Structural basis for a functional antagonist in the transforming growth factor beta superfamily

Within the transforming growth factor beta superfamily, the agonist-antagonist relationship between activin and inhibin is unique and critical to integrated reproductive function. Activin acts in the pituitary to stimulate follicle-stimulating hormone, and is antagonized by endocrine acting, gonadally derived inhibin. We have undertaken a mutational analysis of the activin betaA subunit to determine the precise structural aspects that contribute to inhibin antagonism of activin. By substituting specific amino acid residues in the activin betaA subunit with similarly aligned amino acids from the alpha subunit, we have pinpointed the residues required for activin receptor binding and activity, as well as for inhibin antagonism of activin through its receptors. Additionally, we have identified an activin mutant with a higher affinity for the activin type I receptor that provides structural evidence for the evolution of ligand-receptor interactions within the transforming growth factor beta superfamily.     

Role of inhibin and activin in the modulation of gonadotropin- and steroid-induced oocyte maturation in the teleost Fundulus heteroclitus

Background  

Activin and inhibin are glycoproteins structurally related to the transforming growth factor-beta superfamily. These peptides were first described as factors that regulate the follicle-stimulating hormone (FSH) at the pituitary level. The possible role of inhibin and activin, at the ovarian level, in mediating the stimulatory actions of a Fundulus pituitary extract (FPE) and 17alpha,20beta-dihydroprogesterone (DHP) on oocyte maturation was investigated in this study.     

An activin/furin regulatory loop modulates the processing and secretion of inhibin alpha- and betaB-subunit dimers in pituitary gonadotrope cells

Of all ligands of the transforming growth factor beta superfamily, inhibins and activins are a physiologically relevant pair that are functional antagonists of each other. Activin stimulates whereas inhibin blocks follicle-stimulating hormone biosynthesis and secretion from pituitary gonadotrope cells, and together, inhibin and activin control the pituitary gonadal axis essential for normal reproductive function. Sharing a similar beta-subunit, the secretion of inhibin heterodimers (alpha/beta) or activin homodimers (beta/beta) as mature bioactive ligands depends, in part, on the proteolytic processing of precursor proteins. A short loop regulatory pathway controlling precursor processing and dimer secretion was discovered. Activin stimulates endogenous inhibin alpha- and betaB-subunit mRNA, protein, and proteolytic processing. Simultaneously, activin stimulated the proconvertase furin through a Smad2/3-dependent process. The data provide a mechanism where the regulation of furin and inhibin subunits cooperates in an important positive short feedback loop. This regulatory loop augments the secretion of bioactive mature activin B, as well as inhibin B dimers, necessary for local follicle-stimulating hormone beta regulation.     

Normal reproductive function in InhBP/p120-deficient mice

The inhibins are gonadal transforming growth factor beta superfamily protein hormones that suppress pituitary follicle-stimulating hormone (FSH) synthesis. Recently, betaglycan and inhibin binding protein (InhBP/p120, also known as the product of immunoglobulin superfamily gene 1 [IGSF1]) were identified as candidate inhibin coreceptors, shedding light on the molecular basis of how inhibins may affect target cells. Activins, which are structurally related to the inhibins, act within the pituitary to stimulate FSH production. Betaglycan increases the affinity of inhibins for the activin type IIA (ACVR2) receptor, thereby blocking activin binding and signaling through this receptor. InhBP/p120 may not directly bind inhibins but may interact with the activin type IB receptor, ALK4, and participate in inhibin B's antagonism of activin signaling. To better understand the in vivo functions of InhBP/p120, we characterized the InhBP/p120 mRNAs and gene in mice and generated InhBP/p120 mutant mice by gene targeting in embryonic stem cells. InhBP/p120 mutant male and female mice were viable and fertile. Moreover, they showed no alterations in FSH synthesis or secretion or in ovarian or testicular function. These data contribute to a growing body of evidence indicating that InhBP/p120 does not play an essential role in inhibin biology.     

A Mathematical Model for the Actions of Activin,Inhibin, and Follistatin on Pituitary Gonadotrophs

The timed secretion of the luteinizing hormone (LH) and follicle stimulating hormone (FSH) from pituitary gonadotrophs during the estrous cycle is crucial for normal reproductive functioning. The release of LH and FSH is stimulated by gonadotropin releasing hormone (GnRH) secreted by hypothalamic GnRH neurons. It is controlled by the frequency of the GnRH signal that varies during the estrous cycle. Curiously, the secretion of LH and FSH is differentially regulated by the frequency of GnRH pulses. LH secretion increases as the frequency increases within a physiological range, and FSH secretion shows a biphasic response, with a peak at a lower frequency. There is considerable experimental evidence that one key factor in these differential responses is the autocrine/paracrine actions of the pituitary polypeptides activin and follistatin. Based on these data, we develop a mathematical model that incorporates the dynamics of these polypeptides. We show that a model that incorporates the actions of activin and follistatin is sufficient to generate the differential responses of LH and FSH secretion to changes in the frequency of GnRH pulses. In addition, it shows that the actions of these polypeptides, along with the ovarian polypeptide inhibin and the estrogen-mediated variations in the frequency of GnRH pulses, are sufficient to account for the time courses of LH and FSH plasma levels during the rat estrous cycle. That is, a single peak of LH on the afternoon of proestrus and a double peak of FSH on proestrus and early estrus. We also use the model to identify which regulation pathways are indispensable for the differential regulation of LH and FSH and their time courses during the estrous cycle. We conclude that the actions of activin, inhibin, and follistatin are consistent with LH/FSH secretion patterns, and likely complement other factors in the production of the characteristic secretion patterns in female rats.     

Identification of distinct inhibin and transforming growth factor beta-binding sites on betaglycan: functional separation of betaglycan co-receptor actions

Betaglycan is a co-receptor that mediates signaling by transforming growth factor beta (TGFbeta) superfamily members, including the distinct and often opposed actions of TGFbetas and inhibins. Loss of betaglycan expression, or abrogation of betaglycan function, is implicated in several human and animal diseases, although both betaglycan actions and the ligands involved in these disease states remain unclear. Here we identify a domain spanning amino acids 591-700 of the betaglycan extracellular domain as the only inhibin-binding region in betaglycan. This binding site is within the betaglycan ZP domain, but inhibin binding is not integral to the ZP motif of other proteins. We show that the inhibin and TGFbeta-binding residues of this domain overlap and identify individual amino acids essential for binding of each ligand. Mutation of Val614 to Tyr abolishes both inhibin and TGFbeta binding to this domain. Full-length betaglycan V614Y, and other mutations, retain TGFbeta binding activity via a distinct site, but are unable to bind inhibin-A. These betaglycan mutants fail to mediate inhibin antagonism of activin signaling but can present TGFbeta to TbetaRII. Separating the co-receptor actions of betaglycan toward inhibin and TGFbeta will allow the clarification of the role of betaglycan in disease states such as renal cell carcinoma and endometrial adenocarcinoma.     

Roles of the activin regulatory system in fish reproduction

Activin (betaAbetaA, betaAbetaB, and betaBbetaB) is a dimeric growth factor with diverse biological activities in vertebrate reproduction. Activin exerts its actions by binding to its specific type II and type I receptors. The activity of activin is regulated by follistatin, its binding protein, and the antagonists inhibin and antivin. All major components of the activin-inhibin-follistatin system have been identified in fish except the alpha subunit of inhibin. Using goldfish as a model, we have demonstrated that activin is expressed in the pituitary and the recombinant goldfish activin B has novel inverse effects on the expression of GTH beta subunits. Activin increases the mRNA level of GTH-Ibeta while significantly suppressing the expression of GTH-IIbeta. We have also demonstrated the expression of activin and its receptors in the goldfish and zebrafish ovary. Using an in vitro ovarian follicle incubation as the system, we have investigated the involvement of the activin system in the process of final oocyte maturation. Our evidence clearly indicates that activin has potent effect of promoting final oocyte maturation, and that it may play a role in mediating the stimulatory effect of pituitary gonadotropin in the event of oocyte maturation.     

Expression cloning of an activin receptor, a predicted transmembrane serine kinase.

Activins are involved in the regulation of multiple biological events, ranging from early development to pituitary function. To characterize the cellular mechanisms involved in these processes, cDNAs coding for an activin receptor were cloned from AtT20 mouse corticotropic cells by screening COS cell transfectants for binding of 125I-activin A. The cDNAs code for a protein of 494 amino acids comprising a ligand-binding extracellular domain, a single membrane-spanning domain, and an intracellular kinase domain with predicted serine/threonine specificity. 125I-activin A binds to transfected COS cells with an affinity of 180 pM and can be competed by activin A, activin B, and inhibin A, but not by transforming growth factor beta 1. The kinase domain, but not the extracellular sequence, of the activin receptor is most closely related to the C. elegans daf-1 gene product, a putative transmembrane serine/threonine-specific protein kinase for which the ligand is not known.     

Activin signaling and its role in regulation of cell proliferation, apoptosis, and carcinogenesis

Activins, cytokine members of the transforming growth factor-beta superfamily, have various effects on many physiological processes, including cell proliferation, cell death, metabolism, homeostasis, differentiation, immune responses endocrine function, etc. Activins interact with two structurally related serine/threonine kinase receptors, type I and type II, and initiate downstream signaling via Smads to regulate gene expression. Understanding how activin signaling is controlled extracellularly and intracellularly would not only lead to more complete understanding of cell growth and apoptosis, but would also provide the basis for therapeutic strategies to treat cancer and other related diseases. This review focuses on the recent progress on activin-receptor interactions, regulations of activin signaling by ligand-binding proteins, receptor-binding proteins, and nucleocytoplasmic shuttling of Smad proteins.     

Reconstitution and analysis of soluble inhibin and activin receptor complexes in a cell-free system

Activins and inhibins compose a heterogeneous subfamily within the transforming growth factor-beta (TGF-beta) superfamily of growth and differentiation factors with critical biological activities in embryos and adults. They signal through a heteromeric complex of type II, type I, and for inhibin, type III receptors. To characterize the affinity, specificity, and activity of these receptors (alone and in combination) for the inhibin/activin subfamily, we developed a cell-free assay system using soluble receptor-Fc fusion proteins. The soluble activin type II receptor (sActRII)-Fc fusion protein had a 7-fold higher affinity for activin A compared with sActRIIB-Fc, whereas both receptors had a marked preference for activin A over activin B. Although inhibin A and B binding was 20-fold lower compared with activin binding to either type II receptor alone, the mixture of either type II receptor with soluble TGF-beta type III receptor (TbetaRIII; betaglycan)-Fc reconstituted a soluble high affinity inhibin receptor. In contrast, mixing either soluble activin type II receptor with soluble activin type I receptors did not substantially enhance activin binding. Our results support a cooperative model of binding for the inhibin receptor (ActRII.sTbetaRIII complex) but not for activin receptors (type II + type I) and demonstrate that a complex composed of activin type II receptors and TbetaRIII is both necessary and sufficient for high affinity inhibin binding. This study also illustrates the utility of this cell-free system for investigating hypotheses of receptor complex mechanisms resulting from crystal structure analyses.     

Structural and biophysical coupling of heparin and activin binding to follistatin isoform functions

Follistatin (FS) regulates transforming growth factor-beta 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.     

Role of PCSK5 expression in mouse ovarian follicle development: identification of the inhibin α- and β-subunits as candidate substrates

Inhibin and activin are essential dimeric glycoproteins belonging to the transforming growth factor-beta (TGFβ) superfamily. Inhibin is a heterodimer of α- and β-subunits, whereas activin is a homodimer of β-subunits. Production of inhibin is regulated during the reproductive cycle and requires the processing of pro-ligands to produce mature hormone. Furin is a subtilisin-like proprotein convertase (proconvertase) that activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. We hypothesized that furin-like proconvertases are central regulators of inhibin α- and β-subunit processing within the ovary. We analyzed the expression of the proconvertases furin, PCSK5, PCSK6, and PCSK7 in the developing mouse ovary by real-time quantitative RT-PCR. The data showed that proconvertase enzymes are temporally expressed in ovarian cells. With the transition from two-layer secondary to pre-antral follicle, only PCSK5 mRNA was significantly elevated. Activin A selectively enhanced expression of PCSK5 mRNA and decreased expression of furin and PCSK6 in cultured two-layer secondary follicles. Inhibition of proconvertase enzyme activity by dec-RVKR-chloromethylketone (CMK), a highly specific and potent competitive inhibitor of subtilisin-like proconvertases, significantly impeded both inhibin α- and β-subunit maturation in murine granulosa cells. Overexpression of PC5/6 in furin-deficient cells led to increased inhibin α- and β(B)-subunit maturation. Our data support the role of proconvertase PCSK5 in the processing of ovarian inhibin subunits during folliculogenesis and suggest that this enzyme may be an important regulator of inhibin and activin bioavailability.     

Identification of a binding site on the type II activin receptor for activin and inhibin

Type II activin receptors (ActRII and ActRIIB) are single-transmembrane domain serine/threonine kinase receptors that bind activin to initiate the signaling and cellular responses triggered by this hormone. Inhibin also binds type II activin receptors and antagonizes many activin effects. Here we describe alanine scanning mutagenesis of the ActRII extracellular domain. We identify a cluster of three hydrophobic residues (Phe(42), Trp(60), and Phe(83)) that, when individually mutated to alanine in the context of the full-length receptor, cause the disruption of activin and inhibin binding to ActRII. Each of the alanine-substituted ActRII mutants retaining activin binding maintains the ability to form cross-linked complexes with activin and supports activin cross-linking to the type I activin receptor ALK4. Unlike wild-type ActRII, the three mutants unable to bind activin do not cause an increase in activin signaling when transiently expressed in a corticotroph cell line. Together, our results implicate these residues in forming a critical binding surface on ActRII required for functional interactions with both activin and inhibin. This first identification of a transforming growth factor-beta family member binding site may provide a general basis for characterizing binding sites for other members of the superfamily.     

Paracrine effect of folliculo-stellate cells on the growth factor-like action of activin A in anterior pituitary cultures.

Several studies have shown that pituitary folliculo-stellate (FS) cells exhibit local functions within the pituitary gland. On the other hand, we have shown previously that activin A increases the number of FSH-producing gonadotropes in cultured rat anterior pituitary cells. In this study, we investigated whether FS cells exert an influence on the action of activin A. FS cells were prepared by culturing the dispersed rat anterior pituitary cells in media containing 15% fetal calf serum and 6 mM glutamine for 15 days. Most cells had the morphological characteristics of FS cells and S-100 protein immunoreactivity, a specific marker of FS cells. The number of FSH cells, which was higher in activin A-treated than in control cultures, was reduced to the control level by incubation with activin A plus conditioned media from FS cell-enriched cultures (FSCM). This inhibitory effect of FSCM was neutralized by a follistatin antibody, but not by anti-S-100 protein or anti-basic fibroblast growth factor. Furthermore, follistatin suppressed activin A stimulated increases in the number of FSH cells in a similar inhibitory pattern to that of FSCM. Meanwhile, the number of FSH cells was not affected by FSCM or follistatin in the absence of activin A. These results suggest that FS cells are involved in the regulation of the function and/or the morphogenesis of the FSH cell-lineage by affecting the action of activin A, and that this paracrine effect of FS cells is mediated by follistatin.     

Purification of recombinant activin A using the second follistatin domain of follistatin-related gene (FLRG)

Activins are multifunctional growth factors belonging to the transforming growth factor-beta superfamily. Isolation of activins from natural sources requires many steps and only produces limited quantities. Even though recombinant preparations have been used in recent studies, purification of recombinant activins still requires multiple steps. To purify recombinant activin A, we have developed a simple method using the second follistatin domain of an activin-binding protein follistatin-related gene (FLRG). An affinity column was prepared with a partial FLRG fusion protein. The partial FLRG protein contained the second follistatin domain and the C-terminus acidic domain, and was tagged with six histidine residues at its N-terminus. The fusion protein was expressed in Escherichia coli and purified with nickel affinity column. Thereafter, the purified fusion protein was coupled to NHS-activated column. Recombinant activin A was produced in Chinese hamster ovary (CHO) cells, which were stably transfected with rat inhibin/activin betaA-subunit cDNA. After 48-h suspension culture of the cells in a serum free medium, the culture media was recovered and passed through the FLRG-coupled column. After washing with phosphate-buffered saline, bound protein was eluted out with an acidic buffer. Any significant contaminations were not detected when the purified protein was analyzed by SDS-PAGE. Apparent sizes of the protein were 14 and 28 kDa under the reduced and non-reduced conditions, respectively. Western blot analysis confirmed that the purified protein was activin A. The purified recombinant activin stimulated p3TP-lux reporter activity in CHO cells and follicle-stimulating hormone secretion from rat pituitary cells.     

The isolation and physiology of inhibin and related proteins

Inhibin, a glycoprotein that preferentially suppresses follicle-stimulating hormone (FSH) secretion, has been isolated from follicular fluid as a heterodimer of two dissimilar subunits linked by disulphide bonds. The larger subunit is termed alpha and the smaller is designated beta. Two forms of inhibin termed A and B have been isolated, the differences being due to variations in the amino acid sequence of the beta-subunit; Inhibin A consists of alpha-beta and Inhibin B of alpha-beta B. Dimers of the beta-subunit, termed activins, have also been found in follicular fluid; these stimulate pituitary FSH secretion. Inhibin is produced in the female by the granulosa cell and corpus luteum under the control of FSH and luteinizing hormone (LH), respectively. The levels in serum rise to peak at mid-cycle and in the mid-luteal phase of the human menstrual cycle, and decline prior to menstruation. In pregnancy, the late-luteal phase decline in inhibin does not occur and the levels increase slowly. Studies suggest that the levels in pregnancy arise from an embryonic source, particularly the placenta. In the male, inhibin is produced by the Sertoli cells under the control of FSH by mechanisms involving cyclic adenosine 3', 5'-monophosphate. Testosterone exerts a minor inhibitory control at supraphysiological levels (10(-5) M), but human chorionic gonadotropin stimulation results paradoxically in a rise in serum inhibin levels. Disruption of spermatogenesis in the rat by cryptorchidism, heat treatment, or efferent duct ligation results in a decline in inhibin levels and a rise in FSH levels, findings consistent with the negative feedback action of inhibin on FSH secretion. As well as their roles in the reproductive system, inhibin and activin have more widespread actions in the haemopoietic, immune and nervous systems as evidenced by the finding of mRNA for its subunits in a range of tissues. Other studies have shown actions on erythroid differentiation and on mitotic activity in thymocytes. These actions suggest that inhibin and activin may function as growth factors as well as regulators of FSH.