Phosphorylation of rat melanopsin at Ser-381 and Ser-398 by light/dark and its importance for intrinsically photosensitive ganglion cells (ipRGCs) cellular Ca2+ 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 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.
From the Department of Clinical Biochemistry, Faculty of Health Sciences, Bispebjerg Hospital, DK-2400 Copenhagen NV, Denmark and jan.fahrenkrug@regionh.dk.
Jan
J
Fahrenkrug
From the Department of Clinical Biochemistry, Faculty of Health Sciences, Bispebjerg Hospital, DK-2400 Copenhagen NV, Denmark and.
Birgitte
B
Falktoft
From the Department of Clinical Biochemistry, Faculty of Health Sciences, Bispebjerg Hospital, DK-2400 Copenhagen NV, Denmark and.
Birgitte
B
Georg
From the Department of Clinical Biochemistry, Faculty of Health Sciences, Bispebjerg Hospital, DK-2400 Copenhagen NV, Denmark and.
Jens
J
Hannibal
From the Department of Clinical Biochemistry, Faculty of Health Sciences, Bispebjerg Hospital, DK-2400 Copenhagen NV, Denmark and.
Sarah B
SB
Kristiansen
Department of Biology, Faculty of Science, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Thomas K
TK
Klausen
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000528:radiation effects; D003624:Darkness; D005123:Eye / Q000378:metabolism / Q000528:radiation effects; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D008027:Light; D008297:Male; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation / Q000528:radiation effects; D017433:Protein Structure, Secondary; D017208:Rats, Wistar; D012165:Retinal Ganglion Cells / Q000378:metabolism* / Q000528:radiation effects; D017299:Rod Opsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D000099210:Melanopsin
D002118:Calcium; D017299:Rod Opsins; D012694:Serine; D000099210:Melanopsin
Calcium Imaging; G Protein-coupled Receptor (GPCR); Mutant; Phosphorylation; Photoreceptor; Retinal Ganglion Cells