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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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Purification and characterization of ezrin and moesin  N-ERMADs. (A and B) show the purifications. Samples were  run on a 15% SDS gel and stained with Coomassie blue. Lane 1,  total extract of uninduced bacteria; lane 2, total extract of induced bacteria; lane 3, purified proteins. (C) The recombinant  proteins exhibit ERMAD activity. Blot overlays using biotinyl  ezrin N-ERMAD (E-N) and biotinyl moesin N-ERMAD (M-N)  probes on a mixture of ezrin and moesin are shown. The mobilities of molecular mass standards and of placental ezrin (81) and  moesin (77) are indicated in kD. DF, dye front.
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Figure 1: Purification and characterization of ezrin and moesin N-ERMADs. (A and B) show the purifications. Samples were run on a 15% SDS gel and stained with Coomassie blue. Lane 1, total extract of uninduced bacteria; lane 2, total extract of induced bacteria; lane 3, purified proteins. (C) The recombinant proteins exhibit ERMAD activity. Blot overlays using biotinyl ezrin N-ERMAD (E-N) and biotinyl moesin N-ERMAD (M-N) probes on a mixture of ezrin and moesin are shown. The mobilities of molecular mass standards and of placental ezrin (81) and moesin (77) are indicated in kD. DF, dye front.

Mentions: The N-ERMADs of human ezrin and moesin (residues 1–296) were expressed as soluble, untagged proteins in bacteria, and purified to homogeneity (Fig. 1, A and B). Because N-ERMADs require a native conformation for their activity in a blot overlay assay (Gary and Bretscher, 1995), we tested the ability of these bacterially expressed products to bind purified ezrin and moesin. Both recombinant ERMADs bound specifically to human placental ezrin and moesin that had been electrophoresed and blotted to a membrane (Fig. 1 C). These results suggested that they were native in conformation and therefore suitable for use in the search for binding proteins.


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)

Purification and characterization of ezrin and moesin  N-ERMADs. (A and B) show the purifications. Samples were  run on a 15% SDS gel and stained with Coomassie blue. Lane 1,  total extract of uninduced bacteria; lane 2, total extract of induced bacteria; lane 3, purified proteins. (C) The recombinant  proteins exhibit ERMAD activity. Blot overlays using biotinyl  ezrin N-ERMAD (E-N) and biotinyl moesin N-ERMAD (M-N)  probes on a mixture of ezrin and moesin are shown. The mobilities of molecular mass standards and of placental ezrin (81) and  moesin (77) are indicated in kD. DF, dye front.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Purification and characterization of ezrin and moesin N-ERMADs. (A and B) show the purifications. Samples were run on a 15% SDS gel and stained with Coomassie blue. Lane 1, total extract of uninduced bacteria; lane 2, total extract of induced bacteria; lane 3, purified proteins. (C) The recombinant proteins exhibit ERMAD activity. Blot overlays using biotinyl ezrin N-ERMAD (E-N) and biotinyl moesin N-ERMAD (M-N) probes on a mixture of ezrin and moesin are shown. The mobilities of molecular mass standards and of placental ezrin (81) and moesin (77) are indicated in kD. DF, dye front.
Mentions: The N-ERMADs of human ezrin and moesin (residues 1–296) were expressed as soluble, untagged proteins in bacteria, and purified to homogeneity (Fig. 1, A and B). Because N-ERMADs require a native conformation for their activity in a blot overlay assay (Gary and Bretscher, 1995), we tested the ability of these bacterially expressed products to bind purified ezrin and moesin. Both recombinant ERMADs bound specifically to human placental ezrin and moesin that had been electrophoresed and blotted to a membrane (Fig. 1 C). These results suggested that they were native in conformation and therefore suitable for use in the search for binding proteins.

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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