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Lauren Daniele, Edward Pugh; Probing the sensitivity of rhodopsin expression to chromophore levels in the retina. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1589.
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© ARVO (1962-2015); The Authors (2016-present)
To test the hypothesis that the decrease in rhodopsin expression in young RPE65-/- mice is due to enhanced ER associated degradation (ERAD). Mice with deletions of proteins required for 11-cis retinal synthesis undergo retinal degeneration. While cones are well established to have disrupted opsin expression and trafficking, and degenerate rapidly, rods degenerate more slowly. Nevertheless, rhodopsin levels are reduced early, suggesting 11-cis retinal can also affect rhodopsin expression (Rohrer et al., 2003, IOVS; Feathers et al., 2008, IOVS; Sato et al., 2010, Exp Eye Res)
Western blotting was used to estimate the relative amounts of rhodopsin (Rho) in total retina lysates of WT and Rpe65-/- mice aged 2-3 months. To test for increased association of rhodopsin with proteins involved in ERAD, retina lysates were subject to immunoprecipitation (IP). IP’s were performed with antibodies to VCP/p97 and EDEM1, key proteins known to be involved in ERAD of mutant opsins. IP’s were analyzed by PAGE followed by Western blotting
Western blots reveal Rho expression to be reduced to 71% of WT in 2-3 month old Rpe65-/- mice (n=6 littermate pairs). These results confirm previously published observations (Rohrer et al., 2003, IOVS; Feathers et al., 2008, IOVS; Sato et al., 2010, Exp Eye Res). When a Rho antibody was used for IPs of retina lysate, the amount of VCP detected relative to rhodopsin in the immunoprecipitated fractions was constant for both genotypes. However, when a VCP antibody was used for IP’s, over 10-fold more Rho was detected relative to VCP in WT compared with Rpe65-/- mice. IPs with EDEM1 antisera were unsuccessful
Based on co-immunoprecipitation, a small fraction of total Rho is associated with VCP in WT and in Rpe65-/- retinas, suggestive of active ERAD quality control operative in both genotypes. It is unclear why the Rho/VCP ratio in WT is so much higher relative to Rpe65-/- in VCP pulldowns. The results suggest that the reduction in rhodopsin expression in RPE65-/- is not due to increased ERAD. However it is possible that rhodopsin undergoing ERAD in the RPE65-/- is degraded more rapidly and has a more fleeting interaction with VCP. Alternatively, as supported by evidence that Rho mRNA is reduced to ~ 70% of WT at 3-6 weeks of age in Lrat-/- mice (Sato et al., Exp Eye Res, 2010), transcriptional loss may underlie reduced Rho in the Rpe65-/- mice
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