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D. Deretic, A. Williams, N. Ransom, P.A. Hargrave, A. Arendt; Rhodopsin C–Terminus, the Site of Mutations Causing Autosomal Dominant Retinitis Pigmentosa (adRP), Regulates Trafficking by Binding to ARF4 . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4779.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The severe form adRP mutations occur in the C–terminal of rhodopsin, within the sorting signal VXPX–COOH. To identify proteins that recognize and bind to this sorting motif, we used the established cell free assay which reconstitutes rhodopsin trafficking in vitro, and the synthetic peptide corresponding to the rhodopsin C–terminal, previously shown to inhibit budding of rhodopsin transport carriers (RTCs) from the trans–Golgi network (TGN). Methods: The UV–activatable crosslinker Br–DAP was attached to the N–terminally biotinylated rhodopsin C–terminal peptide. The truncated peptide modeling the Q344Ter adRP mutation, lacking the sorting signal, was synthesized as a control. Peptides were added to the retinal cell–free system and following illumination at 366 nm proteins crosslinked to the peptides were detected by SDS–PAGE and Western blotting with Streptavidin–HRP. Results: The rhodopsin C–terminal peptide specifically crosslinked to a 20 kDa protein identified by MS/MS spectrometry as the small GTPase ARF4. A polyclonal antibody was generated against the peptide obtained by MS/MS, unique to ARF4. Anti–ARF4 specifically recognized a ∼20 kDa cytosolic protein. Although ARF4 is predominantly cytosolic, confocal microscopy showed high immunoreactvity in the vicinity of the Golgi/TGN. Furthermore, ARF4 was crosslinked to newly synthesized rhodopsin during RTC budding in the cell free assay. Both the anti–ARF4 antibody, and the ARF4 peptide used to generate the antibody, inhibited RTC budding to the same extent as the anti–rhodopsin C–terminal antibody and the rhodopsin C–terminal peptide. The effects of blocking ARF4 action were functionally equivalent to the effects of blocking the rhodopsin C–terminal sorting signal. Conclusions: The small GTPase ARF4 specifically recognizes and binds to the VXPX–COOH sorting motif of rhodopsin and regulates budding of RTCs targeted to the rod outer segments. Thus, the severe retinitis pigmentosa alleles that affect the rhodopsin sorting signal interfere with interactions between ARF4 and rhodopsin, leading to aberrant rhodopsin trafficking and initiation of retinal degeneration.
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