Cellular retinaldehyde-binding protein interacts with ERM-binding phosphoprotein 50 in retinal pigment epithelium.
OBJECTIVE
To characterize mechanisms of apical localization of visual cycle components in retinal pigment epithelium (RPE) by the identification of cellular retinaldehyde-binding protein (CRALBP) interaction partners.


METHODS
An overlay assay was used to detect interactions of CRALBP with components of RPE microsomes. Interacting proteins were identified with two-dimensional (2D)-PAGE and liquid chromatography tandem mass spectrometry (LC MS/MS). Protein interactions were characterized by affinity chromatography, peptide competition, and expression of protein domains. Protein colocalization in mouse retina was examined using double-label immunocytochemistry and confocal microscopy.


RESULTS
CRALBP bound to a 54-kDa protein in RPE microsomes, which was identified as ERM (ezrin, radixin, moesin)-binding phosphoprotein 50 (EBP50), a PDZ domain protein, also known as sodium/hydrogen exchanger regulatory factory type 1 (NHERF-1). EBP50 and ezrin in solubilized microsomes bound to CRALBP-agarose but not to a control agarose column. CRALBP bound to both recombinant PDZ domains of EBP50 but not to the C-terminal ezrin-binding domain. In outer retina, EBP50 and ezrin were localized to RPE and Müller apical processes. CRALBP was distributed throughout both RPE and Müller cells, including their apical processes.


CONCLUSIONS
RM proteins are multivalent linkers that connect plasma membrane proteins with the cortical actin cytoskeleton. EBP50 interacts with ERM family members through a C-terminal domain and binds targets such as CRALBP through its PDZ domains, thus contributing to an apical localization of target proteins. Our results provide a structural basis for apical localization of a retinoid-processing complex in RPE cells and offer insight into the cell biology of retinoid processing and trafficking in RPE.
Nawrot
Maria
M
Department of Ophthalmology, University of Washington School of Medicine, Seattle, Washington 98195-6485, USA.
West
Karen
K
Huang
Jing
J
Possin
Daniel E
DE
Bretscher
Anthony
A
Crabb
John W
JW
Saari
John C
JC
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D018613:Microscopy, Confocal; D008861:Microsomes / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012172:Retinaldehyde / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
C020932:11-cis-retinal-binding protein; D002352:Carrier Proteins; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D012172:Retinaldehyde