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J.C. Saari, M. Nawrot, G.G. Garwin, J.W. Crabb; Interaction of CRALBP and EBP50/NHERF–1 . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2970.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the interactions of cellular retinaldehyde–binding protein (CRALBP) and ERM–binding phosphoprotein 50 (EBP50), also known as sodium hydrogen exchanger regulatory factor type 1 (NHERF–1) using solution phase and overlay assays. Both proteins are present in RPE and Müller cell microvilli and could interact in vivo.
For the overlay assay, microsomes from mouse eyes were subjected to SDS–PAGE, incubation with CRALBP, and staining with anti–CRALBP. For the solution phase studies, rCRALBP and/or rEBP50/NHERF–1 were applied to a Pharmacia Superdex 200 gel filtration column. UV/Vis spectrophotometry and Western blotting were used to analyze the fractions.
Anti–EBP50 recognized three components in extracts of mouse RPE/choroid that differed in their electrophoretic mobility. Alkaline phosphatase converted the two slower migrating components into the most rapidly migrating component, suggesting that the components differed in their state of phosphorylation. CRALBP bound only to the faster migrating band. The amounts of the three components did not change as a function of the lighting environment. rCRALBP·9–cRal, rCRALBP·11–cRal, rCRALBP·9–cRol, and rEBP50/NHERF–1 migrated as single components when applied individually to the gel filtration column. A higher MW component was generated when the two proteins were applied together, indicating complex formation. Complex formation did not perturb the absorption spectrum of the ligand bound to CRALBP.
The overlay assay results suggest that phosphorylation of EBP50/NHERF–1 affects its interaction with CRALBP. The solution phase studies indicate that interaction of CRALBP with EBP50/NHERF–1 does not appreciably perturb the ligand binding site of CRALBP. Interaction of CRALBP, a visual cycle component, and EBP50/NHERF–1 within RPE microvilli may be a mechanism for assembly of a complex of visual cycle components.
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