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S.S. Yu, R.N. Fariss, E. Poliakov, A. Boulanger, S. Gentleman, T. Redmond; Interaction of RPE65 with Caveolin-1 . Invest. Ophthalmol. Vis. Sci. 2003;44(13):945.
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Purpose: Known components of the visual cycle in the RPE are membrane bound (LRAT, 11-cis RDH), membrane-associated (RPE65) or soluble (CRALBP) proteins. RPE65, loss of which blocks the visual cycle, has been proposed to be a "scaffolding" protein that might functionally organize the visual cycle proteins. We wish to determine if and how RPE65 might perform such a function. Methods: Bioinformatics methods were used to analyze RPE65 proteins for protein interaction domains. Confocal immunofluorescence microscopy was used to analyze localization of proteins in RPE. RT-PCR and western blotting were used to determine expression of caveolin-1 and other proteins in RPE. A fusion construct for GST-caveolin-1 was generated and expressed protein purified for use in a GST-pulldown protocol. MALDI-TOF mass spectroscopy was used to confirm identity of proteins. Results: Inspection of RPE65 protein sequences reveals the presence of a conserved caveolin-binding domain at residues 414-426 of all known RPE65s. Caveolin-1 was found to be highly expressed at both the mRNA and protein level in bovine and mouse RPE. Immunofluorescence microscopy shows caveolin-1 to co-localize, at least in part, with the distribution of RPE65. GST-pulldown experiments employing expressed GST-caveolin-1 fusion protein and native bovine RPE RPE65 revealed that RPE65 interacts with caveolin-1 in vitro. In addition to RPE65, other visual cycle proteins such as LRAT and 11-cis RDH were detected in the pull-down assemblage, perhaps through interaction with RPE65. Immunoprecipitation of bovine RPE microsomal proteins with anti-RPE65 antibody confirmed these findings by showing the co-sedimentation of RPE65, caveolin-1, and other visual cycle proteins. Conclusions: RPE65 contains a caveolin-interaction domain that is conserved in all species and is functionally active. Since caveolin-1 is a "scaffolding" protein known to be involved in membrane trafficking, lipid transport and in signal transduction, its interaction with RPE65 may involve some or all of these roles. This interaction, along with the previously shown interaction with phospholipids, may allow for the association of RPE65 with membrane anchored visual cycle components and provides a new focus for study of the visual cycle complex. These data demonstrate that RPE65 can interact with a known "scaffolding" protein, although they still leave open the question as to whether RPE65 is a "scaffolding" protein per se.
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