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E. E. Biswas-Fiss, M. B. Bah, S. B. Biswas; Two Hybrid Analysis of Protein Interaction with the Extracellular Domains of Human ABCR. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5426.
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The retina specific ABC transporter, ABCR, is expressed exclusively in retina photoreceptor cells and is thought to mediate the transport of retinal derivatives across the rod outer segment disc membrane. The mechanism of the transport is not completely understood. Mutations in the ABCA4 gene have been linked to several visual diseases including Stargardt disease (STGD) and autosomal recessive cone-rod dystrophy. The purpose of this study was to identify proteins that interact with the extra-cellular domains (ECDs) of the ABCR protein.
Stratagene’s CytoTrap® yeast two-hybrid system was used in combination with yeast mating to screen a human retina cDNA library with ECD2 (1395-1680) as bait.
Approximately one million diploids were screened with the pSOS-ECD2 bait construct. Twenty-five positive clones were identified after several cycles of screening. One gene that was identified multiple times in the screens was sequenced and BLAST searched; this gene was identified as the human smoothened (SMO) gene. This interaction was further analyzed through immunoprecipitation and GST pull-down assay.
This study provides evidence for the interaction of ECD2 domain of ABCR with SMO protein in human retina. These studies point to a possible functional interaction between ABCR and SMO in the retina.
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