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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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Immunoblot of EBP50 in tissues and cells. 25 μg of total proteins from murine tissues, human placenta and JEG-3 cells,  10 ng of recombinant EBP50, and 2 μg of total proteins of isolated human placental microvilli were resolved on a 11.5% gel,  transferred to PVDF, and probed with affinity-purified antibody  to EBP50.
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Figure 8: Immunoblot of EBP50 in tissues and cells. 25 μg of total proteins from murine tissues, human placenta and JEG-3 cells, 10 ng of recombinant EBP50, and 2 μg of total proteins of isolated human placental microvilli were resolved on a 11.5% gel, transferred to PVDF, and probed with affinity-purified antibody to EBP50.

Mentions: To explore the tissue distribution and cellular localization of EBP50, a polyclonal antibody was raised to its COOH-terminal 117 residues, since this region is divergent from human TKA-1. Affinity-purified antibodies were used to probe a blot of SDS-soluble lysates from an assortment of murine and human tissues (Fig. 8). EBP50 was found to varying extents in almost all the tissues examined, except for heart and skeletal muscle. It was also found in cultured JEG-3 human choriocarcinoma cells. In addition to the information gathered from this immunoblot, EST database searches revealed that cDNA clones for EBP50 were also present in breast, white blood cells, and embryo.


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)

Immunoblot of EBP50 in tissues and cells. 25 μg of total proteins from murine tissues, human placenta and JEG-3 cells,  10 ng of recombinant EBP50, and 2 μg of total proteins of isolated human placental microvilli were resolved on a 11.5% gel,  transferred to PVDF, and probed with affinity-purified antibody  to EBP50.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139813&req=5

Figure 8: Immunoblot of EBP50 in tissues and cells. 25 μg of total proteins from murine tissues, human placenta and JEG-3 cells, 10 ng of recombinant EBP50, and 2 μg of total proteins of isolated human placental microvilli were resolved on a 11.5% gel, transferred to PVDF, and probed with affinity-purified antibody to EBP50.
Mentions: To explore the tissue distribution and cellular localization of EBP50, a polyclonal antibody was raised to its COOH-terminal 117 residues, since this region is divergent from human TKA-1. Affinity-purified antibodies were used to probe a blot of SDS-soluble lysates from an assortment of murine and human tissues (Fig. 8). EBP50 was found to varying extents in almost all the tissues examined, except for heart and skeletal muscle. It was also found in cultured JEG-3 human choriocarcinoma cells. In addition to the information gathered from this immunoblot, EST database searches revealed that cDNA clones for EBP50 were also present in breast, white blood cells, and embryo.

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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