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  1. Article ; Online: Probing conformational changes in rhodopsin using hydrogen-deuterium exchange coupled to mass spectrometry.

    Orban, Tivadar / Tsybovsky, Yaroslav

    Methods in molecular biology (Clifton, N.J.)

    2015  Volume 1271, Page(s) 113–121

    Abstract: Hydrogen-deuterium exchange coupled to mass spectrometry is a powerful tool to evaluate changes in protein conformation between two or more states. Here, we describe a complete methodology that can be used to assess conformational changes in rhodopsin ... ...

    Abstract Hydrogen-deuterium exchange coupled to mass spectrometry is a powerful tool to evaluate changes in protein conformation between two or more states. Here, we describe a complete methodology that can be used to assess conformational changes in rhodopsin accompanying its transition from the inactive to activated state upon light exposure. This approach may be employed to investigate the structure and conformational changes of various membrane proteins.
    MeSH term(s) Deuterium/chemistry ; Deuterium Exchange Measurement/methods ; Hydrogen/chemistry ; Mass Spectrometry ; Rhodopsin/chemistry
    Chemical Substances Hydrogen (7YNJ3PO35Z) ; Rhodopsin (9009-81-8) ; Deuterium (AR09D82C7G)
    Language English
    Publishing date 2015
    Publishing country United States
    Document type Journal Article
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-4939-2330-4_8
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  2. Article ; Online: Cytokine production pattern of T lymphocytes in neonatal arterial ischemic stroke during the first month of life-a case study.

    Bajnok, Anna / Berta, László / Orbán, Csaba / Tulassay, Tivadar / Toldi, Gergely

    Journal of neuroinflammation

    2018  Volume 15, Issue 1, Page(s) 191

    Abstract: Background: The perinatal period carries the highest risk for stroke in childhood; however, the pathophysiology is poorly understood and preventive, prognostic, and therapeutic strategies are not available. A new pathophysiological model describes the ... ...

    Abstract Background: The perinatal period carries the highest risk for stroke in childhood; however, the pathophysiology is poorly understood and preventive, prognostic, and therapeutic strategies are not available. A new pathophysiological model describes the development of neonatal arterial ischemic stroke (NAIS) as the combined result of prenatal inflammation and hypoxic-ischemic insult. Neuroinflammation and a systemic inflammatory response are also important features of NAIS. Identifying key players of the inflammatory system is in the limelight of current research.
    Case presentation: We present four NAIS cases, in whom detailed analysis of intracellular and plasma cytokine levels are available from the first month of life. All neonates were admitted with the initial diagnosis of hypoxic ischemic encephalopathy (HIE); however, early MRI examination revealed NAIS. Blood samples were collected between 3 and 6 h of life, at 24 h, 72 h, 1 week, and 1 month of life. Peripheral blood mononuclear cells were assessed with flow cytometry and plasma cytokine levels were measured. Pooled data from the cohort of four NAIS patients were compared to infants with HIE. At 6 and 72 h of age, the prevalence of IL10+ CD8+ lymphocytes remained lower in NAIS. At 6 h, CD8+ lymphocytes in NAIS produced more IL-17. At 72 h, CD8+ cells produced more IL-6 in severe HIE than in NAIS, but IL-6 production remained elevated in CD8 cells at 1 month in NAIS, while it decreased in HIE. At 1 week, the prevalence of TGF-β + lymphocytes prone to enter the CNS was elevated in NAIS. On the other hand, by 1 month of age, the prevalence of TGF-β + CD4+ lymphocytes decreased in NAIS compared to HIE. At 72 h, we found elevated plasma levels of IL-5, MCP-1, and IL-17 in NAIS. By 1 month, plasma levels of IL-4, IL-12, and IL-17 decreased in NAIS but remained elevated in HIE.
    Conclusions: Differences in the cytokine network are present between NAIS and HIE. CD8 lymphocytes appear to shift towards the pro-inflammatory direction in NAIS. The inflammatory response appears to be more pronounced at 72 h in NAIS but decreases faster, reaching lower plasma levels of inflammatory markers at 1 month.
    MeSH term(s) Adolescent ; Cytokines/metabolism ; Female ; Gestational Age ; Humans ; Hypoxia-Ischemia, Brain/complications ; Infant ; Leukocytes, Mononuclear/metabolism ; Leukocytes, Mononuclear/pathology ; Lymphocyte Count ; Magnetic Resonance Imaging ; Male ; Stroke/diagnostic imaging ; Stroke/etiology ; Stroke/pathology ; T-Lymphocytes/metabolism
    Chemical Substances Cytokines
    Keywords covid19
    Language English
    Publishing date 2018-06-22
    Publishing country England
    Document type Case Reports ; Journal Article
    ISSN 1742-2094
    ISSN (online) 1742-2094
    DOI 10.1186/s12974-018-1229-y
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  3. Article ; Online: From atomic structures to neuronal functions of g protein-coupled receptors.

    Palczewski, Krzysztof / Orban, Tivadar

    Annual review of neuroscience

    2013  Volume 36, Page(s) 139–164

    Abstract: G protein-coupled receptors (GPCRs) are essential mediators of signal transduction, neurotransmission, ion channel regulation, and other cellular events. GPCRs are activated by diverse stimuli, including light, enzymatic processing of their N-termini, ... ...

    Abstract G protein-coupled receptors (GPCRs) are essential mediators of signal transduction, neurotransmission, ion channel regulation, and other cellular events. GPCRs are activated by diverse stimuli, including light, enzymatic processing of their N-termini, and binding of proteins, peptides, or small molecules such as neurotransmitters. GPCR dysfunction caused by receptor mutations and environmental challenges contributes to many neurological diseases. Moreover, modern genetic technology has helped identify a rich array of mono- and multigenic defects in humans and animal models that connect such receptor dysfunction with disease affecting neuronal function. The visual system is especially suited to investigate GPCR structure and function because advanced imaging techniques permit structural studies of photoreceptor neurons at both macro and molecular levels that, together with biochemical and physiological assessment in animal models, provide a more complete understanding of GPCR signaling.
    MeSH term(s) Animals ; Humans ; Models, Animal ; Neurons/physiology ; Neurons/ultrastructure ; Receptors, G-Protein-Coupled/chemistry ; Receptors, G-Protein-Coupled/physiology ; Receptors, G-Protein-Coupled/ultrastructure ; Signal Transduction/physiology ; Structure-Activity Relationship
    Chemical Substances Receptors, G-Protein-Coupled
    Language English
    Publishing date 2013-05-15
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Review
    ZDB-ID 282459-0
    ISSN 1545-4126 ; 0147-006X
    ISSN (online) 1545-4126
    ISSN 0147-006X
    DOI 10.1146/annurev-neuro-062012-170313
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    Zs.A 1433: Show issues Location:
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  4. Article ; Online: Different calcium influx characteristics upon Kv1.3 and IKCa1 potassium channel inhibition in T helper subsets.

    Orbán, Csaba / Bajnok, Anna / Vásárhelyi, Barna / Tulassay, Tivadar / Toldi, Gergely

    Cytometry. Part A : the journal of the International Society for Analytical Cytology

    2014  Volume 85, Issue 7, Page(s) 636–641

    Abstract: Functional imbalance between T helper subsets plays important role in the pathogenesis of autoimmune disorders. Transient increase of cytoplasmic calcium level, and sustention of negative membrane potential by voltage sensitive Kv1.3 and calcium- ... ...

    Abstract Functional imbalance between T helper subsets plays important role in the pathogenesis of autoimmune disorders. Transient increase of cytoplasmic calcium level, and sustention of negative membrane potential by voltage sensitive Kv1.3 and calcium-dependent IKCa1 potassium channels are essential for short-term lymphocyte activation, thus present possible target for selective immunomodulation. We aimed to investigate calcium influx sensitivity to the inhibition of potassium channels in the main T helper subsets. Peripheral blood from 11 healthy individuals was drawn and calcium influx kinetics following activation with phytohemagglutinin in Th1, Th2, Th17, and Treg cells were evaluated. Alteration of calcium influx induced by specific inhibitors of Kv1.3 and IKCa1 potassium channels, and the expression of Kv1.3 channels were also assessed. Highest cytoplasmic calcium concentration was observed in stimulated Th1 cells, while the lowest level was measured in Treg cells. In Th1 and Th17 cells, inhibition of both investigated potassium channels decreased calcium influx. In Th2 cells only the inhibitor of Kv1.3 channels, while in Treg cells none of the inhibitors had significant effect. Upon the inhibition of IKCa1 channels, short-term activation of proinflammatory cells was specifically decreased without affecting anti-inflammatory subsets, indicating that selective immunomodulation is possible in healthy individuals.
    MeSH term(s) Adult ; Antibodies, Monoclonal/pharmacology ; Autoimmune Diseases/immunology ; Calcium/metabolism ; Calcium Signaling/immunology ; Female ; Humans ; Intermediate-Conductance Calcium-Activated Potassium Channels/antagonists & inhibitors ; Kv1.3 Potassium Channel/antagonists & inhibitors ; Kv1.3 Potassium Channel/biosynthesis ; Leukocytes, Mononuclear/immunology ; Lymphocyte Activation/immunology ; Male ; Phytohemagglutinins/pharmacology ; Potassium Channel Blockers/pharmacology ; Potassium Channels/metabolism ; Pyrazoles/pharmacology ; Scorpion Venoms/pharmacology ; T-Lymphocytes, Regulatory/immunology ; T-Lymphocytes, Regulatory/metabolism ; Th1 Cells/immunology ; Th1 Cells/metabolism ; Th17 Cells/immunology ; Th17 Cells/metabolism ; Th2 Cells/immunology ; Th2 Cells/metabolism ; Young Adult
    Chemical Substances Antibodies, Monoclonal ; Intermediate-Conductance Calcium-Activated Potassium Channels ; KCNN4 protein, human ; Kv1.3 Potassium Channel ; Phytohemagglutinins ; Potassium Channel Blockers ; Potassium Channels ; Pyrazoles ; Scorpion Venoms ; TRAM 34 ; margatoxin (6197NL836C) ; Calcium (SY7Q814VUP)
    Language English
    Publishing date 2014-07
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2099868-5
    ISSN 1552-4930 ; 0196-4763 ; 1552-4922
    ISSN (online) 1552-4930
    ISSN 0196-4763 ; 1552-4922
    DOI 10.1002/cyto.a.22479
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    Zs.A 1688: Show issues Location:
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  5. Article ; Online: Hydrogen/Deuterium Exchange Mass Spectrometry of Human Green Opsin Reveals a Conserved Pro-Pro Motif in Extracellular Loop 2 of Monostable Visual G Protein-Coupled Receptors.

    Hofmann, Lukas / Alexander, Nathan S / Sun, Wenyu / Zhang, Jianye / Orban, Tivadar / Palczewski, Krzysztof

    Biochemistry

    2017  Volume 56, Issue 17, Page(s) 2338–2348

    Abstract: Opsins comprise the protein component of light sensitive G protein-coupled receptors (GPCRs) in the retina of the eye that are responsible for the transduction of light into a biochemical signal. Here, we used hydrogen/deuterium (H/D) exchange coupled ... ...

    Abstract Opsins comprise the protein component of light sensitive G protein-coupled receptors (GPCRs) in the retina of the eye that are responsible for the transduction of light into a biochemical signal. Here, we used hydrogen/deuterium (H/D) exchange coupled with mass spectrometry to map conformational changes in green cone opsin upon light activation. We then compared these findings with those reported for rhodopsin. The extent of H/D exchange in green cone opsin was greater than in rhodopsin in the dark and bleached states, suggesting a higher structural heterogeneity for green cone opsin. Further analysis revealed that green cone opsin exists as a dimer in both dark (inactive) and bleached (active) states, and that the predicted glycosylation sites at N
    MeSH term(s) Amino Acid Motifs ; Amino Acid Substitution ; Asparagine/metabolism ; Binding Sites ; Computational Biology ; Cone Opsins/chemistry ; Cone Opsins/genetics ; Cone Opsins/metabolism ; Cone Opsins/radiation effects ; Conserved Sequence ; Deuterium Exchange Measurement ; Glycosylation ; Humans ; Ligands ; Light ; Point Mutation ; Proline/chemistry ; Protein Conformation ; Protein Refolding/radiation effects ; Protein Structure, Tertiary ; Receptors, G-Protein-Coupled/chemistry ; Receptors, G-Protein-Coupled/genetics ; Receptors, G-Protein-Coupled/metabolism ; Receptors, G-Protein-Coupled/radiation effects ; Recombinant Proteins ; Rod Opsins/chemistry ; Rod Opsins/genetics ; Rod Opsins/metabolism ; Rod Opsins/radiation effects ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry
    Chemical Substances Cone Opsins ; Ligands ; Receptors, G-Protein-Coupled ; Recombinant Proteins ; Rod Opsins ; middle-wavelength opsin ; Asparagine (7006-34-0) ; Proline (9DLQ4CIU6V)
    Language English
    Publishing date 2017-04-21
    Publishing country United States
    Document type Comparative Study ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 1108-3
    ISSN 1520-4995 ; 0006-2960
    ISSN (online) 1520-4995
    ISSN 0006-2960
    DOI 10.1021/acs.biochem.7b00165
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    Zs.A 217: Show issues Location:
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  6. Article ; Online: Altered activation of peripheral CD8+ T cells in pediatric Crohn's disease.

    Orbán, Csaba / Szabó, Dolóresz / Bajnok, Anna / Vásárhelyi, Barna / Tulassay, Tivadar / Arató, András / Veres, Gábor / Toldi, Gergely

    Immunology letters

    2017  Volume 185, Page(s) 48–51

    Abstract: Aim: Although Crohn's disease (CD) is an extensively investigated autoimmune condition, knowledge on early phase activation of lymphocytes, especially CD8+ Tc cells is scarce. Our aim was to investigate the calcium influx characteristics of CD8+ cells ... ...

    Abstract Aim: Although Crohn's disease (CD) is an extensively investigated autoimmune condition, knowledge on early phase activation of lymphocytes, especially CD8+ Tc cells is scarce. Our aim was to investigate the calcium influx characteristics of CD8+ cells upon activation as well as the expression and function of Kv1.3 and IKCa1 lymphocyte potassium channels.
    Methods: We took peripheral blood from 12 healthy controls, 23 CD children on conventional therapy and 6 severe CD children before and after infliximab therapy. Intracellular calcium levels were monitored in CD8+ lymphocytes using flow cytometry.
    Results: In CD treated with standard therapy calcium response during activation was elevated. This was not affected by the inhibition of Kv1.3 or IKCa1 potassium channels. After the switch to infliximab potassium channel function and expression of CD8+ lymphocytes were comparable to healthy controls in severe CD.
    Conclusion: Calcium handling of CD8+ lymphocytes is altered in pediatric CD, which is normalized by infliximab therapy.
    Language English
    Publishing date 2017-05
    Publishing country Netherlands
    Document type Journal Article
    ZDB-ID 445150-8
    ISSN 1879-0542 ; 0165-2478
    ISSN (online) 1879-0542
    ISSN 0165-2478
    DOI 10.1016/j.imlet.2017.03.009
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  7. Article ; Online: Structural approaches to understanding retinal proteins needed for vision.

    Orban, Tivadar / Jastrzebska, Beata / Palczewski, Krzysztof

    Current opinion in cell biology

    2013  Volume 27, Page(s) 32–43

    Abstract: The past decade has witnessed an impressive expansion of our knowledge of retinal photoreceptor signal transduction and the regulation of the visual cycle required for normal eyesight. Progress in human genetics and next generation sequencing ... ...

    Abstract The past decade has witnessed an impressive expansion of our knowledge of retinal photoreceptor signal transduction and the regulation of the visual cycle required for normal eyesight. Progress in human genetics and next generation sequencing technologies have revealed the complexity behind many inherited retinal diseases. Structural studies have markedly increased our understanding of the visual process. Moreover, technical innovations and improved methodologies in proteomics, macromolecular crystallization and high resolution imaging at different levels set the scene for even greater advances. Pharmacology combined with structural biology of membrane proteins holds great promise for developing innovative accessible therapies for millions robbed of their sight or progressing toward blindness.
    MeSH term(s) Animals ; Arrestins/chemistry ; Arrestins/genetics ; Arrestins/metabolism ; Eye Proteins/chemistry ; Eye Proteins/metabolism ; G-Protein-Coupled Receptor Kinase 1/metabolism ; Humans ; Models, Molecular ; Retina/chemistry ; Retina/metabolism ; Retina/radiation effects ; Retinal Rod Photoreceptor Cells/metabolism ; Retinal Rod Photoreceptor Cells/radiation effects ; Retinaldehyde/biosynthesis ; Retinaldehyde/metabolism ; Retinoids/metabolism ; Rhodopsin/biosynthesis ; Rhodopsin/chemistry ; Rhodopsin/metabolism ; Transducin/chemistry ; Transducin/metabolism ; Vision, Ocular/physiology ; Vision, Ocular/radiation effects ; cis-trans-Isomerases/chemistry ; cis-trans-Isomerases/metabolism
    Chemical Substances Arrestins ; Eye Proteins ; Retinoids ; Rhodopsin (9009-81-8) ; G-Protein-Coupled Receptor Kinase 1 (EC 2.7.11.14) ; retinoid isomerohydrolase (EC 3.1.1.64) ; Transducin (EC 3.6.5.1) ; cis-trans-Isomerases (EC 5.2.-) ; Retinaldehyde (RR725D715M)
    Language English
    Publishing date 2013-11-28
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Review
    ZDB-ID 1026381-0
    ISSN 1879-0410 ; 0955-0674
    ISSN (online) 1879-0410
    ISSN 0955-0674
    DOI 10.1016/j.ceb.2013.11.001
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    Zs.A 2963: Show issues Location:
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  8. Article ; Online: Altered calcium influx of peripheral Th2 cells in pediatric Crohn's disease: infliximab may normalize activation patterns.

    Orbán, Csaba / Szabó, Dolóresz / Bajnok, Anna / Vásárhelyi, Barna / Tulassay, Tivadar / Arató, András / Veres, Gábor / Toldi, Gergely

    Oncotarget

    2016  Volume 7, Issue 29, Page(s) 44966–44974

    Abstract: Objective: Crohn's disease is a chronic inflammation of the gastrointestinal tract with an abnormal immune phenotype. We investigated how intracellular calcium kinetics of Th1 and Th2 lymphocytes alter upon specific inhibition of Kv1.3 and IKCa1 ... ...

    Abstract Objective: Crohn's disease is a chronic inflammation of the gastrointestinal tract with an abnormal immune phenotype. We investigated how intracellular calcium kinetics of Th1 and Th2 lymphocytes alter upon specific inhibition of Kv1.3 and IKCa1 channels in pediatric Crohn's disease.
    Study design: Blood was taken from 12 healthy and 29 Crohn's disease children. Of those, 6 were switched to infliximab and re-sampled after the 4th infliximab treatment. Intracellular calcium levels were monitored using flow cytometry in the presence or absence of specific inhibitors of Kv1.3 and IKCa1 potassium channels.
    Results: In Crohn's disease treated with standard therapy, calcium response during activation was higher than normal in Th2 cells. This was normalized in vitro by inhibition of Kv1.3 or IKCa1 potassium channels. After the switch to infliximab, potassium channel function and expression in Th2 lymphocytes were comparable to those in Th1 cells.
    Conclusion: These results may indicate that potassium channels are potential immune modulatory targets in Crohn's disease.
    Language English
    Publishing date 2016-07-19
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2560162-3
    ISSN 1949-2553 ; 1949-2553
    ISSN (online) 1949-2553
    ISSN 1949-2553
    DOI 10.18632/oncotarget.10036
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  9. Article: Hydrogen/Deuterium Exchange Mass Spectrometry of Human Green Opsin Reveals a Conserved Pro-Pro Motif in Extracellular Loop 2 of Monostable Visual G Protein-Coupled Receptors

    Hofmann, Lukas / Alexander Nathan S / Orban Tivadar / Palczewski Krzysztof / Sun Wenyu / Zhang Jianye

    Biochemistry. 2017 May 02, v. 56, no. 17

    2017  

    Abstract: Opsins comprise the protein component of light sensitive G protein-coupled receptors (GPCRs) in the retina of the eye that are responsible for the transduction of light into a biochemical signal. Here, we used hydrogen/deuterium (H/D) exchange coupled ... ...

    Abstract Opsins comprise the protein component of light sensitive G protein-coupled receptors (GPCRs) in the retina of the eye that are responsible for the transduction of light into a biochemical signal. Here, we used hydrogen/deuterium (H/D) exchange coupled with mass spectrometry to map conformational changes in green cone opsin upon light activation. We then compared these findings with those reported for rhodopsin. The extent of H/D exchange in green cone opsin was greater than in rhodopsin in the dark and bleached states, suggesting a higher structural heterogeneity for green cone opsin. Further analysis revealed that green cone opsin exists as a dimer in both dark (inactive) and bleached (active) states, and that the predicted glycosylation sites at N³² and N³⁴ are indeed glycosylated. Comparison of deuterium uptake between inactive and active states of green cone opsin also disclosed a reduced solvent accessibility of the extracellular N-terminal region and an increased accessibility of the chromophore binding site. Increased H/D exchange at the extracellular side of transmembrane helix four (TM4) combined with an analysis of sequence alignments revealed a conserved Pro-Pro motif in extracellular loop 2 (EL2) of monostable visual GPCRs. These data present new insights into the locus of chromophore release at the extracellular side of TM4 and TM5 and provide a foundation for future functional evaluation.
    Keywords binding sites ; deuterium ; glycosylation ; G-protein coupled receptors ; humans ; loci ; mass spectrometry ; retina ; rhodopsin ; sequence alignment ; solvents
    Language English
    Dates of publication 2017-0502
    Size p. 2338-2348.
    Publishing place American Chemical Society
    Document type Article
    ZDB-ID 1108-3
    ISSN 1520-4995 ; 0006-2960
    ISSN (online) 1520-4995
    ISSN 0006-2960
    DOI 10.1021%2Facs.biochem.7b00165
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  10. Article ; Online: Dephosphorylation by protein phosphatase 2A regulates visual pigment regeneration and the dark adaptation of mammalian photoreceptors.

    Kolesnikov, Alexander V / Orban, Tivadar / Jin, Hui / Brooks, Celine / Hofmann, Lukas / Dong, Zhiqian / Sokolov, Maxim / Palczewski, Krzysztof / Kefalov, Vladimir J

    Proceedings of the National Academy of Sciences of the United States of America

    2017  Volume 114, Issue 45, Page(s) E9675–E9684

    Abstract: Resetting of G-protein-coupled receptors (GPCRs) from their active state back to their biologically inert ground state is an integral part of GPCR signaling. This "on-off" GPCR cycle is regulated by reversible phosphorylation. Retinal rod and cone ... ...

    Abstract Resetting of G-protein-coupled receptors (GPCRs) from their active state back to their biologically inert ground state is an integral part of GPCR signaling. This "on-off" GPCR cycle is regulated by reversible phosphorylation. Retinal rod and cone photoreceptors arguably represent the best-understood example of such GPCR signaling. Their visual pigments (opsins) are activated by light, transduce the signal, and are then inactivated by a GPCR kinase and arrestin. Although pigment inactivation by phosphorylation is well understood, the enzyme(s) responsible for pigment dephosphorylation and the functional significance of this reaction remain unknown. Here, we show that protein phosphatase 2A (PP2A) acts as opsin phosphatase in both rods and cones. Elimination of PP2A substantially slows pigment dephosphorylation, visual chromophore recycling, and ultimately photoreceptor dark adaptation. These findings demonstrate that visual pigment dephosphorylation regulates the dark adaptation of photoreceptors and provide insights into the role of this reaction in GPCR signaling.
    Language English
    Publishing date 2017-11-07
    Publishing country United States
    Document type Journal Article
    ZDB-ID 209104-5
    ISSN 1091-6490 ; 0027-8424
    ISSN (online) 1091-6490
    ISSN 0027-8424
    DOI 10.1073/pnas.1712405114
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