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Submitted on March 28, 2003
Accepted on October 10, 2003
1 Endocrine Unit and Reproductive Endocrine Unit, Massachusetts General Hospital, Boston MA 02114
* To whom correspondence should be addressed. E-mail: Keutmann{at}helix.mgh.harvard.edu.
Follistatin (FS) is an important regulator of pituitary FSH secretion through its potent ability to bind and bioneutralize activin. It also represents a prototype for binding proteins that control bioavailability of other TGF
-related growth factors such as the BMPs. The 288-residue follistatin molecule has a distinctive structure comprised principally of three ten-cysteine "follistatin domains (FSD's)." These are preceded by an N-terminal segment shown by us previously to contain hydrophobic residues essential for activin binding. To establish the contribution of the FS domains themselves to follistatin's bioactivity, we prepared mutants with deleted or exchanged domains and intradomain point mutations. Mutants were expressed from mammalian (CHO) cells and evaluated for activin binding and for biological activity in assays measuring differing aspects of follistation bioactivity: activin-mediated transcriptional activity and suppression of FSH secretion in primary pituitary cell cultures. The N-terminal domain (residues 1--63) alone could not bind activin or suppress activin-mediated transcription, either alone or in combination with the FS-domain region (residues 64--288). Deletion of FS domains 1 or 2 abolished activin binding and biological activity in both assays, whereas deletion of domain 3 was tolerated. Bioactivity was also reduced or eliminated after exchange of domains (FS 2/1/3 and FS 3/1/2) or doubling of domain 1 (FS 1/1/3) or domain 2 (FS 2/2/3). Several hydrophobic residues clustered within the C-terminal region of FSD-1 and FSD-2 are highly conserved among all follistatin domains. Mutation of any of these to Asp or Ala either reduced or eliminated FS bioactivity, and disrupted distant epitopes for heparin binding (FSD-1) or antibody recognition (FSD-2), suggesting their role in maintaining the conformational integrity of the domain and possibly the FS molecule as a whole. These results are consistent with the importance of domain conformation as well as the overall order of the domains in follistatin function. A continuous sequence comprising the N-terminal domain and followed by FS domains 1 and 2 fulfills the minimum structural requirement for activin binding and follistatin bioactivity.
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