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U. Wolfrum, K. Bauß, N. Overlack, E. van Wijk, F. Kersten, R. Roepman, H. Kremer, T. Maerker; Novel SANS Interactors in the Periciliary Usher Syndrome Protein Network of Photoreceptor Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4895.
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For the maintenance and function of vertebrate photoreceptor cells directed transport mechanisms are essential. We have identified a protein network related to the human Usher syndrome (USH) as components for the delivery of cargo vesicles from the inner segment to the outer segment. USH is the most common form of combined deaf-blindness. The purpose of our study was to identify novel interacting proteins to the USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain) which should provide further insights in the organization of USH protein networks, photoreceptor cell function and the pathomechanisms underlying USH.
Yeast-2-hybrid screens of retinal cDNA libraries; protein-protein interaction assays: yeast-two-hybrid, co-transfection of cell lines, GST-pull down assays of recombinant expressed polypeptides; subcellular localization of proteins: Western blots, subcellular fractionation, immunofluorescence and immunoelectron microscopy of mouse and Xenopus retinas; microtubule destabilization assays in organotypic retina cultures.
In yeast-2-hybrid screens with SANS baits, we identified among other proteins the PDZ domain proteins PDZRN4, whirlin (USH2D), and the MAGUK (membrane associated guanylate kinase) protein MAGI-2 as interaction partners. Validation of these interaction showed binding of SANS to PDZ1 and 2 of whirlin and PDZ5 of MAGI-2. Subcellular analyses revealed localization of these proteins in the USH protein network in the periciliary region and the connecting cilium of mammalian and frog photoreceptor cells. Furthermore, our analyses indicated direct association of MAGI-2 with transport vesicles and a dependency of the cellular SANS distribution on the microtubule cytoskeleton.
The USH protein network investigated may play an essential role in the intracellular transport to the outer segment of photoreceptor cells. SANS and their interactors serve as molecular linkers to microtubules and/or vesicular cargo transported through the inner segment. We provide additional evidence that proteins of the network participate in membrane targeting and reloading of cargo on its way to the outer segment. Defects of complex partners should lead to dysfunction of the entire USH network and failure of molecular cargo delivery which may be the cause for photoreceptor degeneration in USH patients.
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