Suppression of Inhibin A Biological Activity by Alterations in the Binding Site for Betaglycan

Abstract

Inhibins A and B negatively regulate the production and secretion of follicle-stimulating hormone from the anterior pituitary, control ovarian follicle development and steroidogenesis, and act as tumor suppressors in the gonads. Inhibins regulate these reproductive events by forming high affinity complexes with betaglycan and activin or bone morphogenetic protein type II receptors. In this study, the binding site of inhibin A for betaglycan was characterized using inhibin A mutant proteins. An epitope for high affinity betaglycan binding was detected spanning the outer convex surface of the inhibin ␣-subunit. Inhibin A and inhibin B, members of the TGF␤ 2 superfamily, are essential regulatory factors in mammalian reproduction. Their expression is critical for the regulation of fertility based on their dual inhibitory actions on follicle-stimulating hormone (FSH) secretion by the pituitary (1, 2) and gametogenesis in the gonads (3, 4). It is recognized that inhibins regulate these reproductive events by inhibiting the stimulatory actions of the structurally related proteins, activins (5, 6). Inhibins are heterodimers of an 18-kDa ␣-subunit disulfide-linked to one of two 13-kDa ␤-subunits (␤A and ␤B), resulting in inhibin A or inhibin B, respectively. Each activin is composed of two ␤-subunits; ␤A-␤A (activin A), ␤A-␤B (activin AB), and ␤B-␤B (activin B). Although the inhibin A and inhibin B crystal structures have not been determined, based upon sequence identity it is expected that the ␤-subunits would retain a similar conformation to that observed in the activin A dimer (7). Thus, within either ␤-subunit, two pairs of anti-parallel ␤-strands, forming a short and a long "finger," stretch outward from the cysteine knot core of the monomer. At the base of the fingers, each ␤-subunit forms an ␣-helix, which together with the prehelix loop, generates the "wrist" region of the molecule (8).An interchain disulfide bond between Cys 80 of the ␤A-subunit (Cys 79 of the ␤B-subunit) and Cys 95 of the ␣-subunit covalently connects the two chains of the inhibin dimers. In humans, unlike other species, two molecular mass isoforms of mature inhibin A and B (31 and 34 kDa) have been identified (9). This molecular mass heterogeneity is due to the presence of two N-linked glycosylation sites at Asn 36 and Asn 70 of the ␣-subunit. Asn 36 is always glycosylated (producing 31-kDa inhibin A or B), whereas glycosylation of Asn 70 is differentially regulated (producing 34-kDa inhibin A or B) (10). The presence of a second carbohydrate moiety significantly reduces the biological activity of inhibin A (9), suggesting that human inhibins may be under more stringent control than inhibins from other species.Inhibins exert their biological effects by antagonizing the actions of TGF␤ ligands that utilize activin (ActRII/IIB) or BMP (BMPRII) type II receptors as part of their signaling complex (11). The inhibin residues involved in binding to the type II receptors can be inferred from the crystal structure of activin A bound to ActRIIB (12). I...

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

“…Nevertheless, our work confirms the findings of several recent papers, which reported that a synthesized peptide representing the inhibin ␣-subunit N-terminal region has biological function in the ovary (33,34). We and others (14,20,52) have previously demonstrated that the antagonistic function of inhibin can be decreased with an antibody directed against the ␣-subunit N-terminal extension region or by deletion of the N-terminal region. Taken together, we propose a model in which the inhibin heterodimer achieves potent antagonism of activin signaling in the pituitary gonadotrope through the interaction of its ␤-subunit with ActRII/IIB enhanced by the co-receptor betaglycan, as well as through the interaction of the ␣-subunit N-terminal extension region with ALK4 (Fig.…”

“…Additional inhibin-binding proteins that increase the affinity of inhibin for the type II receptors are thought to be involved in mediating the antagonistic actions of inhibin. Although several candidate inhibin-binding proteins have been identified to date, only the type III TGF␤ receptor (commonly known as betaglycan) has been shown to increase the binding affinity of inhibin to the type II activin and BMP receptors (6,13,14) (Fig. 1A).…”

Changes in expression of bone morphogenetic proteins (BMPs), their receptors and inhibin co-receptor betaglycan during bovine antral follicle development: inhibin can antagonize the suppressive effect of BMPs on thecal androgen production

“…We confirmed that betaglycan immunoreactivity was present in both the TC and GC layers of bovine antral follicles and in cultured bovine TC. Since betaglycan is considered an essential co-receptor required to facilitate the presentation of inhibin to type II signalling receptors and hence disrupt activin signalling (Lewis et al 2000, Chapman et al 2002, Makanji et al 2008, this observation prompted us to use our bovine TC culture model to evaluate the possibility that GC-derived inhibin functions as a physiological antagonist of BMP signalling in the ovary, in a manner comparable to its welldocumented role as an activin antagonist.…”

A Common Biosynthetic Pathway Governs the Dimerization and Secretion of Inhibin and Related Transforming Growth Factor β (TGFβ) Ligands

“…A, a homology model of the mature inhibin A dimer was generated in a previous study (38). The inhibin ␣-subunit is colored orange, whereas the inhibin ␤ A -subunit is green.…”

“…To identify the binding epitope on inhibin A for the ␣-subunit prodomain, residues through the finger regions of the ␣-subunit were substituted for alanine. In all, a set of 28 variants was generated (38 in the finger region of the ␣-subunit are necessary for inhibin A production and secretion (Fig. 5B, lanes 2, 4, 6, 7, and 9).…”