Follistatin and its role as an activin-binding protein

Follistatin (FS), a specific binding protein for activin, neutralizes the diverse actions of activin by forming an inactive complex with activin. FS is a monomer derived from two polypeptide core sequences of 288 (FS-288) and 315 (FS-315) amino acids originated from alternatively spliced mRNA. We purified six molecular forms of FS from porcine ovaries. Their structural differences were caused by truncation of the COOH-terminal region and/or the presence of carbohydrate chains, resulting in the formation of FS-288, FS-315 and FS composed of 303 amino acids (FS-303) in various forms of glycosylation on the two potential Asn-linked glycosylation sites. All six molecular species have almost the same activin binding activity (Kd=540-680 pM). By contrast, the COOH-terminal truncated form, FS-288, showed much higher affinity for heparan sulfate proteoglycans of the cell surface than FS-303, whereas the intact form of FS, FS-315, had no affinity. Furthermore, FS-288 more effectively blocked the suppression of follicle-stimulating hormone (FSH) secretion from rat pituitary cells by activin. This implies that activin binds to the cell surface through FS-288 which adheres to the cell surface. To clarify the physiological role of cell-associated FS, we then investigated the binding of activin to cell-associated FS and the fate of cell surface-bound activin and FS using primary cultured rat pituitary and ovarian granulosa cells. When the cells were incubated with 125I-activin A in the presence of FS-288 or 315, the binding of activin A to the cell surface was promoted much more markedly by FS-288 than by FS-315. The amounts of radioactivity recovered in trichloroacetic acid-soluble fractions (degraded activin) from the incubation medium were greatly increased by the addition of FS-288. This increase was abolished by heparan sulfate, monensin (an endocytosis inhibitor), chloroquine (a lysosome function inhibitor) and several lysosomal enzyme inhibitors. These results suggest that cell-associated FS-288 accelerates the internalization of activin into the cells, leading to its degradation by lysosomal enzymes, and that cell surface-associated FS therefore plays a role in the clearance system of activin.