MOESIN, EZRIN, AND P205 ARE ACTIN-BINDING PROTEINS ASSOCIATED WITH NEUTROPHIL PLASMA-MEMBRANES

Actin-binding proteins in bovine neutrophil plasma membranes were iden tified using blot overlays with I-125-labeled F-actin. Along with surf ace-biotinylated proteins, membranes were enriched in major actin-bind ing polypeptides of 78, 81, and 205 kDa. Binding was specific for F-ac tin because G-actin did not bind. Further, unlabeled F-actin blocked t he binding of I-125-labeled F-actin whereas other acidic biopolymers w ere relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the a ctin filaments. The 78- and 81-kDa polypeptides were identified as moe sin and ezrin, respectively, by co-migration on sodium dodecyl sulfate -polyacrylamide gel electrophoresis and immunoprecipitation with antib odies specific for moesin and ezrin. Although not present in detectabl e amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound I-125-labeled F-actin on blot o verlays. Experiments with full-length and truncated bacterial fusion p roteins localized the actin-binding site in moesin to the extreme carb oxy terminus, a highly conserved sequence. Immunofluorescence microgra phs of permeabilized cells and cell ''footprints'' showed moesin co-lo calization with actin at the cytoplasmic surface of the plasma membran e, consistent with a role as a membrane-actin-linking protein.