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F. S. Chen, C.-K. Chen; Characterization of a Phospho-Specific Antibody for Autophosphorylated GRK1. Invest. Ophthalmol. Vis. Sci. 2008;49(13):162.
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Autophosphorylation of GRK1 at Ser488 and Thr489 has been suggested to change its affinity for R*. We sought to develop a phospho-specific antibody for autophosphorylated GRK1 to probe its function under physiological and pathological conditions.
A synthetic phosphopeptide, CQDVGAFSpTpVKGVAF, was conjugated to keyhole lamprey hemocyanin and used to immunize rabbits. Phospho-specific immunoglobulin (A4102) against autophosphorylated GRK1 was pre-absorbed with nonphosphopeptide and purified using a phosphopeptide column. Wildtype and mutant bovine GRK1 carrying missense Oguchi mutations were expressed in insect H5 cells using the baculovirus system. The degree of GRK1 autophosphorylation in mouse retina was examined by immunoblotting and immunohistochemistry.
Mutagenesis studies of recombinant GRK1 showed that an intact kinase domain is required for GRK1 autophosphorylation, whereas isoprenylation at the C-terminus is not. Residual autophosphorylation is seen in one missense Oguchi mutation V380D, but completely absent in the P388H mutant. When probing for GRK1 expression in mouse retina at least two species of GRK1 were found. The autophosphorylated form has slower mobility in 12% SDS-PAGE. Prolonged dark- or light-adaptation does not change the level of GRK1, nor the degree of GRK1 autophosphorylation in vivo.
We have generated a useful reagent to study the in vivo role of GRK1 autophosphorylation and demonstrated that autophosphorylation event can be an intrinsic indicator of GRK1 activity. Interestingly, the degree of GRK1 autophosphorylation is comparable under light- and dark-adapted conditions in the retina, suggesting that light-dependent rhodopsin phosphorylation is not regulated by the activation of GRK1 kinase activity.
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