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I. Gosens, A.I. den Hollander, S.J. F. Letteboer, F.P. M. Cremers, R. Roepman; Towards the identification of novel members of the human CRB1 protein network in the retina . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4593.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The human Crumbs homologue 1 (CRB1) protein is a 154 kDa transmembrane protein that is preferentially present in photoreceptors. Its similarity to the fruitfly Drosophila Crumbs suggests a role in cell polarity. Mutations in the CRB1 gene have been found in patients with Leber congenital amaurosis, retinitis pigmentosa (RP) type 12, and RP with Coats exudative vasculopathy. Two homologous proteins were identified, CRB2 and CRB3, which are expressed in the retina as well as other tissues. Nothing is known about the function of CRB2, but CRB3 localizes to tight junctions in human epithelial cells and participates in the establishment of polarity. Our project aims at the identification of the members of the CRB protein complex in the human retina in order to i) obtain functional clues for the retinal pathway(s) in which CRB1 has its function, ii) identify the role of CRB2 and 3 in the retina and iii) obtain novel candidate genes for inherited retina disorders. Methods: We have used GAL4–based interaction trap screens in yeast (yeast two–hybrid system, Y2H) of retina cDNA libraries to search for proteins that interact with the intracellular domain of CRB1 (CRB1intra). Furthermore, human homologues of members of the Drosophila Crumbs protein complex have been isolated by RT–PCR and were fused to both GAL4–AD and GAL4–BD domains of the Y2H system. Putative protein pairs were analyzed for interaction in yeast. Novel interactions are being confirmed using GST pull–downs, co–IPs, and liquid ß–galactosidase assays. Results: Using bovine CRB1intra as a bait in our Y2H screens, we have repeatedly isolated bovine PALS1 as a valid interacting protein. The interacting domain of PALS1 is highly conserved, with a 99% identical protein sequence in human and mouse counterparts. We did not identify the mammalian counterparts of Drosophila moesin and ßH–spectrin, nor any new interactors for CRB1intra. In the protein pair analysis, an interaction was found between the intracellular domain of all three human CRB proteins and the PDZ domain of PALS1. This finding confirms the interaction found in the yeast–2 hybrid screen with bovine CRB1intra, and the known interaction with CRB3. All previously described interactions of the human CRB protein complex have been confirmed using this experimental setup. Furthermore, putative novel protein pairs in the complex were identified, which need to be confirmed. Conclusions: PALS1 has been identified as an interactor of CRB1 in the Y2H screen, and of the human homologues CRB2 and CRB3 by protein pair analysis. Confirmation of novel protein pairs is in progress.
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