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Xiaogang Cheng, Nicholas J. Reish, Alecia K. Gross; Rab11 Is A Component Of Rhodopsin Trafficking In Mouse Retina Through Interaction With Rhodopsin C-terminus. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1585.
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Mislocalization of rhodopsin in patients with retinitis pigmentosa is often due to mutations in the C-terminus of the protein that disrupt the interaction of rhodopsin with its trafficking components. To identify proteins that mediate proper transport, we have conducted protein pull-down experiments on rhodopsin from wild type (WT) and rhodopsin C-terminus mutant knock-in mouse retina. We have identified rab11 as a binding partner of rhodopsin in mouse retina. To confirm this interaction and investigate its possible role in cells, we utilized a GST-rab11 fusion protein in pull-down assays from mouse retinal extracts containing either WT rhodopsin, rhodopsin Q344ter or rhodopsin-EGFP. We also prepared transgenic Xenopus. laevis tadpoles expressing WT and mutant rab11 to monitor protein localization in rod cells.
Rhodopsin protein affinity chromatography was used to isolate potential rhodopsin binding proteins from mouse retinal rod outer segment preparations. Synthetic peptides specific to regions on the cytoplasmic surface of rhodopsin were employed to competitively elute bound proteins from N-terminal tethered mouse rhodopsin, and identities of eluted proteins were revealed using in-gel trypsin digestion and LC-tandem mass spectrometry. Immunoprecipitation was used to confirm the interactions among newly identified proteins and rhodopsin. GST fused rab11 was expressed and purified to examine and confirm the interaction between rab11 and rhodopsin. X. laevis tadpoles carrying transgenic wild type, dominant negative and constitutively active rab11 were generated, and monitored for rhodopsin localization.
Rab11 was identified as a rhodopsin binding protein in mammalian retinas. Reciprocal pull-down experiments confirmed the interaction of rab11 with WT rhodopsin, but not rhodopsin C-terminal rhodopsin mutants from homozygous knock-in Q344Ter or rhodopsin-EGFP mice. GST-rab11 pull down experiments indicated that the active (GTP bound) form of rab11 is required for its binding to rhodopsin. Expression of a dominant-negative mutant rab11 mutation, S25N, in X. laevis rods did not cause significant rhodopsin mislocalization or retinal degeneration but we found S25N rab11 was mislocalized to the inner segment plasma membrane and the outer segment.
We found that Rab11 interacts with rhodopsin in preparations from WT mouse retina. This interaction was abolished in two different mouse lines with rhodopsin carboxy-terminal mutations, indicating that the presence and accessibility of this region of the protein is essential for rab11-dependent trafficking. The binding between rab11 and rhodopsin is mediated by the active form of rab11.
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