Identification of an Inhibin Receptor in Gonadal Tumors from Inhibin α-Subunit Knockout Mice

Abstract

Inhibins and activins are dimeric proteins that are functional antagonists and are structurally related to the transforming growth factor-␤ (TGF␤) family of growth and differentiation factors. Receptors for activin and TGF␤ have been identified as dimers of serinethreonine kinase subunits that regulate cytoplasmic proteins known as Smads. Despite major advances in our understanding of activin and TGF␤ receptors and signaling pathways, little is known about inhibin receptors or the mechanism by which this molecule provides a functionally antagonistic signal to activin. Studies described in this paper indicate that an independent inhibin receptor exists. Numerous tissues were examined for inhibin-specific binding sites, including the developing embryo, in which the spinal ganglion and trigeminal ganglion-bound iodinated inhibin A. Sex cord stromal tumors, derived from male and female inhibin ␣-subunit-deficient mice, were also identified as a source of inhibin receptor. Abundant inhibin and few activin binding sites were identified in tumor tissue sections by in situ ligand binding using iodinated recombinant human inhibin A and 125 I-labeled recombinant human inhibin A. Tumor cell binding was specific for each ligand (competed by excess unlabeled homologous ligand and not competed by heterologous ligand). Based on these results and the relative abundance and homogeneity of tumor tissues versus the embryonic ganglion, tumor tissues were homogenized, membrane proteins were purified, and putative inhibin receptors were isolated using an inhibin affinity column. Four proteins were eluted from the column that bind iodinated inhibin but not iodinated activin. These data suggest that inhibin-specific membrane-associated proteins (receptors) exist. The inhibin and activin ␤-subunits are 30% homologous to TGF␤ based on alignment of conserved cysteine amino acids. Crystal structures of two TGF␤ family members (TGF␤2 and osteogenic protein-2) have been solved, and the overall structural similarity between these proteins implies that a conserved topology exists between members of the superfamily (8, 9). Proteins within the TGF␤ family span multiple species and appear to be central factors in bone formation and morphogenesis of embryos, and the genes that encode them may function as tumor suppressor genes (10).Activin activity is inhibited by interaction with a bioneutralizing binding protein called follistatin (11). Follistatin binds both inhibin and activin through the ␤-subunit; however, the ability of follistatin to neutralize inhibin activity cannot be determined until a cellular activity is described for inhibin that is not confounded by activin. Follistatin is structurally complex and is highly conserved between species with two conserved amino acid differences between rat and human and 87% homology with the Xenopus follistatin homologue (12,13).Cellular response to activin is transduced through two single membrane-spanning serine-threonine kinase subunits (3). The ligand binds a type II receptor (70 -75 kDa) that tran...

Mechanisms of Inhibin Signal Transduction

“…Gonadal tumors derived from inhibin a-subunit knockout mice bind iodinated inhibin A but bind iodinated activin A at much lower levels. This inhibin binding is not displaced by excess unlabeled activin (Draper et al, 1998). These data indicate that the association between inhibin and target cells is mediated by an inhibinspecific receptor or binding protein and not solely via the activin type II receptors, or at least that an accessory protein forms a stable association between inhibin and actRI1 that is not easily disrupted by activin (Lewis et al, 2000).…”

“…First, we discuss differential regulation of inhibin isoforms. (Draper et al, 1998;Hertan ef al., 1999;Lewis et al, 2000;Chung er al, 2000). Next, we detail our current understanding of inhibin signal transduction.…”

A sensitive and specific in vitro bioassay for activin using a mouse plasmacytoma cell line, MPC-11

“…In contrast to follistatin, inhibin A did not antagonize activin's effects, as it does in pituitary cell and K562 cell cultures (Yu et al 1987, Robertson et al 1992. This finding needs to be explored further, particularly in view of the hypothesis that inhibin blocks the binding of activin to its type II receptor (Martens et al 1997), and the proposed existence of a distinct inhibin receptor (Draper et al 1998).…”

Temporal and hormonal regulation of inhibin protein and subunit mRNA expression by post-natal and immature rat ovaries

“…Inhibin is also thought to play local regulatory roles in the ovary since it has been shown to directly modulate thecal cell androgen synthesis (Hillier et al 1991) and oestrogen production (Smyth et al 1994), and it has been postulated to promote follicular growth (Woodruff et al 1990). Recently, binding sites for inhibin were demonstrated on ovarian cells (Draper et al 1998).…”