title: Interactome analysis reveals ezrin can adopt multiple conformational states.
abstract: Ezrin, a member of the ezrin-radixin-moesin family (ERM), is an essential regulator of the structure of microvilli on the apical aspect of epithelial cells. Ezrin provides a linkage between membrane-associated proteins and F-actin, oscillating between active/open and inactive/closed states, and is regulated in part by phosphorylation of a C-terminal threonine. In the open state, ezrin can bind a number of ligands, but in the closed state the ligand-binding sites are inaccessible. In vitro analysis has proposed that there may be a third hyperactivated form of ezrin. To gain a better understanding of ezrin, we conducted an unbiased proteomic analysis of ezrin-binding proteins in an epithelial cell line, Jeg-3. We refined our list of interactors by comparing the interactomes using quantitative mass spectrometry between wild-type ezrin, closed ezrin, open ezrin, and hyperactivated ezrin. The analysis reveals several novel interactors confirmed by their localization to microvilli, as well as a significant class of proteins that bind closed ezrin. Taken together, the data indicate that ezrin can exist in three different conformational states, and different ligands "perceive" ezrin conformational states differently.
authors.affiliation: From the Department of Molecular Biology and Genetics and Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York 14853.
authors.forename: Raghuvir
authors.initials: R
authors.lastname: Viswanatha
authors.forename: Jessica
authors.initials: J
authors.lastname: Wayt
authors.forename: Patrice Y
authors.initials: PY
authors.lastname: Ohouo
authors.forename: Marcus B
authors.initials: MB
authors.lastname: Smolka
authors.forename: Anthony
authors.initials: A
authors.lastname: Bretscher
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D050660:Core Binding Factors; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D008871:Microvilli / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D020543:Proteome; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D000099149:Ezrin
chemical_list: D050660:Core Binding Factors; D003598:Cytoskeletal Proteins; D008024:Ligands; D010750:Phosphoproteins; D020543:Proteome; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D000099149:Ezrin; C093376:sodium-hydrogen exchanger regulatory factor
keywords: Cytoskeleton; Ezrin; Phosphorylation; Plasma Membrane; Proteomics