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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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Nucleotide and derived protein sequence of human  EBP50 cDNA. Residues matching the two placental γ-EBP50 peptide sequences are underlined. These sequence data are available  from EMBL/GenBank/DDBJ under accession number AF015926.
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Figure 4: Nucleotide and derived protein sequence of human EBP50 cDNA. Residues matching the two placental γ-EBP50 peptide sequences are underlined. These sequence data are available from EMBL/GenBank/DDBJ under accession number AF015926.

Mentions: EBP50 was affinity purified from human placenta or bovine brain using N-ERMAD beads in the large-scale, affinity binding assay. Approximately 8 μg of each protein was resolved by preparative SDS-PAGE, and then blotted to PVDF. The membrane was stained with Ponceau-S (Sigma Chemical Co.) and regions containing the desired EBP50 bands were excised and then washed extensively in double-distilled water. Amino acid analysis and peptide microsequencing was performed at Harvard Microchem (Cambridge, MA). Samples were digested in situ with endoproteinase lys-C, subjected to HPLC fractionation, and the peak fractions were analyzed using matrix-assisted laser desorption time-of-flight mass spectrometry. Homogenous fractions were then chosen for automated peptide sequencing. Peptide sequences (see Figs. 4 and 5) were used to query the National Center for Biotechnology Information (Bethesda, MD) nonredundant database using the BLAST program (Altschul et al., 1990).


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)

Nucleotide and derived protein sequence of human  EBP50 cDNA. Residues matching the two placental γ-EBP50 peptide sequences are underlined. These sequence data are available  from EMBL/GenBank/DDBJ under accession number AF015926.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Nucleotide and derived protein sequence of human EBP50 cDNA. Residues matching the two placental γ-EBP50 peptide sequences are underlined. These sequence data are available from EMBL/GenBank/DDBJ under accession number AF015926.
Mentions: EBP50 was affinity purified from human placenta or bovine brain using N-ERMAD beads in the large-scale, affinity binding assay. Approximately 8 μg of each protein was resolved by preparative SDS-PAGE, and then blotted to PVDF. The membrane was stained with Ponceau-S (Sigma Chemical Co.) and regions containing the desired EBP50 bands were excised and then washed extensively in double-distilled water. Amino acid analysis and peptide microsequencing was performed at Harvard Microchem (Cambridge, MA). Samples were digested in situ with endoproteinase lys-C, subjected to HPLC fractionation, and the peak fractions were analyzed using matrix-assisted laser desorption time-of-flight mass spectrometry. Homogenous fractions were then chosen for automated peptide sequencing. Peptide sequences (see Figs. 4 and 5) were used to query the National Center for Biotechnology Information (Bethesda, MD) nonredundant database using the BLAST program (Altschul et al., 1990).

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