Abstract
Abstract: :
Purpose: Analysis of the phenotype of cellular retinaldehyde-binding protein (CRALBP) knockout mice led us to conclude that the visual cycle was delayed at the isomerase step because of the lack of an efficient acceptor for 11-cis-retinol. Earlier studies in vitro demonstrated that 11-cis-retinol bound to CRALBP was a good substrate for a cis-specific dehydrogenase of RPE. Thus, it is likely that apo-CRALBP accepts 11-cis-retinol from an isomerase and facilitates its oxidation to 11-cis-retinal. In order to understand the mechanism of release of 11-cis-retinal from CRALBP and from RPE, we sought proteins that interact with CRALBP in RPE. Methods: Interacting proteins were detected with an overlay assay. RPE microsomes were subjected to 1D or 2D SDS PAGE and blotted to a PVDF membrane, which was blocked, incubated with CRALBP, washed and probed with anti-CRALBP. Proteins were excised from the gel, digested in situ with trypsin and identified by LC MS/MS sequence analysis. Results: CRALBP bound to a protein with an apparent molecular weight of 54 kDa in 1D gels. Other proteins substituted for CRALBP in the assay did not bind, suggesting a specific interaction. The protein was resolved into several components on 2D gels and each was identified by sequence analysis as ERM-binding phosphoprotein 50 (EBP50), also known as sodium/hydrogen exchanger-3 regulatory factor (NHERF-1). Conclusions: ERM (ezrin, radixin, moesin) proteins link plasma membrane proteins with the actin cytoskeleton. EBP50 is a phosphoprotein that binds to ERM proteins through its C-terminal domain. It is known to colocalize with ezrin in apical RPE. It also interacts through its two PDZ-domains with a number of other proteins, including sodium/hydrogen exchanger-3. The functional significance of the affinity of EBP50 for CRALBP is not known, but it may involve recruitment of the binding protein to an RPE apical protein complex involved in releasing or processing 11-cis-retinal.
Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium • cytoskeleton