PARENT SESSION
PLATFORM SESSION 8: REGULATION OF GONADOTROPINS AND THEIR RECEPTORS: I
Chair: Narayan, Prema¹, ¹
Co-chair: Rispoli, Louisa¹, ¹
Dover A-C
4:30 PM-6:30 PM

(58) REGULATION OF ACTIVIN ACTION BY FOLLISTATIN: ROLE OF THE HEPARIN-BINDING REGION .

Sidis, Yisrael¹, Schneyer, Alan¹, Sluss, Patrick¹, Keutmann, Henry¹, ¹ Reproductive Endocrine and Endocrine Units, Massachusetts General Hospital, Boston, MA

ABSTRACT- Follistatin (FS) is an important regulator of FSH secretion through its potent ability to bind and neutralize activin. FS has been postulated to act as a local regulator by means of binding to cell-surface heparan-sulfate proteoglycans. Besides modulating availability of endogenous activin of autocrine origin, the surface-bound follistatin may act as a barrier to access by exogenous activin from the circulation. Surface binding is achieved principally through a lysine-rich consensus heparin-binding sequence (KKCRMNKKNKPR; residues 75-86) within the first of the three 10-cysteine FS domains. We have characterized this interaction through mutational analyses using domain replacements and amino acid substitutions within the heparin-binding sequence. Mutants were tested for activin binding in a solid-phase competition assay, and for biological responses in assays employing exogenous activin (luciferase-reporter transcriptional response) and endogenous activin (suppression of pituitary cell FSH secretion). Substitution of the (75-86) sequence with the homologous segment from FS domain II (STCVVDQTNNAY) markedly diminished the ability of FS to suppress pituitary cell FSH secretion despite retention of activin binding and transcriptional inhibition. This was consistent with loss of cell-surface binding, as demonstrated by loss of binding to either cultured HEK-293 cells or a solid-phase heparin affinity matrix. Decreased pituitary-cell effects relative to binding were also observed after outright replacement of FS domain I by full-length FS domain II, and by FS domain I from a follistatin homolog, FSRP, which also lacks a heparin-binding sequence. Alanine replacement of either pair of lysines (75-76) or (81-82) did not affect bioactivity relative to activin binding, and both mutants bound to HEK-293 cell surfaces and to heparin affinity matrix. Thus, the full-length heparin-binding sequence is essential for cell surface interaction and neutralization of endogenous activin. These results show that the heparin-binding region in FS domain I is important for regulation of endogenous activin bioactivity, but less so for activin binding and exogenous activin bioactivity, strengthening the concept that follistatin is a local regulator of activin and possibly other TGF--related ligands as well.

KEY WORDS: Follistatin, Activin, Pituitary, FSH