Henry T. Keutmann

Activins, inhibins, and follistatins: from endocrinology to signaling. A paradigm for the new millennium.

It has been 70 years since the name inhibin was used to describe a gonadal factor that negatively regulated pituitary hormone secretion. The majority of this period was required to achieve purification and definitive characterization of inhibin, an event closely followed by identification and characterization of activin and follistatin (FS). In contrast, the last 15–20 years saw a virtual explosion of information regarding the biochemistry, physiology, and biosynthesis of these proteins, as well as identification of activin receptors, and a unique mechanism for FS action—the nearly irreversible binding and neutralization of activin. Many of these discoveries have been previously summarized; therefore, this review will cover the period from the mid 1990s to present, with particular emphasis on emerging themes and recent advances. As the field has matured, recent efforts have focused more on human studies, so the endocrinology of inhibin, activin, and FS in the human is summarized first. Another area receiving significant recent attention is local actions of activin and its regulation by both FS and inhibin. Because activin and FS are produced in many tissues, we chose to focus on a few particular examples with the most extensive experimental support, the pituitary and the developing follicle, although nonreproductive actions of activin and FS are also discussed. At the cellular level, it now seems that activin acts largely as an autocrine and/or paracrine growth factor, similar to other members of the transforming growh factor β superfamily. As we discuss in the next section, its actions are regulated extracellularly by both inhibin and FS. In the final section, intracellular mediators and modulators of activin signaling are reviewed in detail. Many of these are shared with other transforming growh factor β superfamily members as well as unrelated molecules, and in a number of cases, their physiological relevance to activin signal propagation remains to be elucidated. Nevertheless, taken together, recent findings suggest that it may be more appropriate to consider a new paradigm for inhibin, activin, and FS in which activin signaling is regulated extracellularly by both inhibin and FS whereas a number of intracellular proteins act to modulate cellular responses to these activin signals. It is therefore the balance between activin and all of its modulators, rather than the actions of any one component, that determines the final biological outcome. As technology and model systems become more sophisticated in the next few years, it should become possible to test this concept directly to more clearly define the role of activin, inhibin, and FS in reproductive physiology.

The glycoprotein hormones: recent studies of structure-function relationships.

The structural features of the heterodimeric glycoprotein hormones (LH, FSH, TSH, and hCG) are briefly reviewed. Removal of carbohydrate chains does not reduce binding of the hormones to membrane receptors, but markedly reduces biological responses. The ∗∗∗glycopeptides from the hormone do not reduce binding of native hormone to receptors but do reduce biological responses. Newer data concerned with replication of different regions of the peptide chains of these molecules using synthetic peptides are reviewed and presented. These studies indicate that two regions on the common α subunit are involved with receptor binding of the LH, hCG, and TSH molecules. These regions are α26 to 46 and α75‐92. Two synthetic disulfide loop peptides from the hCGβ subunit β38‐57 and β93‐100 also block binding of hCG to its receptor. In addition, the β38‐57 peptide stimulates testosterone production by Leydig cells. These data indicate that glycoprotein hormone binding to plasma membrane receptors involves a discontinuous site on the hormone that spans both the α and β subunits, and that the α subunit sites are similar for several hormones.—Ryan, R. J.; Charlesworth, M. C.; McCormick, D. J.; Milius, R. P.; Keutmann, H. T. The glycoprotein hormones: recent studies of structure‐function relationships. FASEB J. 2: 2661‐2669; 1988.

Structure-function relationships of gonadotropins.

Publisher Summary The gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) of pituitary origin, and chorionic gonadotropin (hCG, eCG) of placental origin, along with thyroid-stimulating hormone (TSH) constitute a family of glycoprotein hormones. The α-subunit is common to all of the hormones and shows considerable homology from one species to another. The β-subunits are unique for LH, FSH, and TSH. The s-subunits of hLH and hCG are highly homologous (85%) through the first 114 residues, but hCG β differs in that it has a C-terminus extension rich in serine and proline. This offers a chemical basis for their common biological activities. Baboon chorionic gonadotropin, and eLH and chorionic gonadotropin s-subunits also have C-terminal extensions. The glycoprotein hormone s-subunits also show regions of homology between themselves and among different species. A wide range of chemical and enzymatic modifications have been employed in efforts to define residues and sequences in the s-subunits that may be essential for receptor binding. In general, chemical modifications of reactive amino acids appear to be more tolerated in the s- than in the α-subunits.

Osteoporosis, intestinal lactase deficiency and low dietary calcium intake.

OSTEOPOROSIS (senile and postmenopausal) is a common disorder of the elderly, with an estimated morbidity of at least 20 per cent of persons over the age of fifty years.1 2 3 Despite this high prev...

Improved recovery of methionine after acid hydrolysis using mercaptoethanol

Abstract The variable fate and overall poor recovery of methionine sulfoxide during acid hydrolysis (plus its coelution with aspartic acid in the rapidelution analyzer system) prevent accurate quantitation of the original content of methionine in a protein or peptide. As much as 65% of the methionine sulfoxide is not recovered as methionine or other derivatives that can be adequately quantitated. We have found that addition of mercaptoethanol to the 5.7 N HCl at 1:2000 (v/v) provides essentially quantitative conversion of the sulfoxide to methionine during hydrolysis, thereby providing a reproducible, accurate estimation of the true concentration of the amino acid in a polypeptide. Recovery of the other amino acids commonly found in protein hydrolyzates is not deleteriously affected by mercaptoethanol; in fact, the procedure improves recovery of tyrosine and certain other labile amino acid residues as well as methionine.

Follistatin-related protein (FSRP): a new member of the follistatin gene family

The identification and characterization of follistatin related protein (FSRP) suggests that the follistatin (FS) gene family may actually contain two sub-families. The first includes FS and FSRP by virtue of their high degree of structural homology and comparable activin-binding activity, while the second sub-family contains extracellular matrix proteins that possess one or more 10-cysteine FS domains, but do not bind activin or related TGF-beta family members. Characterization of FSRP indicates that it binds activin with similar affinity and selectivity as FS, but does not bind heparin. Furthermore, although FSRP inhibits activin-mediated gene transcription in heterologous assays, FSRP is much less active than FS in the rat pituitary bioassay. When overexpressed in transgenic mice, FSRP may lead to interruption of follicular development and fertility in females but appears to have only a modest effect on males. These results suggest that FSRP is a structural, but not necessarily a functional homologue of FS.

α-Enolase is restricted to basal cells of stratified squamous epithelium

We have developed a monoclonal antibody against a 50-kDa protein that binds preferentially to basal cells in the limbus of rat, rabbit, and human corneas (J. D. Zieske, G. Bukusoglu, and M. A. Yankauckas, Invest. Ophthalmol. Visual Sci. 33, 143-152, 1992). Here we report on the purification and identification of the antigen. The 50-kDa antigen was purified from rabbit limbal and corneal epithelium using HPLC methodology including anion exchange (DEAE) followed by reverse-phase (C18) chromatography. The purified 50-kDa protein was then digested with endoproteinase Lys-C, and a reproducible profile comprising approximately 20 peptides was observed by reverse-phase HPLC of the digest. Sequence analysis of five peptides ranging in length from 4 to 20 residues revealed that the 50-kDa protein was alpha-enolase, a glycolytic enzyme. Overall, 57 amino acids were identified with a 95% sequence homology. Localization of alpha-enolase in rat epithelium by immunofluorescence microscopy demonstrated that simple epithelium contained low or undetectable levels of the enzyme. Stratified squamous epithelium, however, showed high levels of alpha-enolase, which was localized specifically to cells of the basal layer. Epidermal, corneal limbal, oral mucosal, vaginal, and laryngeal epithelium all showed cytoplasmic binding specific to the basal cells. These data indicate that the glycolytic enzyme alpha-enolase is preferentially localized in the basal cell layer of stratified squamous epithelium and suggest that glycolytic activity is concentrated in these cells. The localization pattern suggests that a major change in metabolism occurs as cells leave the mitotically active basal cell layer and migrate toward terminal differentiation in the suprabasal cell layers.

Differential Antagonism of Activin, Myostatin and Growth and Differentiation Factor 11 by Wild-Type and Mutant Follistatin

Follistatin binds and neutralizes members of the TGFbeta superfamily including activin, myostatin, and growth and differentiation factor 11 (GDF11). Crystal structure analysis of the follistatin-activin complex revealed extensive contacts between follistatin domain (FSD)-2 and activin that was critical for the high-affinity interaction. However, it remained unknown whether follistatin residues involved with myostatin and GDF11 binding were distinct from those involved with activin binding. If so, this would allow development of myostatin antagonists that would not inhibit activin actions, a desirable feature for development of myostatin antagonists for treatment of muscle-wasting disorders. We tested this hypothesis with our panel of point and domain swapping follistatin mutants using competitive binding analyses and in vitro bioassays. Our results demonstrate that activin binding and neutralization are mediated primarily by FSD2, whereas myostatin binding is more dependent on FSD1, such that deletion of FSD2 or adding an extra FSD1 in place of FSD2 creates myostatin antagonists with vastly reduced activin antagonism. However, these mutants also bind GDF11, indicating that further analysis is required for creation of myostatin antagonists that will not affect GDF11 activity that could potentially elicit GDF11-induced side effects in vivo.

Follistatin: Essential Role for the N-terminal Domain in Activin Binding and Neutralization

Follistatin is recognized to be an important regulator of cellular differentiation and secretion through its potent ability to bind and bioneutralize activin with which it is colocalized in many tissue systems. The 288-residue follistatin molecule is comprised of a 63-residue N-terminal segment followed by three repeating 10-cysteine “follistatin domains” also represented in several extracellular matrix proteins. We have used chemical modifications and mutational analyses to define structural requirements for follistatin bioactivity that previously have not been investigated systematically. Mutant follistatins were stably expressed from Chinese hamster ovary cell cultures and assayed for activin binding in a solid-phase competition assay. Biological activities were determined by inhibition of activin-mediated transcriptional activity and by suppression of follicle-stimulating hormone secretion by cultured anterior pituitary cells. Deletion of the entire N-terminal domain, disruption of N-terminal disulfides, and deletion of the first two residues each reduced activin binding to <5 % of expressed wild-type follistatin and abolished the ability of the respective mutants to suppress activin-mediated responses in both bioassay systems. Hence, the three follistatin domains inherently lack the ability to bind or neutralize activin. Activin binding was impaired after oxidation of at least one tryptophan, at position 4, in FS-288. Mutation of Trp to Ala or Asp at either positions 4 or 36 eliminated activin binding and bioactivity. Mutation of a third hydrophobic residue, Phe-52, reduced binding to 20%, whereas substitutions for the individual Lys and Arg residues in the N-terminal region were tolerated. These results establish that hydrophobic residues within the N-terminal domain constitute essential activin-binding determinants in the follistatin molecule. The correlation among the effects of mutation on activin binding, activin transcriptional responses, and follicle-stimulating hormone secretion substantiates the concept that, at least in the pituitary, the biological activity of follistatin is attributable to its ability to bind and bioneutralize activin.

Evidence for a conformational change in deglycosylated glycoprotein hormones

Carbohydrate Glycoprotein Follitropin Choriogonadotropin Conformation Membrane receptor