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A. Adekeye, M. Haeri, J. Chen, B. E. Knox; Mutant Rhodopsin (P23H) Forms Aggregates in the Rod Outer Segment. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4042.
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Mutations in rhodopsin cause inherited retinal degenerative diseases. We characterized the oligomeric state of the P23H rhodopsin mutation in transgenic rods and mammalian cells.
We generated transgenic Xenopus rods that express opsin (Rho-EGFP) or P23H mutant (RhoP23H-EGFP) cDNAs with EGFP fused to its carboxyl terminus. Retinal explants were examined for EGFP expression using quantitative confocal microscopy of live rods. Parallel experiments were performed in transiently transfected mammalian cells. SDS-PAGE was used to study potential oligomeric forms of the fusion proteins.
Rho-EGFP was observed primarily in the rod outer segment. Rods expressing RhoP23H-EGFP exhibited an abnormal fluorescent protein distribution. Fluorescence was observed within the inner segment and in small focal regions in both inner and outer segments. The outer segment protein foci were immobile. When retinal extracts from Rho-EGFP expressing tadpoles were examined by western blot analysis, Rho-EGFP was detected as monomers, homodimers and heterodimers with endogenous rhodopsin. By contrast, retinal extracts from tadpoles expressing RhoP23H-EGFP primarily contained homodimers. Minor amounts of monomers were observed, but unlike Rho-EGFP, dimers with endogenous rhodopsin were undetectable. The aggregation behavior is currently under biochemical analysis.
RhoP23H-EGFP, but not Rho-EGFP, forms aggregates in the rod outer segment. Although both mutant and wild type proteins were able to form detergent resistant dimers, RhoP23H-EGFP selectively formed homodimers but not heterodimers with endogenous rhodopsin. Our results suggest that the P23H mutation may render rhodopsin susceptible to form self-aggregates, which may be cytotoxic. Future studies will determine if mutant rhodopsin forms aggregates in other species.
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