Abstract
Purpose::
Cyclic AMP levels in photoreceptor cells are regulated by acute exposure to light and in a circadian manner in the vertebrate retina. The regulation of cAMP has been most extensively studied in rods, where several substrates of cAMP-dependent protein kinase (PKA) have been identified. Recently we have identified 2 new substrates for PKA in vitro: rhodopsin kinase (GRK1), which is expressed in rods and also in cones in some vertebrates, and GRK7, which is expressed in vertebrate cones, but not rods. In the present study we report the phosphorylation of GRK7 by PKA in retinas from several vertebrate species, using an antibody that selectively recognizes phosphorylated Ser36, the major site of phosphorylation by PKA in GRK7.
Methods::
An antibody was generated against a peptide containing a phosphoserine corresponding to Ser36, the major site of phosphorylation by PKA in GRK7. The antibody was characterized by immunoblot and immunocytochemical analysis of GRK7 expressed in HEK-293 cells. Vertebrates retinas were also dissected for immunoblot analysis or processed for immunocytochemistry and analyzed for the presence of phosphorylated GRK7.
Results::
Immunoblot analysis demonstrates that the anti-phosphoGRK7 antibody recognizes purified GRK7 phosphorylated at Ser36 in vitro and does not recognize nonphosphorylated GRK7. To further evaluate this antibody, GRK7 was expressed in HEK-293 cells, where it exhibited very low levels of phosphorylation. However, exposure to forskolin dramatically increased the levels of phosphorylated GRK7 detected by immunoblot analysis and immunocytochemistry. Treatment of forskolin-stimulated HEK-293 cell extracts with phosphatase eliminated the detection of phosphoGRK7 by this antibody. Detection of GRK7 by immunofluorescence in vertebrate retinas using the anti-phosphoGRK7 antibody could be blocked by incubation with the phosphopeptide but not with a nonphosphopeptide corresponding to the same sequence. Therefore, we have successfully generated an antibody that can be used to probe the phosphorylation status of GRK7 in vivo. This antibody detected phosphorylated GRK7 in retinas from pigs, frogs and zebrafish using immunocytochemical methods.
Conclusions::
These results demonstrate that phosphorylation of GRK7 by PKA is conserved among vertebrates as diverse as mammals, amphibians and fish, implying an essential function for this phosphorylation. We are presently characterizing the phosphorylation of GRK1 by PKA in vivo. The function of GRK phosphorylated by PKA, and its regulation in photoreceptor cells, will also be investigated.
Keywords: photoreceptors • phosphorylation • signal transduction