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P. Liu, E.R. Weiss, S. Osawa; Cone Opsin Phosphorylation in Mammalian Retinas . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1509.
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Purpose: Rhodopsin phosphorylation is a critical step in desensitization during phototransduction in mammalian rods. Although there have been reports of cone opsin phosphorylation in lower vertebrates, the occurrence of phosphorylation during phototransduction in mammalian cones has not been addressed. This study investigates whether the cone opsins are phosphorylated in 13-lined ground squirrel (GS) and pig retinas, which are cone- and rod-dominant, respectively. The role of GRK7, a potential cone opsin kinase, is also addressed. Methods: Retinas from dark-adapted GS were incubated with [32P]H3PO4 for 1 h prior to continuous intense illumination or incubation in the dark. Incorporation of radioactivity into cone opsin was analyzed by rapid quenching with 8 M urea, followed by immunoprecipitation of cone opsins with anti-GS green opsin antibody. To determine whether GRK7 phosphorylates cone opsins in GS and pig retinas, homogenates prepared from dark-adapted retinas of these 2 species were incubated with [γ-32P]ATP during exposure to light in the presence of anti-GRK7 antibodies. Phosphorylation of the cone opsins was determined by immunoprecipitation using anti-cone opsin antibodies. Results: Light-dependent phosphorylation was detected in intact GS retinas by immunoprecipitation with anti-GS cone opsin antibody. Cone opsin phosphorylation reached maximum levels within 1 min. In GS retina homogenates, the addition of anti-GS GRK7 blocked cone opsin phosphorylation by approximately 70%, compared to the addition of rabbit IgG as a negative control. In contrast, phosphorylation of pig cone opsin in the presence of anti-pig GRK7 antibody was inhibited by 20%. The inefficient block of cone opsin phosphorylation by GRK7 antibody in pig retina homogenates may be due to phosphorylation by GRK1 released from rods during homogenization. Conclusions: Cone opsin is rapidly phosphorylated in a light-dependent manner in mammalian retinas. GRK7 appears to be the major kinase responsible for cone opsin phosphorylation in GS retinas. The presence of GRK7 and the absence of GRK1 in pig cones suggest that GRK7 is also responsible for cone opsin phosphorylation in this rod-dominant mammal.
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