Structural and Biophysical Coupling of Heparin and Activin Binding to Follistatin Isoform Functions

Abstract

Follistatin (FS) regulates transforming growth factor-␤ superfamily ligands and is necessary for normal embryonic and ovarian follicle development. Follistatin is expressed as two splice variants (FS288 and FS315). Previous studies indicated differences in heparin binding between FS288 and FS315, potentially influencing the physiological functions and locations of these isoforms. We have determined the structure of the FS315-activin A complex and quantitatively compared heparin binding by the two isoforms. The FS315 complex structure shows that both isoforms inhibit activin similarly, but FS315 exhibits movements within follistatin domain 3 (FSD3) apparently linked to binding of the C-terminal extension. Surprisingly, the binding affinities of FS288 and FS315 for heparin are similar at lower ionic strengths with FS315 binding decreasing more sharply as a function of salt concentration. When bound to activin, FS315 binds heparin similarly to the FS288 isoform, consistent with the structure of the complex, in which the acidic residues of the C-terminal extension cannot interact with the heparin-binding site. Activin-induced binding of heparin is unique to the FS315 isoform and may stimulate clearance of FS315 complexes.Ligands of the transforming growth factor-␤ (TGF␤) 3 superfamily regulate a diverse set of cellular and physiological functions, including early embryonic development, cellular growth and proliferation, and reproduction (1-3). Activin A, a member of the TGF␤ superfamily, is necessary to the regulation of both the male and the female reproductive axes by stimulating the release of follicle-stimulating hormone from the pituitary (4, 5). Activin A also has other important physiological roles, because activin A-deficient mice die shortly after birth and exhibit multiple defects in craniofacial development (6, 7). Comparative studies of the similarities and differences between TGF␤ ligands have lead to a better understanding of the many physiological effects of this multipotent ligand superfamily.TGF␤ superfamily members typically exist as covalently linked dimers and fall into three main sub-categories: TGF␤s, activins/inhibins/nodals, and bone morphogenetic proteins (BMPs) (8). Structurally, these proteins all share a cysteine-knot fold and signal by engaging type II and type I receptors (9). Activin and BMPs bind to their type II receptors through a structural feature known as the "knuckle" region (10 -12). They have similar type I receptor binding sites on the concave surfaces of the molecule, which span both monomers of the ligands (13-16). Although specific ligand:receptor pairs have been characterized, multiple ligands share several receptors providing complexity and precise control to the system. The members of this superfamily are produced as prepro-proteins, and the pro-domain often regulates biological functions. In the absence of the pro-domain, TGF␤-1 and -2, as well as BMP-2, -4, and -7, can bind directly to the cell surface through heparin binding sites (17,18). This provides a mechanism f...

Inhibin α-Subunit N Terminus Interacts with Activin Type IB Receptor to Disrupt Activin Signaling

“…Materials-Purified human activin A, inhibin A, the extracellular domain of mouse ALK4 (ALK4-ECD), ActRIIB (ActRIIB-ECD), and mouse follistatin 288 (Fst 288) were produced by our laboratory (35,36). The CHO-Flp-in cell line and pcDNA5 plasmid used for cloning and establishment of isogenic stable cell lines were purchased from Invitrogen (Carlsbad, CA).…”

Clinical and Therapeutic Implications of Follistatin in Solid Tumours

“…Along with FST, activin acts as a pleiotropic growth factor system, which is involved in proliferation, differentiation, and apoptosis of a number of cell types (23)(24)(25)(26)(27)(28). As such, the functions of the FST isoforms are therefore linked to their binding affinity for activin (6,10,11,(29)(30)(31)(32)(33)(34)(35)(36)(37). In fact, this critical binding and neutralisation of activin, either partial or complete, is based on the order of at least two of the FST cysteine domains and its Nterminal (38), conferring a difference in affinities for activin between the FST isoforms.…”

Follistatin is essential for normal postnatal development and function of mouse oviduct and uterus

“…FST315 is the main circulating protein and, while FST288 also circulates, its major role is in the interaction with activin at cell surfaces since it is bound to heparin sulfate proteoglycans, facilitating the degradation of activin via a lysosomal pathway (Hashimoto et al 1997). Following FST315-activin complex formation, the complex is capable of binding to the cell surface and activin degradation ensues (Lerch et al 2007). A third isoform also exists, FST303, which is derived from proteolytic cleavage of FST315 and has weaker cell-surface binding properties compared with FST288 (Sugino et al 1993).…”