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T. L. Whitcomb, J. Young, E. Kasperek, C. Dlugos, S. C. Khani; GRK1 Expression and Photoreceptor Susceptibility to Light. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4404.
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Efficient deactivation and recovery of light-exposed photoreceptors hinges on phosphorylation of visual pigments by GRK1 followed by trapping of the phosphorylated opsin adduct into complex. While absence of the GRK activity has been associated with poor recovery of photoreceptors and increased propensity towards light-induced apoptosis, there is evidence that excess phosphorylation and stable arrestin binding of some opsin mutants may also lead to photoreceptor degeneration from drosophila to man. The study below was to further define the impact of GRK1 on photoreceptor susceptibility.
Bacterial artificial chromosome mediated transgenesis was used to express excess GRK1 in mouse rod and cone photoreceptors. Expression was analyzed by quantitative RT-PCR, western blotting and high-resolution confocal microscopy. Genotyping was performed using PCR using BAC sequence primer to verify presence of additional GRK1 locus and at RPE65 codon 450 using restriction fragment length polymorphism. Age and sex matched wild type and GRK1+ transgenic lines were exposed to cyclic room light (80 lux) or 10,000 lux of white light and the photoreceptor damage was compared by retinal morphometry, TUNEL and other apoptotic assays.
Two transgenic lines expressed 3-6 fold excess of full length GRK1 in their retina based on quantitative analysis of GRK1 RNA and proteins in mouse eyes. GRK1 was abundantly and specifically expressed both by rods and cones in transgenic lines verified by N- and C-terminal specific GRK1 antibodies. The photoreceptors retained normal morphology for at least 4 months when reared at cyclic room light in both homozygous Met450 and Leu450 RPE65 backgrounds. No spontaneous apoptosis was detected among the photoreceptors of cyclic light reared mice with excess GRK1 based on TUNEL staining. Intense light treatments led to apoptosis in both transgenic and wild type strains in RPE65 background heterozygous and homozygous for Leu450.
BAC mediated transgenesis led to excess GRK1 expression in mouse retina. Overexpression was specific to photoreceptors and at least as prominent in cones as in rods. The photoreceptors retained normal morphology in cyclic room light. The quantitative comparison of the damage between the wild type and GRK1+ strain is currently underway.
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