The carboxyl-terminal region of EBP50 binds to a site in the amino-terminal domain of ezrin that is masked in the dormant molecule.
EBP50 (ezrin-radixin-moesin-binding phosphoprotein 50) was recently identified by affinity chromatography on the immobilized NH2-terminal domain of ezrin. Here we map and characterize the regions in EBP50 and ezrin necessary for this association. Using blot overlays and in solution binding assays, the COOH-terminal 30 residues of EBP50 were found to be sufficient for an association with residues 1-286 of ezrin. EBP50 did not bind to full-length (1-585) ezrin, indicating that the EBP50 binding site is masked in the full-length molecule. Ezrin contains two complementary self-association domains known as N- and C-ERMADs (ezrin-radixin-moesin-association domains), encompassing residues 1-296 and 479-585, respectively. An ezrin 1-583 construct lacking the two terminal residues necessary for this association was found to have an unmasked EBP50 binding site. Moreover, binding of EBP50 and the C-ERMAD to ezrin residues 1-296 was found to be mutually exclusive, with the C-ERMAD having a higher affinity. These results suggest that in full-length ezrin, the binding site for EBP50 is masked through an intramolecular N/C-ERMAD association. Based on these and additional results, we propose a model whereby dormant ezrin can be activated to bind EBP50 on its NH2-terminal end and F-actin on its COOH-terminal end. Since EBP50 is proposed to bind membrane proteins through its PDZ domains, this provides a molecular description of the regulated linkage of microfilaments to membranes in cell surface microvilli.
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Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
Bretscher
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003598:Cytoskeletal Proteins; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017923:Sodium-Hydrogen Exchangers; D013329:Structure-Activity Relationship; D000099149:Ezrin
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; D000099149:Ezrin; C093376:sodium-hydrogen exchanger regulatory factor