Follistatin is an important regulator of reproductive function at the pituitary and ovarian level through its potent ability to bind and neutralize activin. The 288-residue follistatin molecule (FS-288) is comprised of three unique 75-residue, 10-cysteine follistatin domains preceded by a 63-residue N-terminal extension or N-domain.

As an initial approach to exploring the role of the different regions in follistatin's function, we have used a combination of limited protein cleavage and site-directed mutagenesis to determine the importance of the three methionine residues located, respectively, in the N-domain (Met-50), and in the first (Met-79) and third (Met-268) FS domains. After treatment with cyanogen bromide (130mM, 20°C, 18 hr, pH 2.0), cleavage at these residues was confirmed by sequence analysis. In a competition assay for labeled activin binding to solid-phase follistatin, the CNBr-cleaved follistatin retained activity identical to a control sample treated with solvent alone.

Since our previous studies by peptide mapping have implicated the N-domain in activin binding, we confirmed the results of follistatin chain cleavage by mutagenesis of Met-50 to a polar glutamic acid. The expression product from stably-transfected mammalian (CHO) cells bound activin comparably to wild-type FS-288. Activity was also retained after mutation of adjacent residues Lys-48 and Trp-49 to alanine.