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T.J. Horner, S. Osawa, E.R. Weiss; Comparison of GRK7 and GRK1: In vitro Phosphorylation of Opsin Substrates and Regulation by cAMP-dependent Protein Kinase (PKA) . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1511.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: G protein-coupled receptor kinase 1 (GRK1) is essential for desensitization of the visual signaling pathway in rods. GRK7, a novel cone-specific GRK, is coexpressed with GRK1 in human and monkey cones. However, the contributions of these kinases to desensitization in cones are not well characterized. The goal of this study is to compare the activities of GRK7 and GRK1, using rhodopsin as a model substrate, and to demonstrate phosphorylation of human green opsin in vitro. We also determined that GRK7 and GRK1 are substrates for PKA and have investigated the influence of phosphorylation on the activities of these GRKs. Methods: FLAG-tagged GRK7 and GRK1 were expressed in HEK-293 cells and purified by affinity chromatography. Using urea-stripped bovine rod outer segment membranes as a substrate, a filter assay was established to measure initial rates of rhodopsin phosphorylation by GRK7 and GRK1. Human green opsin expressed in HEK-293 cells was also used as a substrate for phosphorylation by these GRKs. Phosphorylation of GRK7 and GRK1 by PKA was measured in vitro and examined for its influence on rhodopsin phosphorylation. In situ phosphorylation by PKA was also evaluated in forskolin-treated HEK-293 cells by immunoprecipitation with FLAG antibody followed by SDS-PAGE and phosphorimage analysis. Results: The Km for rhodopsin phosphorylation is similar for GRK7 and GRK1. The Vmax for GRK1 is somewhat higher than for GRK7. GRK7 and GRK1 also phosphorylated human green opsin expressed in HEK-293 cells. GRK7 and GRK1 are substrates for PKA in vitro and in forskolin-treated HEK-293 cells. Phosphorylation by PKA significantly inhibits the ability of GRK7 and GRK1 to phosphorylate rhodopsin. Conclusion: Biochemical comparison of GRK7 and GRK1 suggests that their intrinsic activities are similar. Since these kinases are coexpressed in human cones, they may compete for the same substrate. Phosphorylation of GRK7 and GRK1 effectively inhibits their ability to phosphorylate the model substrate, rhodopsin. Whether phosphorylation by PKA regulates the function of these kinases in the retina will be addressed in future studies.
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