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Xiaogang Cheng, Nicholas Reish, Alecia Gross; Identification Of Rhodopsin Interacting Components. Invest. Ophthalmol. Vis. Sci. 2011;52(14):518.
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A cause of retinitis pigmentosa is the mislocalization of rhodopsin in rod cells. This mislocalization can be due to abnormal protein interactions between mutant rhodopsin and its trafficking components. To identify proteins that mediate proper transport and disk membrane biogenesis, we have conducted protein pull-down experiments on rhodopsin from bovine as well as wild type and rhodopsin carboxy-terminus mutant knock-in mouse retina to isolate proteins specifically bound to rhodopsin carboxyl-terminus sorting motif. In addition, we are investigating the importance of these proteins in polarized IMCD cells and transgenic Xenopus laevis tadpoles. These studies may provide a better understanding of the molecular mechanism of retinal degeneration.
Rhodopsin protein affinity chromatography was employed to isolate rhodopsin binding proteins. Synthetic peptides specific to regions on the cytoplasmic surface of rhodopsin were used to competitively elute bound proteins from N-terminal tethered bovine or mouse rhodopsin. Eluted protein identities were revealed using in-gel trypsin digestion and LC-tandem mass spectrometry. Immunoblot was used to examine the interactions among newly identified proteins and rhodopsin. The localization of the expressed proteins in IMCD cells was monitored using fluorescence protein fusions and immunohistochemistry. Mutant versions of identified proteins were introduced into X. laevis tadpoles via the I-SceI meganuclease method to monitor their localization as well as rhodopsin localization in retinal cryosections via immunohistochemistry.
Rab11, adenine transporter, and several other proteins with unknown function were discovered as rhodopsin binding proteins from mammalian retinas. Immunoblot analysis confirms that rab11 binds to wild type rhodopsin rather than the C-terminal early truncation mutant Q344Ter rhodopsin. When expressed in IMCD cells, fluorescence images show that rab11 does not interfere with proper localization of rhodopsin in transport, while a dominant negative mutant of rab11 causes mislocalization of rhodopsin.
We have adapted an approach to identify several rhodopsin interacting proteins responsible for rhodopsin transport and localization in mammals. These proteins will give us a broader scope to study the underlying mechanism of rhodopsin trafficking, disk membrane biogenesis, and retinal degenerations.
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