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S. M. Conley, D. Chakraborty, M. I. Naash; Differential Composition of the RDS Tetraspanin Web in Rod- vs. Cone-Dominant Retinas. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2725.
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Rods and cones have a differential requirement for the photoreceptor tetraspanin protein, RDS (retinal degeneration slow). In Rod-dominant mouse retinas (wild-type, WT), rods without RDS fail to form outer segments (OSs) and undergo degeneration while in cone-dominant Nrl-/- retinas, cones without RDS retain functional, albeit dysmorphic OSs. Tetraspanins are typically part of a large complex called the tetraspanin web and exert their function partially based on its composition. The purpose of this study was to determine whether differences exist in the RDS tetraspanin web in rod-dominant vs. cone-dominant retinas.
To study differences in RDS protein partner binding in rod- vs. cone-dominant retinas, we took a multi-pronged approach. First, immunoprecipitation/Western blotting were used to determine whether known RDS binding partners in rods (WT) also bind RDS in Nrl-/- retinas. Second, RDS complexes were immunoprecipitated from metabolically radiolabeled [35S] WT and Nrl-/- retinas and patterns of RDS binding partners were examined. Third, RDS protein complexes were affinity purified from WT and Nrl-/- retinas in order to isolate binding partners using 2D liquid chromatography tandem MS (2D-LC-LC-MS-MS).
First we examined interactions between RDS and the cyclic nucleotide gated channel (CNG). In rods, RDS is known to interact with the GARP subunit of CNG. In cones we did not detect any association between RDS and either subunit of cone CNG. Second, radiolabeled WT and Nrl-/- immunoprecipitants underwent autoradiography. Distinct specific binding patterns (with the exception of the RDS band) were observed. Third, after optimizing affinity purification and one-shot 2D-LC-LC-MS-MS protocols on WT retinas, RDS binding partners are being identified in WT and Nrl-/- retinas (pending).
Our results suggest that the RDS tetraspanin web may have a different protein composition in rod-dominant vs. cone-dominant retinas and that these differences in binding partners may underlie the divergences in the role of RDS in the two photoreceptor types. These results enhance our understanding of the role of RDS, and further our knowledge of the inherent differences in rod vs. cone OS biogenesis.
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