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J.W. Crabb, S.K. Bhattacharya, J.S. Crabb, K.A. West, J. Sun, J.C. Saari, V.L. Bonilha; Support For a Retinoid–Processing Complex In Apical RPE . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4589.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The interaction of cellular retinaldehyde binding protein (CRALBP) with ERM (ezrin, radixin, moesin) binding phosphoprotein 50 (EBP50) in RPE microsomes has lead to the hypothesis of a retinoid–processing complex in apical retinal pigment epithelium (RPE) (Nawrot et al., 2004 IOVS). To test this hypothesis, we have pursued proteomic analysis of RPE apical processes and anti–ezrin immunoprecipitation products. Methods: RPE apical processes were isolated from mouse eyecups on lectin–coated agarose beads essentially according to McLaughlin and co–workers (1987). Proteins bound to the beads were solubilized in SDS, separated by SDS–PAGE, excised from the gel, digested in situ with trypsin and identified by capillary LC MS/MS. Protein interactions were also sought in human and bovine RPE microsomes by immunoprecipitation and proteomic analysis. Results: Morphological analysis of the bead–bound RPE apical processes support a highly purified preparation. CRALBP, EBP50, 11–cis–retinol dehydrogenase (RDH5) and ezrin were identified among the proteins bound to the lectin beads. Anti–ezrin immunoprecipitation products contained CRALBP and RDH5 and reciprocal immunoprecipitations contained ezrin. Conclusions: The results further support a retinoid–processing complex in the apical RPE. CRALBP binds to N–terminal PDZ domains in EBP50 which contains a C–terminal ezrin–binding domain. ERM proteins like ezrin connect the actin cytoskeleton with plasma membrane proteins. CRALBP also interacts directly with RDH5, which has been found in the RPE plasma membrane. Other proteins that function in this complex remain to be determined. Supported in part by NIH grants EY06603, EY14239, EY02317, EY01730, The Foundation Fighting Blindness and The Cleveland Clinic Foundation.
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