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Joshua D. Sammons, TJ Hollingsworth, Alecia K. Gross; Monitoring Consequences of Altering Rhodopsin’s C-terminus. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3331.
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C-terminal obstructions of rhodopsin lead to photoreceptor degeneration in humans and mice. To help understand the mechanism for this degeneration, we have expressed two rhodopsin fusion proteins, rhopaGFP and rhopaGFP-1D4, in which photo-activatable GFP (paGFP) is fused to the C-terminus of rhodopsin with or without the C-terminal 8 amino acids of rhodopsin (1D4 epitope) amended to paGFP. We also expressed the human disease-causing rhodopsin read-through mutation, Ter349Glu, which has an additional 51 amino acids at the C-terminus of the wild-type (WT) protein and its 1D4 variant, Ter349Glu-1D4. We determined the functional properties and sub-cellular localization of these mutant proteins to test possible mechanisms by which C-terminal occlusions cause photoreceptor degeneration.
Mutant rhodopsins were expressed in COS cells, reconstituted with 11-cis retinal, solubilized and subjected to UV/Vis spectroscopy in the dark and after light exposure. Activation of the G-protein transducin was tested by uptake of GTPγ35S. The proteins were transiently or stably transfected into polarized mouse Inner Medullary Collecting Duct (IMCD) cells. Immunocytochemistry was performed on transiently transfected IMCD cells and those with mutant cDNA stably.integrated. Transgenic Xenopus laevis were created expressing each protein under the Xenopus opsin promoter to examine in vivo localization.
With 11-cis retinal bound, all proteins absorbed light maximally at 500nm similarly to WT rhodopsin. Ter349Glu activation of transducin was similar to that of WT. In IMCD cells, the paGFP fusion proteins localized correctly to the primary cilium in transient and stably transfected cells. Ter349Glu-1D4 localized correctly in transiently transfected cells while Ter349Glu localized apically, basolaterally, and perinuclearly, but appeared to localize correctly to the primary cilium in stably transfected cells. In transgenic X. laevis all four proteins localized to the rod outer segment.
All C-terminal mutants tested bind 11-cis retinal and fold correctly. Additional amino acids do not appear to affect transducin activation or correct localization in low-expressing IMCD cells or rods of transgenic X. laevis. These results suggest that misfolding or simple failure to be recognized by IFT complexes responsible for ciliary targeting cannot explain why photoreceptors die in knock-in mice or human patients with C-terminal obstructions.
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