Document Detail

Phosphorylation of rat melanopsin at Ser-381 and Ser-398 by light/darkness and its importance for ipRGCs cellular Ca2+ signalling.
MedLine Citation:
PMID:  25378407     Owner:  NLM     Status:  Publisher    
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 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 recognising 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 darkness and dephosphorylated when light is turned on. On the contrary, we found that melanopsin at Ser-398 was unphosphorylated in darkness 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 hour experimental period. Furthermore, phosphorylation at Ser-381 and Ser-398 were independent of each other. The changes in phosphorylation were confirmed in vivo by immunohistochemical staining of rat retinas during light and darkness. We further demonstrated that mutation of Ser-381 and Ser-398 in melanopsin expressing HEK-293 cells affected the light- induced Ca2+ response being significantly reduced as compared to wild type. Examining the light-evoked Ca2+ response in a melanopsin Ser-381 plus Ser-398 double mutant provided evidence that the phosphorylation events were independent.
Jan Fahrenkrug; Birgitte Falktoft; Birgitte Georg; Jens Hannibal; Sarah B Kristiansen; Thomas K Klausen
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2014-11-6
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  -     ISSN:  1083-351X     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2014 Nov 
Date Detail:
Created Date:  2014-11-7     Completed Date:  -     Revised Date:  2014-11-8    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
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