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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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Coimmunoprecipitation of EBP50 and ezrin from human placental microvilli. Extracts of isolated microvilli were subjected to immunoprecipitation with antibodies to EBP50 (A) and  ezrin (B). Lanes 1 show a control with antibody but no extract;  lanes 2 show a control with extract but no specific antibody; and  lanes 3 show the complete reaction. The immunoprecipitates  were resolved on a 10% gel, transferred to PVDF, and then  probed with biotinylated ezrin antibody (A) or biotinylated  EBP50 antibody (B), followed by avidin-peroxidase. In B, lane 4,  excess ezrin was added to the lysate immediately before immunoprecipitation. The migration positions of ezrin (A) and EBP50  (B) are indicated by arrows.
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Figure 12: Coimmunoprecipitation of EBP50 and ezrin from human placental microvilli. Extracts of isolated microvilli were subjected to immunoprecipitation with antibodies to EBP50 (A) and ezrin (B). Lanes 1 show a control with antibody but no extract; lanes 2 show a control with extract but no specific antibody; and lanes 3 show the complete reaction. The immunoprecipitates were resolved on a 10% gel, transferred to PVDF, and then probed with biotinylated ezrin antibody (A) or biotinylated EBP50 antibody (B), followed by avidin-peroxidase. In B, lane 4, excess ezrin was added to the lysate immediately before immunoprecipitation. The migration positions of ezrin (A) and EBP50 (B) are indicated by arrows.

Mentions: To assess whether EBP50 and ezrin associate in vivo, lysates of isolated human placental microvilli were subjected to immunoprecipitation with EBP50 and ezrin antibodies and the immunoprecipitates examined for the presence of ezrin and EBP50, respectively (Fig. 12). Immunoblot analysis showed that ezrin was present in the EBP50 immunoprecipitate (Fig. 12 A, lane 3). In the converse experiment, EBP50 was evident in the ezrin immunoprecipitate, although it was difficult to discern precisely which of the multiple species (α, β, or γ) was present (Fig. 12 B, lane 3). Neither ezrin nor EBP50 was detected in the corresponding protein A control precipitates (Fig. 12 A and B, lane 2) or preimmune serum control precipitates (not shown). Additional support for the existence of an ezrin–EBP50 complex came from the ability to compete away the EBP50 in ezrin immunoprecipitates by the addition of excess purified human ezrin to the reaction (Fig. 12 B, lane 4).


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)

Coimmunoprecipitation of EBP50 and ezrin from human placental microvilli. Extracts of isolated microvilli were subjected to immunoprecipitation with antibodies to EBP50 (A) and  ezrin (B). Lanes 1 show a control with antibody but no extract;  lanes 2 show a control with extract but no specific antibody; and  lanes 3 show the complete reaction. The immunoprecipitates  were resolved on a 10% gel, transferred to PVDF, and then  probed with biotinylated ezrin antibody (A) or biotinylated  EBP50 antibody (B), followed by avidin-peroxidase. In B, lane 4,  excess ezrin was added to the lysate immediately before immunoprecipitation. The migration positions of ezrin (A) and EBP50  (B) are indicated by arrows.
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Figure 12: Coimmunoprecipitation of EBP50 and ezrin from human placental microvilli. Extracts of isolated microvilli were subjected to immunoprecipitation with antibodies to EBP50 (A) and ezrin (B). Lanes 1 show a control with antibody but no extract; lanes 2 show a control with extract but no specific antibody; and lanes 3 show the complete reaction. The immunoprecipitates were resolved on a 10% gel, transferred to PVDF, and then probed with biotinylated ezrin antibody (A) or biotinylated EBP50 antibody (B), followed by avidin-peroxidase. In B, lane 4, excess ezrin was added to the lysate immediately before immunoprecipitation. The migration positions of ezrin (A) and EBP50 (B) are indicated by arrows.
Mentions: To assess whether EBP50 and ezrin associate in vivo, lysates of isolated human placental microvilli were subjected to immunoprecipitation with EBP50 and ezrin antibodies and the immunoprecipitates examined for the presence of ezrin and EBP50, respectively (Fig. 12). Immunoblot analysis showed that ezrin was present in the EBP50 immunoprecipitate (Fig. 12 A, lane 3). In the converse experiment, EBP50 was evident in the ezrin immunoprecipitate, although it was difficult to discern precisely which of the multiple species (α, β, or γ) was present (Fig. 12 B, lane 3). Neither ezrin nor EBP50 was detected in the corresponding protein A control precipitates (Fig. 12 A and B, lane 2) or preimmune serum control precipitates (not shown). Additional support for the existence of an ezrin–EBP50 complex came from the ability to compete away the EBP50 in ezrin immunoprecipitates by the addition of excess purified human ezrin to the reaction (Fig. 12 B, lane 4).

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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