Abstract
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.
Highlights
Melanopsin is a retinal light-sensitive receptor involved in non-image forming functions including circadian timing
Light Dephosphorylates Melanopsin at Ser-381—Because melanopsin is activated by light, we examined whether exposure to light or dark caused changes in phosphorylation at Ser381 or Ser-398 in rat melanopsin
The predicted secondary structure of melanopsin indicates a long cytoplasmatic tail with many potential phosphorylation sites suggesting that this effector domain of melanopsin could be highly regulated, and recently a phosphorylation cassette was identified in mouse and zebrafish melanopsin [15]
Summary
Melanopsin is a retinal light-sensitive receptor involved in non-image forming functions including circadian timing. The G protein-coupled light-sensitive receptor melanopsin is involved in non-image-forming light responses including circadian timing. The melanopsin-expressing cells respond to light stimulation with an absorption maximum around 480 nm, which elicits a sluggish depolarization of the membrane potential [5], an increase in intracellular Ca2ϩ (6 – 8), and a sustained expression of the protein encoded by the immediate early response gene Fos [9, 10]. The first evidence was provided that mouse melanopsin is phosphorylated in the C-terminal tail in a light-dependent manner [14] and subsequently a cluster of Ser and Thr residues in the region between amino acid 386 to 396 was shown to be involved in mediating deactivation upon light stimulation [15]. We demonstrated that elimination of phosphorylation by Ser to Ala mutations at these sites significantly reduced the intracellular Ca2ϩ response upon light stimulation
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