Two types of cells, rods and cones, mediate the response to light in the mammalian retina. Rods are used primarily in dim light, whereas the cones mediate vision in bright light. Electrophysiological studies have determined that cones respond to light more rapidly and recover more quickly than rods. The reasons for these differences are not well understood, but may be due in part to altered kinetics of interaction between the proteins that participate directly in the visual signaling cascade or in regulatory mechanisms that further modulate the response to light. Most of our knowledge of phototransduction in mammals is derived from studies of the rod cells, primarily because most mammalian retinas are 90 to 95 percent rods. Although a number of the cone-specific proteins have been cloned, there have been very few in vitro reconstitution studies to determine whether these proteins interact similarly to their rod cell counterparts. Recently, we cloned a novel kinase, GRK7, a G protein-coupled receptor kinase (GRK), from the 13-lined ground squirrel, a cone-dominant mammal whose retina is 94 percent cones, and also established its existence in the retina of the pig, a rod-dominant mammal. In situ hybridization showed that GRK7 is located specifically in the photoreceptor cell layer of the ground squirrel retina. Immunofluorescence, using an antibody directed against GRK7, demonstrates that this kinase is localized specifically in cones. These results suggest that GRK7 may be a cone homologue of GRK1, the rod-specific rhodopsin kinase. This grant begins to address the reasons for the differences in signal termination observed for rods and cones by comparing the ability of GRK7 and GRK1 to phosphorylate the cone opsins and rhodopsin. The specific locations of GRK7 and GRK1 and their colocalization with the cone opsins in pig retinas will be determined using indirect immunofluorescence. Biochemical assays will be established to study cone opsin and rhodopsin phosphorylation in vitro using recombinant preparations of GRK7 and GRK1. The type of isoprenyl modification in the GRK7 expressed in HEK-293 or COS cells and the role of isoprenylation will be investigated. The sites of autophosphorylation and their influence on GRK7 activity will be studied. The influence of the calcium binding proteins, calmodulin and recoverin, on the activity of GRK7 will also be investigated and the substrate sites for this kinase on the cone opsins will be analyzed. The new information provided by these studies will aid in understanding the regulation of visual signaling in cone cells and cone-related disease processes.
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