely reversed by ERK1/2 inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126). Alternatively, treatment with U0126 partially suppressed the neuroprotective of LAP4, suggesting that other mechanisms may be implicated in this process. Further investigation indicated that, in the scenario of sevoflurane neurotoxicity, application of LAP4 (but not AMN082) increased the interaction of β-arrs with transcriptional factors CREB binding protein (CBP) and p300. LAP4 also enhanced the β-arr1-dependent H3 and H4 acetylation in

  3. The studying of washing of arsenic and sulfur from coals having different ranges of arsenic contents

    USGS Publications Warehouse

    Wang, M.; Song, D.; Zheng, B.; Finkelman, R.B.

    2008-01-01

    To study the effectiveness of washing in removal of arsenic and sulfur from coals with different ranges of arsenic concentration, coal was divided into three groups on the basis of arsenic content: 0-5.5 mg/kg, 5.5 mg/kg-8.00 mg/kg, and over 8.00 mg/kg. The result shows that the arsenic in coals with higher arsenic content occurs mainly in an inorganic state and can be relatively easily removed. Arsenic removal is very difficult and less complete when the arsenic content is lower than 5.5 mg/kg because most of this arsenic is in an organic state. There is no relationship between washing rate of total sulfur and arsenic content, but the relationship between the washing rate of total sulfur and percent of organic sulfur is very strong. ?? 2008 New York Academy of Sciences.

  4. The studying of washing of arsenic and sulfur from coals having different ranges of arsenic contents

    SciTech Connect

    Mingshi Wang; Dangyu Song; Baoshan Zheng; R.B. Finkelman

    2008-10-15

    To study the effectiveness of washing in removal of arsenic and sulfur from coals with different ranges of arsenic concentration, coal was divided into three groups on the basis of arsenic content: 0-5.5 mg/kg, 5.5 mg/kg-8.00 mg/kg, and over 8.00 mg/kg. The result shows that the arsenic in coals with higher arsenic content occurs mainly in an inorganic state and can be relatively easily removed. Arsenic removal is very difficult and less complete when the arsenic content is lower than 5.5 mg/kg because most of this arsenic is in an organic state. There is no relationship between washing rate of total sulfur and arsenic content, but the relationship between the washing rate of total sulfur and percent of organic sulfur is very strong.

  5. Developing fair tests for mathematics curriculum comparison studies: the role of content analyses

    NASA Astrophysics Data System (ADS)

    Chávez, Óscar; Papick, Ira; Ross, Daniel J.; Grouws, Douglas A.

    2011-12-01

    This article describes the process of development of assessment instruments for a three-year longitudinal comparative study that focused on evaluating American high school students' mathematics learning from two distinct approaches to content organization: curriculum built around a sequence of three full-year courses (Algebra 1, Geometry, and Algebra 2) and a sequence of integrated mathematics courses (algebra and geometry content, together with functions, data analysis, and discrete mathematics is integrated each year). The study was conducted in six school districts in five states involving over 4,000 students from schools that were using both curricular approaches but with different groups of students. In order to develop assessment instruments that were not biased towards either of the two curriculum programs (Fair Tests), an iterative process of content analyses, identification of common topics, internal and external reviews, pilot tests, and revisions was followed, resulting in five tests that were used in the three years of the study. Results indicate that these tests have solid discrimination properties and address adequately mathematics content common to both secondary curriculum programs. The corresponding scoring rubrics are highly reliable, with interrater reliability above 94% for all tests. Mathematics education researchers involved in curriculum comparison studies need to conduct content analyses of the curriculum materials under study in order to identify salient relationships between curriculum programs and student outcomes.

  6. Genome-Wide Association Study Identifies Candidate Genes for Starch Content Regulation in Maize Kernels.

    PubMed

    Liu, Na; Xue, Yadong; Guo, Zhanyong; Li, Weihua; Tang, Jihua

    2016-01-01

    Kernel starch content is an important trait in maize (Zea mays L.) as it accounts for 65-75% of the dry kernel weight and positively correlates with seed yield. A number of starch synthesis-related genes have been identified in maize in recent years. However, many loci underlying variation in starch content among maize inbred lines still remain to be identified. The current study is a genome-wide association study that used a set of 263 maize inbred lines. In this panel, the average kernel starch content was 66.99%, ranging from 60.60 to 71.58% over the three study years. These inbred lines were genotyped with the SNP50 BeadChip maize array, which is comprised of 56,110 evenly spaced, random SNPs. Population structure was controlled by a mixed linear model (MLM) as implemented in the software package TASSEL. After the statistical analyses, four SNPs were identified as significantly associated with starch content (P ≤ 0.0001), among which one each are located on chromosomes 1 and 5 and two are on chromosome 2. Furthermore, 77 candidate genes associated with starch synthesis were found within the 100-kb intervals containing these four QTLs, and four highly associated genes were within 20-kb intervals of the associated SNPs. Among the four genes, Glucose-1-phosphate adenylyltransferase (APS1; Gene ID GRMZM2G163437) is known as an important regulator of kernel starch content. The identified SNPs, QTLs, and candidate genes may not only be readily used for germplasm improvement by marker-assisted selection in breeding, but can also elucidate the genetic basis of starch content. Further studies on these identified candidate genes may help determine the molecular mechanisms regulating kernel starch content in maize and other important cereal crops. PMID:27512395

  7. Genome-Wide Association Study Identifies Candidate Genes for Starch Content Regulation in Maize Kernels

    PubMed Central

    Liu, Na; Xue, Yadong; Guo, Zhanyong; Li, Weihua; Tang, Jihua

    2016-01-01

    Kernel starch content is an important trait in maize (Zea mays L.) as it accounts for 65–75% of the dry kernel weight and positively correlates with seed yield. A number of starch synthesis-related genes have been identified in maize in recent years. However, many loci underlying variation in starch content among maize inbred lines still remain to be identified. The current study is a genome-wide association study that used a set of 263 maize inbred lines. In this panel, the average kernel starch content was 66.99%, ranging from 60.60 to 71.58% over the three study years. These inbred lines were genotyped with the SNP50 BeadChip maize array, which is comprised of 56,110 evenly spaced, random SNPs. Population structure was controlled by a mixed linear model (MLM) as implemented in the software package TASSEL. After the statistical analyses, four SNPs were identified as significantly associated with starch content (P ≤ 0.0001), among which one each are located on chromosomes 1 and 5 and two are on chromosome 2. Furthermore, 77 candidate genes associated with starch synthesis were found within the 100-kb intervals containing these four QTLs, and four highly associated genes were within 20-kb intervals of the associated SNPs. Among the four genes, Glucose-1-phosphate adenylyltransferase (APS1; Gene ID GRMZM2G163437) is known as an important regulator of kernel starch content. The identified SNPs, QTLs, and candidate genes may not only be readily used for germplasm improvement by marker-assisted selection in breeding, but can also elucidate the genetic basis of starch content. Further studies on these identified candidate genes may help determine the molecular mechanisms regulating kernel starch content in maize and other important cereal crops. PMID:27512395

  8. Structural and Biochemical Basis for Ubiquitin Ligase Recruitment by Arrestin-related Domain-containing Protein-3 (ARRDC3)*

    PubMed Central

    Qi, Shiqian; O'Hayre, Morgan; Gutkind, J. Silvio; Hurley, James H.

    2014-01-01

    After protracted stimulation, the β2-adrenergic receptor and many other G-protein-coupled receptors are ubiquitinated and down-regulated. Arrestin-related domain-containing protein-3 (ARRDC3) has been proposed to recruit the ubiquitin ligase Nedd4 to the β2-adrenergic receptor. ARRDC3 contains two PPXY motifs that could potentially interact with any of the four WW domains of Nedd4. Here we dissect the interaction determinants. ARRDC3 PPXY-Nedd4 WW dissociation constants vary from unmeasurable to Kd = 3 μm for the third WW domain of Nedd4 binding to the first PPXY motif of ARRDC3. Structures of the uncomplexed and PPXY1-bound WW3 domain were determined at 1.1 and 1.7 Å resolution. The structures revealed conformational changes upon binding and the hydrogen bonding network in exquisite detail. Tight packing of ARRDC3 Val-352′, part of a 310 helix at the C terminus of PPXY1, is important for high affinity binding to WW3. Although no single WW domain is strictly essential for the binding of Nedd4 and ARRDC3 expressed in HEK293 cells, high affinity binding of full-length ARRDC3 and Nedd4 is driven by the avid interaction of both PPXY motifs with either the WW2-WW3 or WW3-WW4 combinations, with Kd values as low as 300 nm. PMID:24379409

  9. Structural and biochemical basis for ubiquitin ligase recruitment by arrestin-related domain-containing protein-3 (ARRDC3).

    PubMed

    Qi, Shiqian; O'Hayre, Morgan; Gutkind, J Silvio; Hurley, James H

    2014-02-21

    After protracted stimulation, the β2-adrenergic receptor and many other G-protein-coupled receptors are ubiquitinated and down-regulated. Arrestin-related domain-containing protein-3 (ARRDC3) has been proposed to recruit the ubiquitin ligase Nedd4 to the β2-adrenergic receptor. ARRDC3 contains two PPXY motifs that could potentially interact with any of the four WW domains of Nedd4. Here we dissect the interaction determinants. ARRDC3 PPXY-Nedd4 WW dissociation constants vary from unmeasurable to Kd = 3 μM for the third WW domain of Nedd4 binding to the first PPXY motif of ARRDC3. Structures of the uncomplexed and PPXY1-bound WW3 domain were determined at 1.1 and 1.7 Å resolution. The structures revealed conformational changes upon binding and the hydrogen bonding network in exquisite detail. Tight packing of ARRDC3 Val-352', part of a 310 helix at the C terminus of PPXY1, is important for high affinity binding to WW3. Although no single WW domain is strictly essential for the binding of Nedd4 and ARRDC3 expressed in HEK293 cells, high affinity binding of full-length ARRDC3 and Nedd4 is driven by the avid interaction of both PPXY motifs with either the WW2-WW3 or WW3-WW4 combinations, with Kd values as low as 300 nM. PMID:24379409

  10. Astroglial β-Arrestin1-mediated Nuclear Signaling Regulates the Expansion of Neural Precursor Cells in Adult Hippocampus

    PubMed Central

    Tao, Yezheng; Ma, Li; Liao, Zhaohui; Le, Qiumin; Yu, Jialing; Liu, Xing; Li, Haohong; Chen, Yuejun; Zheng, Ping; Yang, Zhengang; Ma, Lan

    2015-01-01

    Adult hippocampal neurogenesis is crucial for preserving normal brain function, but how it is regulated by niche cells is uncertain. Here we show that β-arrestin 1 (β-arr1) in dentate gyrus (DG) regulates neural precursor proliferation. β-arr1 knockout (KO) mice show reduced neural precursor proliferation in subgranular zone (SGZ) which could be rescued by selective viral expression of β-arr1 but not its nuclear-function-deficient mutants under control of hGFAP promotor in DG. Compared with wild type astrocytes, β-arr1 KO astrocytes nurture less neurospheres, and this may be attributed to changed activity of soluble, heat-sensitive excretive factors, such as BMP2. RNA-sequencing reveals that β-arr1 KO DG astrocytes exhibit an aberrant gene expression profile of niche factors, including elevated transcription of Bmp2. Taken together, our data suggest that β-arr1 mediated nuclear signaling regulates the production of excretive factors derived from niche astrocytes and expansion of neural precursors in DG, thus maintaining homeostasis of adult hippocampal neurogenesis. PMID:26500013