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Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family.

Reczek D, Berryman M, Bretscher A - J. Cell Biol. (1997)

Bottom Line: Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast.These findings show that EBP50 is a physiologically relevant ezrin binding protein.Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

View Article: PubMed Central - PubMed

Affiliation: Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
Members of the ezrin-radixin-moesin (ERM) family of membrane-cytoskeletal linking proteins have NH2- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH2-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin-association domain (N-ERMAD). A collection of polypeptides at 50-53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs. The 50-53-kD placental proteins migrated as a major 50-kD species after phosphatase treatment, indicating that the heterogeneity is due to different phosphorylation states. We refer to these polypeptides as ERM-binding phosphoprotein 50 (EBP50). Sequence analysis of human EBP50 was used to identify an approximately 2-kb human cDNA that encodes a 357-residue polypeptide. Recombinant EBP50 binds tightly to the N-ERMADs of ezrin and moesin. Peptide sequences from the brain candidate indicated that it is closely related to EBP50. EBP50 has two PSD-95/DlgA/ZO-1-like (PDZ) domains and is most likely a homologue of rabbit protein cofactor, which is involved in the protein kinase A regulation of the renal brush border Na+/H+ exchanger. EBP50 is widely distributed in tissues, and is particularly enriched in those containing polarized epithelia. Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast. Moreover, EBP50 and ezrin can be coimmunoprecipitated as a complex from isolated human placental microvilli. These findings show that EBP50 is a physiologically relevant ezrin binding protein. Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

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Localization of EBP50 (A) and ezrin (B) in human  JEG-3 cells. The plane of focus was adjusted to the microvilli-rich  apical surface of the cells. Bar, 10 μm.
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Figure 10: Localization of EBP50 (A) and ezrin (B) in human JEG-3 cells. The plane of focus was adjusted to the microvilli-rich apical surface of the cells. Bar, 10 μm.

Mentions: In cryosections of human placenta, specific EBP50 staining was seen in the apical region of the syncytiotrophoblast (Fig. 9 A). Double labeling with rhodamine phalloidin showed that EBP50 was colocalized with actin in areas with abundant microvilli (Fig. 9, A and B, arrowheads). In addition, the distribution of EBP50, like ezrin, was highly polarized to the microvilli of the intestinal epithelial brush border (Fig. 9, C and D). The specific localization of EBP50 in surface microvilli was most clearly revealed by immunofluorescence microscopy of human JEG-3 cells (Fig. 10 A). The pattern of EBP50 staining in microvilli was very similar to that seen for ezrin (Fig. 10 B).


Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family.

Reczek D, Berryman M, Bretscher A - J. Cell Biol. (1997)

Localization of EBP50 (A) and ezrin (B) in human  JEG-3 cells. The plane of focus was adjusted to the microvilli-rich  apical surface of the cells. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139813&req=5

Figure 10: Localization of EBP50 (A) and ezrin (B) in human JEG-3 cells. The plane of focus was adjusted to the microvilli-rich apical surface of the cells. Bar, 10 μm.
Mentions: In cryosections of human placenta, specific EBP50 staining was seen in the apical region of the syncytiotrophoblast (Fig. 9 A). Double labeling with rhodamine phalloidin showed that EBP50 was colocalized with actin in areas with abundant microvilli (Fig. 9, A and B, arrowheads). In addition, the distribution of EBP50, like ezrin, was highly polarized to the microvilli of the intestinal epithelial brush border (Fig. 9, C and D). The specific localization of EBP50 in surface microvilli was most clearly revealed by immunofluorescence microscopy of human JEG-3 cells (Fig. 10 A). The pattern of EBP50 staining in microvilli was very similar to that seen for ezrin (Fig. 10 B).

Bottom Line: Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast.These findings show that EBP50 is a physiologically relevant ezrin binding protein.Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

View Article: PubMed Central - PubMed

Affiliation: Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
Members of the ezrin-radixin-moesin (ERM) family of membrane-cytoskeletal linking proteins have NH2- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH2-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin-association domain (N-ERMAD). A collection of polypeptides at 50-53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs. The 50-53-kD placental proteins migrated as a major 50-kD species after phosphatase treatment, indicating that the heterogeneity is due to different phosphorylation states. We refer to these polypeptides as ERM-binding phosphoprotein 50 (EBP50). Sequence analysis of human EBP50 was used to identify an approximately 2-kb human cDNA that encodes a 357-residue polypeptide. Recombinant EBP50 binds tightly to the N-ERMADs of ezrin and moesin. Peptide sequences from the brain candidate indicated that it is closely related to EBP50. EBP50 has two PSD-95/DlgA/ZO-1-like (PDZ) domains and is most likely a homologue of rabbit protein cofactor, which is involved in the protein kinase A regulation of the renal brush border Na+/H+ exchanger. EBP50 is widely distributed in tissues, and is particularly enriched in those containing polarized epithelia. Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast. Moreover, EBP50 and ezrin can be coimmunoprecipitated as a complex from isolated human placental microvilli. These findings show that EBP50 is a physiologically relevant ezrin binding protein. Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

Show MeSH