3 DETERMINANTS IN EZRIN ARE RESPONSIBLE FOR CELL EXTENSION ACTIVITY

The ERM proteins-ezrin, radixin, and moesin-are key players in membran e-cytoskeleton interactions. In insect cells infected with recombinant baculoviruses, amino acids 1-115 of ezrin were shown to inhibit an ac tin-and tubulin-dependent cell-extension activity located in ezrin C-t erminal domain (ezrin(310-586)), whereas full-length ezrin(1-586) did not induce any morphological change. To refine the mapping of function al domains of ezrin, 30 additional constructs were overexpressed in Sf 9 cells, and the resulting effect of each was qualitatively and semiqu antitatively compared. The removal of amino acids 13-30 was sufficient to release a cell-extension phenotype. This effect was abrogated if t he 21 distal-most C-terminal amino acids were subsequently deleted (ez rin(31-565)), confirming the existence of a head-to-tail regulation in the T whole molecule. Surprisingly, the deletion in full-length ezrin of the same 21 amino acids provided strong cell-extension competence to ezrin(1-565), and this property was recovered in N-terminal constru cts as short as ezrin(1-310). Within ezrin(1-310), amino acid sequence s 13-30 and 281-310 were important determinants and acted in cooperati on to induce cytoskeleton mobilization. In addition, these same residu es are part of a new actin-binding site characterized in vitro in ezri n N-terminal domain.