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S.C. Khani, J.E. Young, E.M. Kasparak; Factors Controlling Expression of Rhodopsin Kinase . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3077.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Rhodopsin kinase (Rk) is a key photoreceptor–restricted G–protein dependent receptor kinase (GRK1) whose expression is essential for the efficient recovery and protection of light exposed photoreceptors. The purpose of this study is to identify and characterize the factors involved in the regulation of Rk expression. Methods: Relative Rk and GFP transcript levels were examined in transgenic mouse eyes carrying the crucial highly conserved 0.2–kb human Rk enhancer/promoter region upstream of the GFP reporter using QRT–PCR in Crx–positive and null backgrounds. Sequences in the enhancer/promoter region were further tested for binding to specific nuclear proteins using EMSA in conjunction with antibody–induced depletion or supershift experiments. Yeast one–hybrid assays were used to identify the interacting transcription factors with a crucial homeodomain binding site in enhancer/promoter region. Results: The transcriptional activity of both the transgenic and endogenous Rk promoter was reduced but not abolished in the absence of Crx. EMSA showed binding of a number of nuclear proteins to the sequences within the critical conserved 0.2–kb enhancer/promoter. Supershift was noted with Crx and Otx2 antibody when the putative homeodomain recognition sequence was tested in EMSA assay. Several Crx and Otx clones were obtained with the recognition sequence as bait in yeast one–hybrid assay. Conclusions: Rhodopsin kinase is likely to be regulated by a number of generalized and photoreceptor–specific transcription factors including homeodomain proteins. Identification of additional factors interacting with the region upstream of the transcription start sites is currently in progress.
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