Abstract
Abstract: :
Purpose: To study the phosphorylation of a green sensitive opsin. The major pigment of the Gecko gecko rod is more homologous to human green cone opsin (76%) than to the rod opsin (43%). This pigment belongs to the L/MWS subfamily of opsins and therefore presents a readily available model for study of cone opsin phosphorylation. Methods: Gecko retinae were excised from animals after overnight dark adaptation or during daylight lab conditions. Some excised dark–adapted eyecups were exposed to controlled light levels. Single retinae were triturated in 8 M urea, washed with water, reduced with tributylphosphine, alkylated with 4–vinylpyridine, and digested with cyanogen bromide. The samples were then dried under vacuum and solubilized in the initial HPLC solvent buffer [2.5% isopropanol/acetonitrile (2:1) in 0.05% aqueous trifluoroacetic acid]. The digest peptides were separated with 2.5–97.5% linear gradient of this mobile phase. The eluent of the HPLC column was analyzed with an LCQ ion trap mass spectrometer. Both mass spectral and sequence data were automatically collected on the most abundant ions. Results: The gecko green sensitive pigment was mapped and post–translational modifications identified. The pigment does not contain the fatty acid modification serving as a membrane anchor which is present in rhodopsin. There are 11 potential sites of phosphorylation on the C–terminus. Light–exposed retinae show high levels of multiple phosphorylations. Within one minute, 0.3 phosphates per opsin are present, with 80% of the phosphorylated products being in the monophosphorylated form. Multiple phosphoryation rises rapidly with a hexaphosphorylated form being detected after 10 minutes illumination. After 10 minutes, little additional phosphorylation was detected. Conclusions: Unlike rod opsin, this cone opsin exhibits high and rapid levels multiphosphorylation. This difference in phosphoryation may correlate with the lack of a membrane anchor which could allow additional flexibility to the C–terminal tail of the protein.
Keywords: opsins • phosphorylation • photoreceptors