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F. F. Kersten, E. van Wijk, L. Hetterschijt, K. Bauss, U. Wolfrum, J. E. E. Keunen, R. Roepman, H. Kremer; A Microtubule-Associated Protein Links to the Usher Protein Network. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2595.
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Usher syndrome is the most common form of combined hereditary deaf-blindness in man. It is caused by mutations in genes encoding proteins with a variety of functions, but that all closely associate in a protein network. Nevertheless, the molecular disease mechanisms of Usher syndrome still remain largely elusive. To gain insight into the pathogenic pathways underlying Usher type 2A (USH2A), we have set out to determine the interacting repertoire of the USH2A isoform B protein in the retina.
Interactors were identified in a yeast two-hybrid (Y2H) screen of a human retinal cDNA library, using the intracellular domain of USH2A as a bait. Putative interactions were validated by glutathione S-transferase (GST) pull-down assays and by (co-) localization studies in the rat retina, employing immunofluorescence and immunoelectron microscopy.
The Y2H screen revealed several interesting interactors, including a microtubule-associated protein of 134 kDa (indicated as MAP134). The intracellular domain of USH2A specifically interacts with the two coiled coil domains of this protein. The interaction was confirmed by GST pull-down assays and these proteins co-localize at several sub-cellular sites in photoreceptor cells, including the basal body. Immunoelectron microscopy showed presence of both USH2A and MAP134 in the apical inner segment, connecting cilium, basal body and centriole of photoreceptor cells.
Our data indicate that USH2A specifically interacts with MAP134 and that the two proteins co-localize in the photoreceptor cells. We thus introduce a novel member of the Usher protein network that is known to play a role in microtubule stabilization. This permits the hypothesis that MAP134 is involved in the structural maintenance of the connecting cilium and/or in the microtubule-based transport in the photoreceptor cells. Its direct association with USH2A indicates that the gene encoding MAP134 is a functional candidate for Usher syndrome or related retinal ciliopathies.
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