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1From the Department of Ophthalmology, University of Washington School of Medicine, Seattle, Washington; 2Cole Eye Institute and Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio; 3Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York; and the 4Department of Biochemistry, University of Washington, Seattle, Washington.
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Abstract |
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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. ERM 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. (247 words)
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Metabolic processes are often organized into complexes of several proteins for efficiency of enzymatic processing and to route intermediates into dedicated metabolic pathways. Work in our laboratories, focused on identifying interaction partners of CRALBP, has provided evidence for a retinoid-processing complex in RPE.20 Here, we demonstrate with two in vitro assays that CRALBP interacts with ERM (ezrin, radixin, moesin)-binding phosphoprotein50 (EBP50), a well-characterized PDZ domain protein.21 EBP50 was independently discovered as a regulatory factor for sodium-hydrogen exchanger type 3 (NHE3) and is also known as sodium-hydrogen exchanger regulatory factor type 1 (NHERF-1).22 We will refer to the protein as EBP50 in this communication. The PDZ domain protein is responsible for localization of proteins to the apical plasma membrane of polarized epithelia,23 24 but has not been previously associated with the visual process. Furthermore, we confirm that EBP50, CRALBP, and ezrin, an interaction partner of EBP50, are present in apical processes of RPE cells and in apical microvilli of Müller cells, the major glial cell of the retina. Our findings provide a structural basis for localization of a retinoid-processing complex to the apical membranes of RPE and Müller cells and offer mechanistic insight into the cell biology of retinoid processing in RPE.
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Materials and Methods |
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Antibodies
The following antibodies were used: Anti-CRALBP (mAb B2), mouse monoclonal antibody to human recombinant CRALBP (rCRALBP);25 anti-CRALBP (UW55), rabbit polyclonal antibody to human rCRALBP; anti-interphotoreceptor retinoid-binding protein (IRBP) (mAb F7), mouse monoclonal antibody to bovine IRBP (Huang J, and Saari J, unpublished data, 1995); anti-EBP50, rabbit polyclonal antibody to human EBP50 C terminus21 ; anti-ezrin, rabbit polyclonal antibody26 ; anti-RDH5 (UW41), rabbit polyclonal antibody made with a peptide epitope from RDH5 (SKYGVEAFSDSLRREL) (Huang J, Palczewski K, Saari J, unpublished data, 1995).
Preparation of RPE Microsomes
Bovine eyes were purchased from Schenk Packing Company (Stanwood, WA) and dissected to yield RPE cell suspensions from which microsomes were prepared as previously described.27 28
Purification of RPE Microsomes
For identification of EBP50, microsomes were washed with 0.6 M NaCl, 20 mM MOPS (3-(N-morpholino)propanesulfonic acid [pH 7]), 0.1 mM dithiothreitol (DTT), and protease inhibitors (HALT, 1:100 dilution of stock; Pierce Biotechnology, Rockford, IL) by homogenizing the pellet in a glass–glass homogenizer and centrifuging at 50,000g for 10 minutes. The resultant pellet was dissolved in 2 mM dodecylmaltoside (DM), 0.5% polyoxyethylene10 dodecylether X-080 (Genapol; Clariant Functional Chemicals, Mount Holly, NC), 0.1% Tween 80, 20 mM MOPS (pH 7), and centrifuged at 100,000g for 10 minutes. The supernatant was applied to a Dspin (Maxi/H) column (Vivascience AG, Hannover, Germany) and centrifuged at 2000g. The column was then washed at least 3 times with 0.5% polyoxyethylene10 dodecylether X-080 (Genapol), 0.1% Tween 80 in MOPS-DTT buffer. A purified microsomal membrane fraction was eluted from the column with two applications of the initial buffer containing 300 mM NaCl. For some procedures, the purified fraction was desalted on spin mini columns (catalog no. 89849; Pierce Biotechnology) or dialyzed.
Preparation of Bovine CRALBP, Recombinant his-Tagged Human CRALBP, and IRBP
Bovine CRALBP complexed with 11-cis-retinal was purified from bovine retinas as described.28 rCRALBP was prepared and purified as described.29 The apo-protein was either used as such or reconstituted with 11-cis-retinal, by using methods described previously.28 29 IRBP was purified from bovine retinas as described.28 30 All procedures involving 11-cis-retinal/CRALBP were performed in dim red light.
Preparation of Recombinant EBP50 and EBP50 Domains
Plasmid constructs were transformed into Escherichia coli Bl21, and protein expression was amplified in liquid cultures (Luria-Bertani [LB] broth plus ampicillin, 100 µg/mL) followed by induction with isopropylthio-ß-D-galactoside. Cells were harvested by centrifugation at 10,000g for 10 minutes. Bacterial lysates were prepared by suspending the cells in 10 mM phosphate buffer (pH 7.2), containing 100 mM NaCl and protease inhibitors (HALT; Pierce Biotechnology), followed by sonication. Lysates were centrifuged at 60,000g for 10 minutes. Domains of EBP50 were expressed as glutathione S-transferase (GST) fusions and purified with GST columns (GST-Trap; Amersham Biosciences, Piscataway, NJ), according to the manufacturer’s instructions. Proteins were eluted with 10 mM glutathione and 50 mM Tris (pH 8) and equilibrated with an appropriate buffer by dialysis or gel filtration.
Gel Overlay Assays
Washed RPE microsomes were dissolved in SDS sample application buffer, subjected to SDS-PAGE, and transferred to an Immobilon-P membrane (Millipore Corp., Bedford, MA) using a specialized device (Mighty Small SE250; Amersham Biosciences). After the membrane was blocked with milk proteins (3% wt/vol) for 1 hour at room temperature, the blot was incubated for 2 hours at 5°C in the dark with purified bovine 11-cis-retinal/CRALBP (5–10 µg/mL) in 10 mM Tris-HCl (pH 8), 150 mM NaCl, and 0.05% Tween-20. The membrane was washed and incubated with anti-CRALBP (either UW55 or mAb B2), washed, and then incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG or rabbit anti-mouse IgG. Incubation with a chromogenic substrate for alkaline phosphatase (NBT, BCIP; Promega, Madison, WI) completed the assay.
Peptide Competition Experiments
The synthetic peptides AEVENTAF, AEVENTAL, EVENAAG, and EVENAAD were obtained from GenMed Synthesis, Inc. (San Francisco, CA). Solutions of the peptides in 50 mM MOPS (pH 7) were diluted into 10 mM Tris (pH 8) 150 mM NaCl, and 0.05% Tween 20, to a final concentration of 150 µM. Peptides were incubated with the PVDF membrane in the CRALBP overlay assay for 5 minutes before addition of CRALBP (see description of overlay assay).
Two-Dimensional Gel Electrophoresis of RPE Microsomes
Two-dimensional (2D) gel electrophoresis was performed with a commercial system (Protean; Bio-Rad, Hercules, CA), according to the manufacturer’s instructions. A sample of purified RPE microsomes (100–200 µg protein) was dialyzed against 10 mM MOPS (pH 7) and 2 mM DM and mixed with 130 µL of isoelectric focusing (IEF) sample buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.2% ampholytes [BioLyte 4/7; Bio-Rad], and a trace of bromophenol blue) and left for 10 minutes at room temperature. The sample was passively absorbed to a gel strip (pH 4–7; DryStrip; Amersham Biosciences) overnight at room temperature, and IEF was performed for 8 to 12 hours according to the manufacturer’s instructions. After IEF, proteins were resolved by SDS-PAGE on 10% gels (Ready Gels, catalog no. 161-1137; Bio-Rad). 2D gels were electroblotted to polyvinylidene difluoride (PVDF) and stained (Gelcode Blue Stain; Pierce Biotechnology) or analyzed with the CRALBP overlay assay.
Protein Identification by Mass Spectrometry
Identification of EBP50 by liquid chromatography electrospray tandem mass spectrometry LC MS/MS used methods described in detail elsewhere.31 32 Briefly, stained gel spots or bands were excised, the stain washed away, the proteins digested in gel with trypsin, and the peptides extracted for mass spectrometric analysis. LC MS/MS was performed with a commercial system (CapLC; Micromass, Beverly, MA) and a quadrupole time-of-flight mass spectrometer (QTOF2; Micromass). Protein identifications from MS/MS data were performed with the system software (ProteinLynx Global Server, MassLynx, ver. 3.5; Micromass), and the Swiss-Prot and NCBI protein sequence databases (http://expasy.hcuge.ch/ provided in the public domain by the Swiss Institute of Bioinformatics, Geneva, Switzerland; and www.ncbi.nlm.nih.gov/ provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD).
Preparation of CRALBP Agarose
Agarose beads (1 mL; Aminolink; Pierce Biotechnology) were washed with 30 mL of 100 mM sodium phosphate (pH 7.2) 150 mM NaCl (coupling buffer) and gently agitated with 2 mL of 11-cis-retinal/CRALBP (2.6 mg/mL). Sodium cyanoborohydride was added to give a final concentration of 50 mM. After 1 hour at room temperature, the incubation was continued with gentle mixing at 5°C for 15 hours and then centrifuged (500g, 2 minutes). The beads were washed with 4 mL of coupling buffer. Residual reactive groups were blocked by incubation with 2 mL of 1 M Tris-HCl (pH 7.4) and 50 mM Na-cyanoborohydride, for 60 minutes at room temperature with gentle agitation. Finally, the beads were washed three times with 5 mL 1 M NaCl and three times with 5 mL immunoprecipitation buffer (8 mM sodium phosphate, 2 mM potassium phosphate [pH 7], 140 mM NaCl, 10 mM KCl). IRBP-agarose was prepared according to the same protocol.
Immunocytochemistry
The methods we used for tissue fixation, immunocytochemistry, and laser scanning confocal microscopy have been described.12 25
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36 kDa). Proteolysis fragments are also usually present (Fig. 2 , bracket, lanes 1 and 2). After overlaying with CRALBP, we also detected CRALBP at an apparent mass of 54 kDa, indicating that a component in RPE microsomes bound the protein (Fig. 2A , lane 2, arrowhead). Both native and recombinant CRALBP bound the 54-kDa component in their apo- and holo(11-cis-retinal) forms. IRBP, another retinoid-binding protein, did not bind to the 54-kDa component after overlay with IRBP and probing with anti-IRBP (results not shown). This suggests that interaction of the 54-kDa protein with CRALBP is relatively specific.
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2D Gel Electrophoresis of RPE Microsomes
Attempts to identify the 54-kDa interaction partner by sequence analysis of the excised band from a 1D gel were not successful because of the presence of comigrating proteins such as tubulin and cytokeratins. However, we were able to separate the 54-kDa component from other proteins by 2D gel electrophoresis. Purified RPE microsomes were subjected to 2D gel electrophoresis, and the resultant blot was stained with Coomassie blue. A limited number of components were stained, including a prominent set of approximately five isoelectric variants with apparent molecular masses of 54 kDa (Fig. 3A , arrows). A duplicate gel was transferred to PVDF film and analyzed with the CRALBP overlay assay. Components corresponding to the five isoelectric variants plus others of slightly lower mass were evident as CRALBP interaction components (Fig. 3B) . Five Coomassie blue–stained components (arrows) were individually excised from the gel, digested in situ with trypsin, and identified by LC MS/MS sequence analysis. Sequences exactly matching those of a mouse homologue of rabbit NHERF-1, also known as EBP50, were identified in each of the excised spots. Overall, we identified six peptides comprising 60 residues or 18% of the total 358 amino acids. The identification suggests that the multiple 2D gel spots detected with the overlay assay represent charge variants of EBP50. Because EBP50 is a phosphoprotein,21 it is possible the electrophoretic variants result from the various amounts of phosphate linked to the protein.
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Binding of CRALBP
To confirm the identification of EBP50 as a CRALBP-interaction partner, we examined the ability of human recombinant (rEBP50) to bind CRALBP. rEBP50 was subjected to the CRALBP overlay assay and the results (Fig. 2B , lane 3) demonstrate clear interaction with CRALBP. Anti-EBP50 labeled a component in bovine RPE microsomes with a migration identical with that of the component labeled in the CRALBP-overlay assay (Fig. 2B , lanes 1 and 2). The apparent mass of bovine EBP50 (54 kDa) was larger than that of the human recombinant protein (50 kDa), as was noted earlier.33
Binding of EBP50 to CRALBP-Agarose
The overlay assay requires that the interaction partner of CRALBP retain some of its structure in the presence of SDS or that its structure renatures after electrophoresis and transfer to PVDF. Although unlikely, interaction of CRALBP and EBP50 in the overlay assay may result artificially from this denaturation–renaturation process. To examine this possibility more thoroughly, we determined whether RPE microsomal EBP50, dissolved in a nonionic detergent, would interact with CRALBP attached to a solid support. Solubilized RPE microsomes were applied to CRALBP-agarose, which was then extensively washed with 0.3 M NaCl. The remaining bound proteins were eluted with 2 M NaCl, analyzed by SDS-PAGE, transferred to PVDF membranes, and probed with the CRALBP overlay assay or with anti-EBP50. The same procedure was performed with IRBP-agarose as a control for the experiment. The results, shown in Figure 4 , indicate that EBP50 was detectable in the eluate from CRALBP-agarose by both the CRALBP-overlay assay and anti-EBP50 antibody (Fig. 4A , lanes 2 and 4). The labeled components of lower molecular weight in Figure 4A , lane 2, have not been identified. Thus, interaction of CRALBP and EBP50 is not dependent on the procedures used in the overlay assay. The same assays failed to detect EBP50 in the eluate from IRBP-agarose (Fig. 4B , lane 3), indicating that the affinity of EBP50 for CRALBP is not related to the derivatized-agarose used in construction of the affinity absorbent.
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CRALBP, EBP50, and Ezrin in RPE and Müller Cells
We examined frozen sections of retina from dark-adapted mice with antibodies to CRALBP, EBP50, and ezrin using laser scanning confocal microscopy. Low magnification showed CRALBP to be distributed throughout the retina from the inner limiting membrane to the RPE, as noted earlier (Figs. 7A 7G) .9 This distribution is due to its presence in Müller cells, which span nearly the complete thickness of the retina, and in RPE cells. At higher magnification, CRALBP was widely distributed within RPE from the basal infoldings to the tips of the apical processes (Figs. 7D 7J) . RPE nuclei were seen as dark objects surrounded by fluorescence. At low magnification, EBP50 was evident in inner retina in a radial orientation resembling a Müller cell distribution (Fig. 7B) . Identification of these inner retinal cells requires more detailed studies. At higher magnification, diffuse labeling of EBP50 within RPE cells was largely confined to the apical processes and terminated abruptly at their bases (Fig. 7E) . A similar distribution was observed in Müller apical processes (Figs. 7B 7E) . Distribution of EBP50 clearly overlapped that of CRALBP in RPE apical processes, as evident in the merged images (Figs. 7C 7F) . EBP50 was also present along the basal RPE membrane and in a punctate distribution pattern over RPE and Müller apical processes and weakly over Müller cells as they traversed the outer nuclear layer (Fig. 7E , described later). Ezrin appeared to fill RPE apical processes from their tips to their bases and to be largely absent in RPE soma (Figs. 7H 7K) . A weaker signal was detected over Müller cell apical processes, which was more evident at higher microscope sensitivity settings. Ezrin distribution overlapped that of CRALBP in RPE apical processes, as indicated by the merged images (Figs. 7I 7L) . Both ezrin,11 35 36 and EBP5036 had been noted in RPE microvilli, and ezrin was reported in Müller microvilli.35 37
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Given the complexity of the visual cycle and the multiple functions that can arise from all-trans-retinol, one of the cycle intermediates, we sought and obtained evidence that some of the components of the cycle in RPE are organized into a functional complex.20 In this study, we found that CRALBP interacts with a well-known scaffold protein, EBP50, which had not been previously associated with the visual cycle. We identified the EBP50–CRALBP interaction with RPE microsomes by using an overlay assay and confirmed the interaction by showing that recombinant human EBP50 bound CRALBP and that a CRALBP-affinity column bound EBP50, ezrin, and RDH5 in RPE microsomes. Analysis of recombinant truncations of EBP50 and of peptide competition experiments indicated that CRALBP interacts with both PDZ domains of EBP50. Furthermore, we showed that interaction of these components is likely in vivo because they occur within the apical processes of RPE cells11 35 36 and of Müller cells, which project into the same extracellular compartment in the retina, bordered by photoreceptor and RPE cells. The presence of CRALBP, EBP50, and ezrin in Müller apical processes is intriguing in light of the considerable indirect evidence supporting a role for these glial cells in cone visual pigment regeneration.6 8
EBP50 has two related PDZ domains that interact with a number of soluble and membrane-bound proteins in various tissues21 22 23 24 and a C-terminal domain that binds proteins of the ERM family, such as ezrin.21 ERM proteins are multivalent linkers that connect plasma membrane proteins with the cortical actin cytoskeleton. An actin-binding domain near the C terminus establishes liaison with the F-actin, and N-terminal FERM (4.1 ERM) domains mediate interaction with plasma membrane proteins, either directly or indirectly, by binding a scaffold protein such as EBP50 or the related exchanger 3 kinase A regulatory protein (E3KARP). EBP50 thus contributes to the development of epithelial polarity by tethering plasma membrane proteins to the actin bundle within apical microvilli or at the apical side of an epithelial cell. Considerable evidence has accumulated in vitro that suggests that EBP50 plays multiple functional roles in the regulation of the transmembrane receptors and transporters, of recycling endosomes, and of other proteins localized to apical plasma membranes of polarized epithelia.21 22 23 24 The initial characterization of NHERF-1 knockout mice revealed a renal phosphate wasting phenotype probably resulting from inappropriate accumulation of sodium-phosphate cotransporter type IIa (Npt2) at internal sites of the glomerular epithelial cell instead of its normal localization in the apical plasma membrane.24 Thus, EBP50 has the potential to cross-link CRALBP to the actin cytoskeleton in RPE apical processes.
Structural Basis for the Interaction of CRALBP and EBP50
PDZ domains interact with their targets by a ß-sheet augmentation mechanism in which the C-terminal four amino acids of the target orient antiparallel to the ß2 strand of the PDZ domain and thereby extend or augment the ß-sheet.38 39 Based on comparisons of all known targets of EBP50, the consensus C-terminal sequence for PDZ1 is X(S/T)XL, where X is any amino acid.34 The C-terminal sequence of mouse CRALBP (NTAL) conforms to this consensus40 ; however, the C terminus of bovine CRALBP used in these studies is NTAF.40 Specificity for C-terminal leucine has been suggested to arise from insertion of its side chain into a tight hydrophobic pocket of PDZ1.41 However, the investigators drew their inference from a crystal structure of a PDZ1 dimer in which the C-terminal leucine of one rPDZ1 molecule inserted into the PDZ1 domain of another at an angle that precluded ß-sheet augmentation. Thus, it is possible that the hydrophobic cavity accommodates another hydrophobic residue such as phenylalanine, if the C-terminal peptide is oriented to allow formation of a classic ß-sheet. No structural information is available regarding the PDZ2 domain of EBP50.
PDZ Domains of EBP50 and CRALBP
Proteins with multiple PDZ domains frequently recognize several targets, allowing the assembly of multiprotein complexes.38 Of the targets identified for EBP50, most bind to PDZ1 and only YAP65 and PLC-ß3 have been identified as targets of PDZ2.34 However, our studies indicate that both individual PDZ domains of EBP50 bind CRALBP when tested with the overlay assay. Given the relative abundance of CRALBP in RPE cells, it is likely that EBP50 tethers a complex of two CRALBPs to the actin cytoskeleton (Fig. 8) . CRALBP, in turn, could interact with other visual cycle components such as RDH5,20 a fraction of which has been localized to the plasma membrane of RPE cells (Fig. 8) .42 Recent proteomic and kinetic evidence further supports the interaction of CRALBP and RDH5.18 20 However, three caveats are in order. First, the overlay assays are not quantitative, and we do not know whether both PDZ domains can bind CRALBP simultaneously. Second, the presence of other target proteins in RPE with higher affinities for the PDZ domain and/or in large amounts could preclude the binding of CRALBP in vivo. Finally, other proteins that bind to CRALBP20 could mask sites important for interaction with EBP50.
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In summary, the interactions of EBP50, ezrin, actin, and CRALBP provide a structural basis for the formation of a retinoid-processing complex localized to the apical membranes of RPE and Müller cells. These interactions offer mechanistic insight into the cell biology of retinoid processing in RPE cells and suggest that cellular processing and intercellular diffusion of the lipophilic retinoids will be as complex as those described for cholesterol.
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Acknowledgements |
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Footnotes |
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Submitted for publication September 8, 2003; revised Ocotber 9, 2003; accepted October 10, 2003.
Disclosure: M. Nawrot, None; K. West, None; J. Huang, None; D.E. Possin, None; A. Bretscher, None; J.W. Crabb, None; J.C. Saari, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: John C. Saari, Department of Ophthalmology, Box 356485, University of Washington School of Medicine, Seattle, WA 98195-6485; jsaari{at}u.washington.edu.
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