Follistatin: Essential role for the N-terminal domain in activin binding and neutralization

Follistatin is recognized to be an important regulator of cellular differen tiation and secretion through its potent ability to bind and bioneutralize activin with which it is colocalized in many tissue systems. The 288-residu e follistatin molecule is comprised of a 63-residue N-terminal segment foll owed by three repeating 10-cysteine "follistatin domains" also represented in several extracellular matrix proteins. We have used chemical modificatio ns and mutational analyses to define structural requirements for follistati n bioactivity that previously have not been investigated systematically. Mu tant follistatins were stably expressed fi om Chinese hamster ovary cell cu ltures and assayed for activin binding in a solid-phase competition assay. Biological activities were determined by inhibition of activin-mediated tra nscriptional activity and by suppression of follicle-stimulating hormone se cretion by cultured anterior pituitary cells. Deletion of the entire N-term inal domain, disruption of N-terminal disulfides, and deletion of the first two residues each reduced activin binding to <5 % of expressed wild-type f ollistatin and abolished the ability of the respective mutants to suppress activin-mediated responses in both bioassay systems. Hence, the three folli statin domains inherently lack the ability to bind or neutralize activin. A ctivin binding was impaired after oxidation of at least one tryptophan, at position 4, in FS-288, Mutation of Trp to Ala or Asp at either positions 4 or 36 eliminated activin binding and bioactivity, Mutation of a third hydro phobic residue, Phe-52, reduced binding to 20% whereas substitutions for th e individual Lys and Arg residues in the N-terminal region were tolerated. These results establish that hydrophobic residues within the N-terminal dom ain constitute essential activin-binding determinants in the follistatin mo lecule. The correlation among the effects of mutation on activin binding, a ctivin transcriptional responses, and follicle-stimulating hormone secretio n substantiates the concept that, at least in the pituitary, the biological activity of follistatin is attributable to its ability to bind and bioneut ralize activin.