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Phosphorylation of Rat Melanopsin at Ser-381 and Ser-398 by Light/Dark and Its Importance for Intrinsically Photosensitive Ganglion Cells (ipRGCs) Cellular Ca(2+) Signaling

The G protein-coupled light-sensitive receptor melanopsin is involved in non-image-forming light responses including circadian timing. The predicted secondary structure of melanopsin indicates a long cytoplasmic tail with many potential phosphorylation sites. Using bioinformatics, we identified a nu...

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Autores principales: Fahrenkrug, Jan, Falktoft, Birgitte, Georg, Birgitte, Hannibal, Jens, Kristiansen, Sarah B., Klausen, Thomas K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271233/
https://www.ncbi.nlm.nih.gov/pubmed/25378407
http://dx.doi.org/10.1074/jbc.M114.586529
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author Fahrenkrug, Jan
Falktoft, Birgitte
Georg, Birgitte
Hannibal, Jens
Kristiansen, Sarah B.
Klausen, Thomas K.
author_facet Fahrenkrug, Jan
Falktoft, Birgitte
Georg, Birgitte
Hannibal, Jens
Kristiansen, Sarah B.
Klausen, Thomas K.
author_sort Fahrenkrug, Jan
collection PubMed
description The G protein-coupled light-sensitive receptor melanopsin is involved in non-image-forming light responses including circadian timing. The predicted secondary structure of melanopsin indicates a long cytoplasmic tail with many potential phosphorylation sites. Using bioinformatics, we identified a number of amino acids with a high probability of being phosphorylated. We generated antibodies against melanopsin phosphorylated at Ser-381 and Ser-398, respectively. The antibody specificity was verified by immunoblotting and immunohistochemical staining of HEK-293 cells expressing rat melanopsin mutated in Ser-381 or Ser-398. Using the antibody recognizing phospho-Ser-381 melanopsin, we demonstrated by immunoblotting and immunohistochemical staining in HEK-293 cells expressing rat melanopsin that the receptor is phosphorylated in this position during the dark and dephosphorylated when light is turned on. On the contrary, we found that melanopsin at Ser-398 was unphosphorylated in the dark and became phosphorylated after light stimulation. The light-induced changes in phosphorylation at both Ser-381 and Ser-398 were rapid and lasted throughout the 4-h experimental period. Furthermore, phosphorylation at Ser-381 and Ser-398 was independent of each other. The changes in phosphorylation were confirmed in vivo by immunohistochemical staining of rat retinas during light and dark. We further demonstrated that mutation of Ser-381 and Ser-398 in melanopsin-expressing HEK-293 cells affected the light-induced Ca(2+) response, which was significantly reduced as compared with wild type. Examining the light-evoked Ca(2+) response in a melanopsin Ser-381 plus Ser-398 double mutant provided evidence that the phosphorylation events were independent.
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spelling pubmed-42712332014-12-19 Phosphorylation of Rat Melanopsin at Ser-381 and Ser-398 by Light/Dark and Its Importance for Intrinsically Photosensitive Ganglion Cells (ipRGCs) Cellular Ca(2+) Signaling Fahrenkrug, Jan Falktoft, Birgitte Georg, Birgitte Hannibal, Jens Kristiansen, Sarah B. Klausen, Thomas K. J Biol Chem Signal Transduction The G protein-coupled light-sensitive receptor melanopsin is involved in non-image-forming light responses including circadian timing. The predicted secondary structure of melanopsin indicates a long cytoplasmic tail with many potential phosphorylation sites. Using bioinformatics, we identified a number of amino acids with a high probability of being phosphorylated. We generated antibodies against melanopsin phosphorylated at Ser-381 and Ser-398, respectively. The antibody specificity was verified by immunoblotting and immunohistochemical staining of HEK-293 cells expressing rat melanopsin mutated in Ser-381 or Ser-398. Using the antibody recognizing phospho-Ser-381 melanopsin, we demonstrated by immunoblotting and immunohistochemical staining in HEK-293 cells expressing rat melanopsin that the receptor is phosphorylated in this position during the dark and dephosphorylated when light is turned on. On the contrary, we found that melanopsin at Ser-398 was unphosphorylated in the dark and became phosphorylated after light stimulation. The light-induced changes in phosphorylation at both Ser-381 and Ser-398 were rapid and lasted throughout the 4-h experimental period. Furthermore, phosphorylation at Ser-381 and Ser-398 was independent of each other. The changes in phosphorylation were confirmed in vivo by immunohistochemical staining of rat retinas during light and dark. We further demonstrated that mutation of Ser-381 and Ser-398 in melanopsin-expressing HEK-293 cells affected the light-induced Ca(2+) response, which was significantly reduced as compared with wild type. Examining the light-evoked Ca(2+) response in a melanopsin Ser-381 plus Ser-398 double mutant provided evidence that the phosphorylation events were independent. American Society for Biochemistry and Molecular Biology 2014-12-19 2014-11-06 /pmc/articles/PMC4271233/ /pubmed/25378407 http://dx.doi.org/10.1074/jbc.M114.586529 Text en © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version full access. Creative Commons Attribution Unported License (http://creativecommons.org/licenses/by/3.0/) applies to Author Choice Articles
spellingShingle Signal Transduction
Fahrenkrug, Jan
Falktoft, Birgitte
Georg, Birgitte
Hannibal, Jens
Kristiansen, Sarah B.
Klausen, Thomas K.
Phosphorylation of Rat Melanopsin at Ser-381 and Ser-398 by Light/Dark and Its Importance for Intrinsically Photosensitive Ganglion Cells (ipRGCs) Cellular Ca(2+) Signaling
title Phosphorylation of Rat Melanopsin at Ser-381 and Ser-398 by Light/Dark and Its Importance for Intrinsically Photosensitive Ganglion Cells (ipRGCs) Cellular Ca(2+) Signaling
title_full Phosphorylation of Rat Melanopsin at Ser-381 and Ser-398 by Light/Dark and Its Importance for Intrinsically Photosensitive Ganglion Cells (ipRGCs) Cellular Ca(2+) Signaling
title_fullStr Phosphorylation of Rat Melanopsin at Ser-381 and Ser-398 by Light/Dark and Its Importance for Intrinsically Photosensitive Ganglion Cells (ipRGCs) Cellular Ca(2+) Signaling
title_full_unstemmed Phosphorylation of Rat Melanopsin at Ser-381 and Ser-398 by Light/Dark and Its Importance for Intrinsically Photosensitive Ganglion Cells (ipRGCs) Cellular Ca(2+) Signaling
title_short Phosphorylation of Rat Melanopsin at Ser-381 and Ser-398 by Light/Dark and Its Importance for Intrinsically Photosensitive Ganglion Cells (ipRGCs) Cellular Ca(2+) Signaling
title_sort phosphorylation of rat melanopsin at ser-381 and ser-398 by light/dark and its importance for intrinsically photosensitive ganglion cells (iprgcs) cellular ca(2+) signaling
topic Signal Transduction
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271233/
https://www.ncbi.nlm.nih.gov/pubmed/25378407
http://dx.doi.org/10.1074/jbc.M114.586529
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