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E. R. Weiss, R. Jo, S. Osawa; GRK1 Is Phosphorylated in Dark-Adapted Mice in vivo. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1668.
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The retina-specific G protein-coupled receptor kinase, GRK1, is expressed in rods in all vertebrates and in cones in several species, including humans, mice and frogs. Our laboratory has previously shown that GRK1 can be phosphorylated in vitro on Ser-21 by cAMP-dependent protein kinase (PKA). Phosphorylation of GRK1 decreases the efficiency of rhodopsin phosphorylation by approximately 50%. In the present study, we demonstrate the development of an antibody that selectively recognizes mouse GRK1 phosphorylated on Ser-21 and use it to investigate the conditions under which phosphorylation occurs in vivo.
An antibody that recognizes GRK1 phosphorylated on Ser-21 (anti-pGRK1) was generated against a synthetic peptide of the corresponding sequence containing a phosphoserine. The antibody was characterized by immunoblot analysis of phosphorylated and unphosphorylated, recombinant GRK1. To determine whether phosphorylation of GRK1 occurs in vivo, mice were dark- or light-adapted and the retinas dissected for immunoblot analysis and immunocytochemistry using the anti-pGRK1 antibody.
Anti-pGRK1 specifically recognizes recombinant, phosphorylated GRK1 by immunoblot analysis. The binding of the antibody to phosphorylated GRK1 was blocked by incubation with the corresponding synthetic peptide phosphorylated on Ser-21, but not by the equivalent unphosphorylated peptide. Treatment of phosphorylated GRK1 with phosphatase blocked the recognition of GRK1 by anti-pGRK1 on immunoblots, also indicating that the antibody is highly specific for the phosphorylated form of the protein. To determine whether mouse GRK1 is phosphorylated in vivo, mice were dark-adapted overnight, followed either by euthanasia and dissection of the retinas under infrared light, or exposure of the mice to bright light (1,500 lux) for 1 h before euthanasia and dissection. Immunoblot analysis demonstrated that GRK1 is phosphorylated in dark-adapted mice. Phosphorylated GRK1 is barely detectable in mice exposed to light for 1 h. Phosphorylation of GRK1 can be detected in both rods and cones by immunocytochemistry in retinas from dark-adapted mice.
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