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J. Isosomppi, A. Aittakorpi, H. Västinsalo, S.F. Geller, E.M. Sankila, J.G. Flannery; A Yeast Two–Hybrid Screen for Clarin–1 (USH3A) Interacting Proteins . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2063.
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To search for proteins that bind to the Usher syndrome type 3 protein Clarin–1.
Yeast two–hybrid screening (Field’s method) was performed using three baits representing the two predicted extra–cellular loops and the intracellular C–terminal region of the mouse Clarin–1 protein. The expression of recombinant GAL4–baits was confirmed by Western blot analysis. Suitability of the baits for screening was additionally confirmed by transactivation activity tests. Each of the baits was used to screen approximately 1.5 million mouse brain cDNAs for interaction. Isolated positive clones were identified by DNA sequencing and database searching. Potential interaction partners of Clarin–1 were subjected to the mating assay.
Screening the mouse cDNA library with three GAL4–Clarin–1 baits identified 125 positive clones that potentially bind to Clarin–1. 110 of the clones were sequenced, of which 41 were in the correct reading frame with GAL4. Most of those 41 clones were considered to be false positives. One of the potential Clarin–1 interacting proteins encodes the ELMO1 protein (Engulfment and Cell Motility protein 1).
Our yeast two–hybrid screen revealed that ELMO1 is a potential interaction partner for Clarin–1, the protein product of the USH3A gene. In mammalian cells, several lines of evidence suggest a role for ELMO proteins in phagocytosis and cell migration. ELMO1 contributes to several Rho GTPase (Rac1) dependent signaling pathways, including actin remodeling and engulfment of apoptotic cells. This association of Clarin–1 with ELMO1 suggests a possible role for Clarin–1 in membrane transport and/or phagocytosis in the retina. Thus, a defective interaction between Clarin–1 and ELMO1 may have a role in the pathogenesis of Usher syndrome type 3 by eliminating a normal binding partner for ELMO1, and thereby altering Rho/Rac1 signaling.
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