Purchase this article with an account.
A.K. Gross, F. Chan, J.J. Mancuso, I.M. Sandoval, K. Kyle, J.H. Wilson, T.G. Wensel; Effects of Light on Phosphorylation and Degeneration in Rhodopsin–EGFP Knock–In Mice . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2295.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To test for altered phosphorylation by rhodopsin kinase in rod outer segments from heterozygous knock–in mice expressing a human rhodopsin–EGFP fusion (+/hRhoG), and to determine whether light exposure is involved in the mechanism of degeneration in homozygotes (hRhoG/hRhoG). The heterozygotes maintain healthy photoreceptors throughout life (Chan et al., 2004 PNAS, 101: 9109), but have altered photoresponse kinetics. Methods: Retinas isolated from +/+ and +/hRhoG mice were incubated with 32P in the dark or light, and phosphorylation levels were measured by phosphorimaging. Levels were normalized by total levels of rhodopsin or rho–EGFP quantified using an antibody that recognizes both. Homozygotes were reared under dark or standard animal facility lighting conditions, and frozen retinal sections were inspected by laser–scanning confocal fluorescence microscopy for retinal degeneration at ages ranging from 6 to 16 weeks. Results: In retinal samples from all genotypes, under continuous illumination over one hour, phosphorylation levels rose rapidly, declined, and then rose again. The maximal level of 32P incorporation per mole of rhodopsin was not significantly different for rhodopsin and rho–EGFP. Homozygous (hRhoG/hRhoG) mice reared in the dark had slightly longer outer segments than those exposed to light cycles from birth, but their retinas degenerated with similar rates. The distribution of rho–EGFP in homozygotes differed from that in heterozygotes; a heavier concentration was observed in the distal, as compared to the proximal, part of the outer segments, and the outer segments had altered morphology. Conclusions: When examined on a time scale of minutes, light–induced phosphorylation of rho–EGFP is not reduced relative to phosphorylation of wildtype rhodopsin. Thus the EGFP–fusion at the carboxyl terminus does not block the action of rhodopsin kinase. Light is not required for the degeneration in (hRhoG/hRhoG) retinas, suggesting that photoresponse defects are not fundamental to the mechanism of degeneration. In contrast, aberrant rho–EGFP within the rod outer segment suggests that wildtype rhodopsin is necessary for proper organization of rho–EGFP , and disruption of this organization may play a role in cell death.
This PDF is available to Subscribers Only