Three determinants in ezrin are responsible for cell extension activity.
The ERM proteins--ezrin, radixin, and moesin--are key players in membrane-cytoskeleton interactions. In insect cells infected with recombinant baculoviruses, amino acids 1-115 of ezrin were shown to inhibit an actin- and tubulin-dependent cell-extension activity located in ezrin C-terminal domain (ezrin310-586), whereas full-length ezrin1-586 did not induce any morphological change. To refine the mapping of functional domains of ezrin, 30 additional constructs were overexpressed in Sf9 cells, and the resulting effect of each was qualitatively and semiquantitatively compared. The removal of amino acids 13-30 was sufficient to release a cell-extension phenotype. This effect was abrogated if the 21 distal-most C-terminal amino acids were subsequently deleted (ezrin31-565), confirming the existence of a head-to-tail regulation in the whole molecule. Surprisingly, the deletion in full-length ezrin of the same 21 amino acids provided strong cell-extension competence to ezrin1-565, and this property was recovered in N-terminal constructs as short as ezrin1-310. Within ezrin1-310, amino acid sequences 13-30 and 281-310 were important determinants and acted in cooperation to induce cytoskeleton mobilization. In addition, these same residues are part of a new actin-binding site characterized in vitro in ezrin N-terminal domain.
Martin
M
M
Centre National de la Recherche Scientifique, Université Montpellier II, France.
Roy
C
C
Montcourrier
P
P
Sahuquet
A
A
Mangeat
P
P
D000818:Animals; D000918:Antibody Specificity; D016367:Baculoviridae; D015153:Blotting, Western; D018929:Cell Culture Techniques; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000502:physiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008840:Microfilament Proteins / Q000502:physiology; D008855:Microscopy, Electron, Scanning; D010641:Phenotype; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology*; D018411:Spodoptera / Q000235:genetics / Q000821:virology; D000099149:Ezrin
D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D000099149:Ezrin