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A. K. Gross, T. Hollingsworth, S. D. McAlear; Defective Membrane Trafficking of Autosomal Dominant Retinitis Pigmentosa Mutant Ter349Glu Rhodopsin Is Rescued by C-Terminal Addition of 1D4 Epitope. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2300.
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One of the most severe forms of Autosomal Dominant Retinitis Pigmentosa (ADRP) observed in a family with a rhodopsin mutation is caused by a read-through mutation of rhodopsin, Ter349Glu. We have expressed the recombinant protein with and without addition of rhodopsin’s C-terminal 8 amino acids (1D4 epitope), and studied its localization, biochemical and spectral properties in order to understand the molecular mechanisms underlying the disease pathophysiology.
A synthetic gene encoding rhodopsin Ter349Glu was transfected into COS cells or into IMCD cells, a polarized ciliated mouse cell line derived from the inner medullary collecting duct. The mutant protein was purified from COS cells, reconstituted with 11-cis retinal and solubilized. UV/Visible spectra were taken in the dark and after exposure to light. The ability of the mutant protein to activate transducin was measured by following [35S]GTPγS uptake before and after exposure to light. The localization of the expressed protein in both COS and IMCD cells was monitored using immunofluorescence via the rhodopsin N-terminal antibody B6-30N and an anti-mouse secondary antibody coupled to either Alexa Fluor 488 or Rhodamine.
Ter349Glu rhodopsin binds 11-cis retinal and absorbs light with a maximum at 500 nm, and it activates transducin with a similar efficiency to that of wild type rhodopsin. When expressed in both COS and IMCD cells, immunofluorescence shows wild type rhodopsin on the membrane surface when probed with either the N-terminal antibody B6-30N or the C-terminal antibody 1D4. In contrast, only perinuclear staining is observed in cells expressing Ter349Glu, presumably due to occlusion of the C-terminal sorting signal (VXPX) by the additional 51 amino acids. When the 1D4 epitope was added to the C-terminus, membrane trafficking was restored to Ter349Glu.
These data suggest that it is not misfolding, but specifically the accessibility of the sorting signal that confers proper cellular localization in cultured cells. These results set the stage for future experiments testing the localization in rod cells in vivo.
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