A DUAL INVOLVEMENT OF THE AMINO-TERMINAL DOMAIN OF EZRIN IN F-ACTIN AND G-ACTIN BINDING

Human recombinant ezrin, or truncated forms, were coated in microtiter plate and their capacity to bind actin determined. F-actin bound ezri n with a K-d of 504 +/- 230 nM and a molecular stoichiometry of 10.6 a ctin per ezrin. Ezrin bound both alpha- and beta/gamma-actin essential ly as F-form. F-actin binding was totally prevented or drastically red uced when residues 534-586 or 13-30 were deleted, respectively. An act in binding activity was detected in amino-terminal constructs (ezrin 1 -310 and 1-333) provided the glutathione S-transferase moiety of the f usion protein was removed, Series of carboxyl-terminal truncations con firmed the presence of this actin-binding site which bound both F- and G-actin, The F- and G-actin-binding sites were differently sensitive to various chemical effecters and distinct specific ezrin antibodies. The internal actin-binding Site Was mapped between residues 281 and 33 3. The association of ezrin amino-terminal fragment to full-length ezr in blocked F-actin binding to ezrin. It is proposed that, in full-leng th ezrin, the F-actin-binding site required the juxtaposition of the d istal-most amino- and carboxyl-terminal residues of the ezrin molecule .