Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 organizes ERM proteins at the apical membrane of polarized epithelia

Abstract

Ezrin-radixin-moesin (ERM) proteins regulate the organization and function of specific cortical structures in polarized epithelial cells by connecting filamentous (F)-actin to plasma membrane proteins. The contribution of ERM proteins to these structures depends on a conformational change to an active state in which the C-terminal region interacts with F-actin and the N-terminal domain interacts with membrane ligands. The specific ligands necessary for stabilizing ERM proteins at the membrane are not known. By generating mice deficient for ERM-binding phosphoprotein 50/Na(+)/H(+) exchanger regulatory factor 1 (EBP50/NHERF1), which binds the N-terminal domain of ERM proteins, we found that EBP50 is required for the maintenance of active ERM proteins at the cortical brush border membranes (BBM) of polarized epithelia. In EBP50(-/-) mice, ERM proteins were significantly decreased specifically in BBM from kidney and small intestine epithelial cells, whereas they remained unchanged in the cytoplasm. In wild-type animals, EBP50 was localized to the BBM compartment where it was processed by cleavage of the ERM-binding motif. In BBM, active ERM proteins formed distinct complexes with full-length EBP50 and with F-actin, suggesting a switch mechanism in which proteolytically processed EBP50 would release ERM proteins to complex with F-actin. The structural defects found in the EBP50(-/-) intestinal microvilli were reminiscent of those described in ezrin(-/-) mice, suggesting a role for EBP50 in organizing apical epithelial membranes.

Fig. 1.

Generation of EBP50-deficient mice. (A) Targeted disruption of the EBP50 locus. A fragment of the wild-type locus (Top) containing the first and last two exons (solid boxes) and the polyadenylation signal (pA) of EBP50 is shown. The targeting construct (Middle) consisting of a 4.0-kb 5′ genomic fragment, a 1.8-kb 3′ genomic fragment, and the neo^R gene without the polyadenylation signal (NeoΔpA) replaces a 16-kb genomic fragment containing exons 1–4 of the EBP50 gene in the targeted mutated locus (Bottom). The solid arrowheads flanking the neo^R gene represent LoxP sites. The positions of the Southern blot probe (hatched boxes), EcoRI sites (E), and the EcoRI wild-type and mutated genomic fragments (double-pointed arrows) are shown. The Southern analysis is shown beneath. (B) Schematic representation of the EBP50 domain structure. The two PDZ domains (1 and 2) and the C-terminal ERM-binding (EB) region are shown with the respective boundaries in amino acids. Solid arrowheads indicate EBP50 mRNA splice sites that delimit EBP50's six exons. Some known interacting proteins for the various domains are listed in italics above each domain. The dimerization with itself or NHERF2 and the interaction with NHE3 most likely extend through both PDZ domains. (C) Western blot analysis of EBP50 from whole organs homogenized in TNN buffer. The organs were collected from two mice per each genotype.

Fig. 2.

Decreased expression levels of ERM proteins in EBP50(–/–) BBM. (A) Schematic protocol of BBM preparation. The protein concentration was measured in each boxed fraction, and samples containing 30 μg of proteins [except from S4 (supernatant 4), which contained only 1.5 μg of proteins] were loaded on polyacrylamide gels. TL, total tissue lysates. P4 (pellet 4) corresponds to the BBM fraction. (B) Western blot analysis of kidney and small intestine BBM fractionation samples from EBP50 (+/+) and (–/–) mice was performed with the indicated antibodies. The quantification analysis (Right) was performed with the imagej program (National Institutes of Health) and shows the expression levels of ezrin and NHE3 normalized to vinculin in kidney fractions. For ezrin, only the upper bands, which represent ezrin, were quantified. Similar results were obtained at least seven times for BBM prepared from either male or female mice. (C–F) The expression of phosphorylated ezrin was analyzed with phospho-ERM antibody by immunofluorescence of kidney (C and D) and jejunum (E and F) sections from EBP50(+/+) (C and E) and (–/–) (D and F) 5-week-old littermates. The samples were imaged with a Zeiss LSM 510 confocal microscope by using the HeNe 543-nm laser and identical imaging parameters. Note the higher apical expression of phosphorylated ezrin (arrows) in wild-type kidney tubules and intestinal villi.

Fig. 3.

The EB region of EBP50 is cleaved in BBM. (A) The Western blot analysis of EBP50 in BBM fractions from EBP50(+/+) and (–/–) mice was carried out on the same samples as in Fig. 2B. Note the presence of EBP50 full-length and low-MW products in membrane fractions from EBP50(+/+) mice. (B)(Upper) EBP50 splice isoforms (I1 to I4) are schematically drawn, and the corresponding size in amino acids is indicated on the left. The black bar indicates the PCR used to identify the isoforms, and the arrowhead marks the position of the EcoRI site used for restriction-fragment-length polymorphism. The PCR on reverse transcribed mRNA extracted from various human tissues and cell lines is shown (Lower Left). The PCR fragments corresponding to the isoforms indicated on the left were subcloned from the U937 PCR product (Middle Right) and analyzed for RFLP with EcoRI (Bottom Right). FL, control full-length EBP50. (C) Pull-down assay with GST ezrin-NT and Npt2-CT fusion proteins (5 μg) of proteins (300 μg) from pooled P3 and P4 EBP50(+/+) or (–/–) kidney BBM fractions. Thirty micrograms of proteins from P3 and P4 fractions are included (TL). Note the specific precipitation of EBP50 FL (arrowhead) by ezrin-NT and of low-MW products (bracket) by Npt2-CT. (D) Western analysis showing EBP50 FL (arrow) and low-MW products (bracket) in human tissue lysates (huTL) in comparison with mouse P4 kidney fraction.

Fig. 4.

EBP50 interacts with phosphorylated ERM proteins. (A) Coimmunoprecipitation of phosphorylated ERM proteins (P-ERM) with EBP50 from EBP50(+/+) and (–/–) kidney BBM P4 fractions. Full-length EBP50, FL and arrowhead; cleavage products, bracket. (B) Gel-filtration analysis of protein complexes from EBP50(+/+) kidney BBM P4 fractions with antibodies indicated on the left. Arrowhead indicates FL EBP50 and TL, total lysate. Note coelution of phosphorylated ERM proteins with FL EBP50 in a low-MW peak and with actin in a high-MW peak.

Fig. 5.

Morphology of intestinal villi in EBP(–/–) mice. (A and B) periodic acid/Schiff reagent (PAS) staining of intestinal villi from EBP50(+/+)(A) and (–/–)(B) littermates showing a higher number of PAS⁺ goblet cells in (–/–) villi. (×100.) (C) Transmission electron microscopy of ileum epithelial cells from 5-week-old littermates shows EBP50(+/+) ordered, rod-like microvilli emerging from a structured terminal web region (bracket) contrasting with the EBP50(–/–) disorganized microvilli and thick terminal web. (×12,000.)