Cone cells are responsible for daylight vision, making up only 5-6% of the photoreceptor cell population in humans. The remaining cells, the rod cells, mediate dim light vision. Because the majority of mammals have very few cones, most of our knowledge regarding phototransduction comes from the study of rods. Significant differences between rods and cones have been observed in the sensitivity and kinetics of the response to light. Cones are less sensitive to light than rods and recovery from light exposure occurs more rapidly. Differences at multiple steps, including G protein activation, receptor deactivation and downstream signaling events may be partly responsible for the observed differences between rod and cone phototransduction. Our laboratory has cloned a novel, retina-specific G protein-coupled receptor kinase (GRK), GRK7. GRK7 is expressed exclusively in cones in mammals we have examined, except in mouse and rat, which, instead express GRK1 (the rod cell GRK) in cones. Our laboratory has demonstrated that GRK7 phosphorylates cone opsins in intact retinas from the 13-lined ground squirrel, a cone-dominant mammal, suggesting that GRK7 is involved in deactivation of cone opsin by phosphorylation. Since GRK7 and GRK1 are co-expressed in human cones, they may both play a role in cone opsin deactivation but may be regulated differently. The biochemical properties of these enzymes will be compared in vitro and the role of posttranslational modifications in their activity investigated. Phosphorylation of cone opsins in situ will be examined in two animal models, the Nrl-/-GRK1 -/- mouse, which has an """"""""all cone"""""""" retina, and the cone-rich retina of Xenopus laevis, the South African clawed frog. GRK7 will be introduced into the Nrl-/-GRK1 -/- mice to determine whether this kinase can substitute for GRK1 in biochemical assays for phosphorylation and electrophysiological studies. PKA and autophosphorylation site mutants of GRK7 and GRK1 will also be introduced into these mice and similarly characterized. We have found that Xenopus retinas express GRK7 and GRK1. Therefore, we will characterize the cellular expression pattern for these 2 kinases and design an antisense strategy using morpholinos to target GRK7 in Xenopus. Using this """"""""knockdown"""""""" approach, we plan to establish a model that can be used to determine whether GRK7 is essential for normal phototransduction in vertebrate cones. ERG measurements on cones will be performed to analyze the functional consequences of GRK """"""""knockdown."""""""" ? ?
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