Reviews of Reproduction (2000) 5, 131–135

Is the action of inhibin mediated via a unique receptor?

David M. Robertson, Ruth Hertan and Paul G. Farnworth

Prince Henry’s Institute of Medical Research, PO Box 5152, Clayton, Victoria, 3168, Australia

The receptor system and the molecular mechanisms by which inhibin acts on its target cells are poorly understood, in contrast to the situation for the structurally related molecule, activin. On the basis of evidence that the biological action of inhibin in a number of systems resembles that of an activin antagonist, it has been contended that inhibin operates by competition for the rather than through a specific inhibin receptor. However, mounting evidence indicates that inhibin also interacts with high affinity and specificity with membrane-binding proteins that are likely to be the putative inhibin receptor.

Inhibin, like other members of the transforming growth factor β accessory binding protein, (Barbara et al., 1999) binds (TGF-β) superfamily, is a disulphide-linked dimer of homol- the TGF-β, BMP-7 and activin type II receptor complexes, facili- ogous subunits that contain a characteristic set of seven cys- tating binding of each complex to its corresponding type I re- teine residues that form a cystine knot within each subunit. ceptor. As a variation, endoglin also binds BMP-2 in association Inhibin consists of α and either βA or βB subunits, while with its type I receptor. activins are dimers of the β subunits, which potentially include A contrasting example is an activin–BMP type I-like recep- other β subunit forms (that is, βC, βD and βE) (Woodruff and tor, which lacks the cytoplasmic tail and, hence, phosphokinase Mather, 1995). activity (BAMBI) (Onichtchouk et al., 1999). BAMBI is able to bind to an appropriate type II receptor–activin–BMP complex Receptors and binding proteins for members of the but is unable to transduce a signal and thus antagonises the actions of activin and BMP. TGF-β family In addition to binding proteins involved in signal transduc- A series of binding proteins has been associated with signalling tion, a number of soluble, secreted binding proteins have been by TGF-β, bone morphogenetic proteins (BMPs), Müllerian identified; these proteins show high affinity and specificity for inhibitory substance and activins (Gaddy-Kurten et al., 1995). particular TGF-β superfamily members and neutralize their Some of these binding proteins are involved directly with the biological activities. Binding proteins identified include the process, but the way in which the proteins latency-associated protein, which binds TGF-β; and interact is proving to be highly complex. It is generally agreed , which bind activin; and , gremlin, , cer- that two groups of binding proteins, called type I and type II berus, follistatin and DAN, which bind BMP (Hsu et al., 1998). receptors, can bind weakly or strongly to their respective TGF-β The action of each is attributable to competition for binding of superfamily member, but the combination is critical to trans- the respective ligand to its receptors. ducing a signal. In the case of activin, the ligand first binds A separate signalling mechanism is used by a distant to an activin type II receptor that incorporates a constitutively group of the TGF-β superfamily. Glial cell line-derived neuro- active serine–threonine kinase domain. The complex then inter- trophic factor (GDNF), persephin, artemin and neurturin acts with an activin type I receptor, which itself has low intrin- bind to specific accessory proteins devoid of phosphokinase sic affinity for activin. The union of type I and II receptors activity, termed GFRα1–3 (Soler et al., 1999). Each complex results in phosphorylation of the type I receptor which, thus then binds to a common receptor, RET. GFRα1–3 proteins activated, phosphorylates nuclear activating factors (Smads) differ from the recognized TGF-β family type II receptors specific to the pathway (Kawabata and Miyazono, 1999), lead- in their lack of structural homology and their glycolipid ing to nuclear activation. anchorage to the cell membrane. However, the most sig- In addition to type I and II receptors, studies with members nificant difference is that the ligand–GFRα–RET complex of the TGF-β superfamily have identified a number of associ- signals through the intrinsic tyrosine kinase activity of RET ated binding proteins thought to modify the interaction of each rather than through the serine–threonine kinase activity char- family member with its putative receptors. These associated acteristic of type I receptors for most other TGF-β super- proteins are localized to the cell surface and apparently lack family members. This finding establishes a precedent for signal transducing activity, but exhibit both high affinity and alternative signalling within the superfamily, raising the possi- specificity for the ligand. As an example, a large molecular bility that inhibin receptors differ from other TGF-β receptor weight protein called TGF-β receptor type III (or β-glycan; family members. Lopez-Casillas et al., 1993) has been identified which is thought These examples indicate many ways in which inhibin might to concentrate or orientate TGF-β at the cell surface. Another act, each involving one or more novel accessory binding © 2000 Journals of Reproduction and Fertility 1359-6004/2000 Downloaded from Bioscientifica.com at 09/25/2021 12:58:29PM via free access 132 D. M Robertson et al.

Model 1 Model 2

Activin Inhibin Inhibin β α α

β β Accessory protein β Competition

Recruitment ?? Recruitment

Inhibited by inhibin

Stimulatory Inhibitory Smad or Independent pathway Smad

Antagonism Inhibition Independent action

Fig. 1. Two proposed models of inhibin action. The first model involves competition between inhibin and activin β subunits for binding to activin type II receptor. As a consequence of inhibin binding, activin type I receptor recruitment is inhibited, and phosphorylation of stimu- latory Smads is prevented (Lebrun et al., 1999). The second model involves inhibin binding to an inhibin-preferring type II receptor that instead recruits an inhibin specific type I receptor. A number of inhibitory signals might then arise, for example, enhancement of inhibitory Smad action (Lebrun et al., 1999), or activation of a totally independent pathway. Accessory proteins may enhance the interaction of inhibin with receptors in either model.

proteins or specific classical type I and II receptors similar to the signalling type I receptor (Xu et al., 1995). Thus, inhibin those identified for other members of the TGF-β superfamily. may be viewed as a strong or weak competitive antagonist of activin, depending on the activin type II receptor subtype involved (type IIB > type IIB > type II; Martens et al., Models of the action of inhibin 2, 4 1, 3 1997). Irrespective of the final biological response, it follows There are two main hypotheses concerning the mechanism of from this hypothesis that inhibin should antagonise the action action of inhibin (summarized in Fig. 1). of activin, and other superfamily members (for example BMP-7; Yamashita et al., 1995) when they use activin type II Model 1: inhibin competes with activin for the activin receptor receptors, but that inhibin by itself should be inactive. Since inhibin and activin share a common βA or βB subunit, Model 2: inhibin binds specifically to its own receptor the β subunit of inhibin should bind to the activin type II re- ceptor. This hypothesis has been explored by several groups Several research groups have attempted to identify the who have shown that the interaction of inhibin with subtypes inhibin receptor by searching for proteins with sequence of the activin type II receptor (in particular type IIB) results in similarities in domains likely to be common to the TGF-β re- up to 40% competition for activin-binding (Lebrun and Vale ceptor family. Searches focused on either the serine–threonine et al., 1997; Martens et al., 1997). However, the α subunit, kinase domain, in which high homology between TGF-β re- although it is similar to the β subunits, is unable to recruit ceptor family members is evident, or the transmembrane or

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Table 1. Examples of the proposed mechanisms of action of inhibin

Proposed mechanism Cell system Action of Inhibin EC50 Action of Activin EC50 Activin + Reference of inhibin action inhibin alone (pmol l–1) activin alone (pmol l–1) inhibin

Via competition with DNA synthesis by None – Suppression 100 Antagonism Xu et al., 1995 activin receptor liver (HepG2) cells DNA synthesis by None – Suppression 120 Antagonism Gonzalez-Manchon ovarian (CHO-K1) and Vale, 1989 cells ACTH secretion by None – Suppression 20–30 No change Bilezikjian et al., 1991 pituitary (AtT20) cells

DNA synthesis by Limited 300 (EC25) Suppression 9 (EC25) Synergism Wang et al., 1996 prostate (LNCaP) suppression 16 (EC50) cells

Via a specific inhibin FSH release by Suppression 30 Stimulation 50 Antagonism P. Farnworth, C. Cahir receptor pituitary cells and P. Leembruggen, personal observations Follistatin synthesis Suppression 20–40 Stimulation 20–40 Antagonism Bilezikjian et al., 1996 by pituitary cells LH-induced androgen Stimulation 60 Suppression 50 Antagonism Hseuh et al., 1987 production by rat testicular and ovarian thecal cells Androgen production Stimulation 30–300 Suppression 300 Antagonism Hillier, 1991 by human thecal cells

EC50 value: concentration of ligand causing 50% of the maximum response. extracellular ligand-binding domains have not been fruitful to family. These binding proteins, although purified to homo- date (Woodruff, 1999). The failure to find a candidate inhibin geneity, were N-terminally blocked and, thus, it was not receptor by these approaches emphasizes the ability of inhibin possible to determine the sequences. Wang et al. (1999) identi- to compete for the activin receptor. fied an inhibin-binding protein with high specificity for inhibin An inhibin receptor would be characterized by interactions in bovine pituitary membranes; this protein may be a compo- with inhibin with high affinity and high specificity in the nent of the inhibin–receptor complex. Overall, these data picomolar concentration range with binding sites on target cells support the presence of high affinity and specific binding to generate a biological response. proteins for inhibin, although the studies concerned did not address the mechanism of action of inhibin. Inhibin-binding proteins Proposed mechanisms of action of inhibin Evidence of specific inhibin binding has been provided using autoradiography (Woodruff and Mather, 1995). Binding of On the basis of the above binding data, it appears that inhibin iodinated inhibin to ovarian, testicular and pituitary sections can act either as an activin receptor antagonist or by interaction is competed for by unlabelled inhibin but not activin. Studies with a specific inhibin receptor. The actions of inhibin in eight of inhibin binding to cells from the ovine anterior pituitary, inhibin and activin responsive systems are presented (Table 1) a known site of inhibin action, indicate the presence of two to characterize which mechanism applies in biological systems. –1 –1 inhibin-binding sites [Kd(1) = 0.3 nmol l ; Kd(2) = 4 nmol l ], In four of the chosen cell systems, inhibin alone is inactive or of β –1 showing low crossreactivity with activin (< 2%) and TGF- 1 limited activity (EC50 value > 300 pmol l ), activin is active (EC50 (< 0.2%) (Hertan et al., 1999). Other cell types not expected to value = 20–120 pmol l–1) and inhibin is able to antagonise the respond to inhibin show low inhibin binding. action of activin. Presumably, this pattern reflects competition Further evidence for the existence of inhibin receptors has for the activin receptor (Xu et al., 1995; Lebrun and Vale, 1997; been obtained by Draper et al. (1998) who isolated a series of Martens et al., 1997). In four other systems, inhibin is active –1 such proteins from ovarian cancer cells in mice in which the alone in the picomolar range (EC50 value = 30–300 pmol l ), α –1 inhibin subunit gene had been deleted. The molecular sizes is at least as potent as activin (EC50 value = 20–300 pmol l ), of the inhibin cross-linked binding proteins were reminiscent and also antagonises activin, which is consistent with the of those observed with other members of the TGF-β receptor involvement of an inhibin receptor. It is important to note

Downloaded from Bioscientifica.com at 09/25/2021 12:58:29PM via free access 134 D. M Robertson et al. that activin expression is widespread, implying that inhibin Work in the authors’ laboratories was funded by a Program Grant may inhibit the biological effects of endogenous activin as the (number 983212) of the National Health and Medical Research Council basis of an apparent inhibin-specific action. As yet, there is little of Australia. evidence of an inhibin action that is truly independent of activin. References Thus, in almost all circumstances in which inhibin acts, it is counteracted by and can counteract activin, which is consistent, Key references are identified by asterisks at least in part, with both proposed mechanisms of action. Barbara NP, Wrana JL and Letarte M (1999) Endoglin is an accessory protein that interacts with the signalling receptor complex of multiple members of However, the potency of inhibin mostly exceeds that expected the transforming growth factor-β superfamily Journal of Biological to arise from simple competition with activin for cell surface re- Chemistry 274 584–594 ceptors. It has been proposed that ancillary inhibin-binding Bilezikjian LM, Blount AL Campen CA, Gonzalez-Manchon C and Vale W proteins can intercede and increase the interaction of inhibin (1991) Activin-A inhibits proopiomelanocortin messenger RNA accumu- lation and adrenocorticotropin secretion of AtT20 cells Molecular with activin type II receptors, thus facilitating the competitive Endocrinology 5 1389–1395 mechanism (Fig. 1, Model 1; for example, see Lebrun and Vale, Bilezikjian LM, Corrigan AZ, Blount AL and Vale WW (1996) Pituitary fol- 1997). An example of such a mechanism was described by listatin and inhibin subunit messenger ribonucleic acid levels are differen- Lewis et al. (2000) in which β-glycan functions as a co-receptor tially regulated by local and hormonal factors Endocrinology 137 4277–4284 with activin type II receptor for inhibin binding. The affinity *Draper LB, Matzuk MM, Roberts VJ, Cox E, Weiss J, Mather JP and Woodruff TK (1998) Identification of an inhibin receptor in gonadal –1 of the resulting complex for inhibin (0.2 nmol l ) exceeds tumors from inhibin α-subunit knockout mice Journal of Biological that of either binding partner alone (0.6 and 6.3 nmol l–1, re- Chemistry 273 398–403 spectively). Activin competes poorly with inhibin for binding Gaddy-Kurten D, Tsuchida K and Vale WW (1995) Activins and receptor- to both β-glycan and the β-glycan–activin type II receptor com- serine kinase superfamily Recent Progress in Hormone Research 50 109–129 β Gonzalez-Manchon C and Vale W (1989) Activin-A, inhibin and trans- plex. Expression of -glycan by activin responsive cells, which forming growth factor-β modulate growth of two gonadal cell lines were previously insensitive to inhibin, results in a potent inhi- Endocrinology 125 1666–1672 bition of activin action. *Hertan R, Farnworth PG, Fitzsimmons KL and Robertson DM (1999) Alternatively, with the inhibin receptor mechanism (Fig. 1, Identification of high affinity binding sites for inhibin on ovine pituitary Model 2), mutual antagonism by inhibin and activin may cells in culture Endocrinology 140 6–12 Hillier SG (1991) Regulatory functions for inhibin and activin in human reflect crosstalk between separate but converging signal ovaries Journal of Endocrinology 131 171–175 transduction pathways. Since Smads identified to date can Hsueh AJW, Dahl KD, Vaughan J, Tucker E, Rivier J, Bardin CW and be stimulatory (Smads 1–3, 5 or 8 in combination with Smad 4) Vale W (1987) Heterodimers and homodimers of inhibin subunits have or inhibitory (Smads 6 and 7), inhibin may oppose the action different paracrine action in the modulation of luteinizing hormone- stimulated androgen biosynthesis Proceedings National Academy of Sciences of activin by using inhibitory Smads (Kawabata and Miyazono, USA 84 5082–5086 1999), or by dephosphorylating or differentially phosphoryl- Hsu DR, Economides AN, Wang X, Eimon PM and Harland PM (1998) The ating stimulatory Smads. A model in which inhibin binds an Xenopus dorsalizing factor Gremlin identifies a novel family of secreted activin type II receptor and recruits an inhibin-specific type I proteins that antagonise BMP activities Molecular Cell 1 673–683 β receptor represents a hybrid of the two proposed mechanisms. Kawabata M and Miyazono K (1999) Signal transduction of the TGF- super- family by Smad proteins Journal of Biochemistry 125 9–16 It is also conceivable that binding of inhibin to a distinct type II *Lebrun JJ and Vale WW (1997) have antagonistic effects receptor allows recruitment of the activin type I receptor to on ligand-dependent heteromerization of the type I and type II activin re- phosphorylate a unique inhibin signalling molecule. Finally, ceptors and human erythroid differentiation Molecular and Cellular Biology the case described by Wang et al. (1996) in which inhibin and 17 1682–1691 Lebrun JJ, Takabe K, Chen Y and Vale W (1999) Role of pathway-specific activin share the same action (Table 1) provides a challenge to and inhibitory Smads in activin receptor signaling Molecular Endocrinology existing models of inhibin action. 13 15–23 *Lewis KA, Gray PC, Blount AL, MacConell LA, Wiater E, Bilezikjian LM and Vale W (2000) Betaglycan binds inhibin and can mediate functional Conclusion antagonism of activin signalling Nature 404 411–414 Lopez-Casillas F, Wrana JL and Massague J (1993) Betaglycan presents Overall, the above data indicate that inhibin operates by two ligand to the TGFβ signalling receptor Cell 73 1435–1444 mechanisms: the first is based on competition of inhibin for *Martens JWM, de Winter JP, Timmerman MA, McLuskey A, van activin receptors, which results in an antagonistic action; the Schaik RHN, Themmen APN and de Jong FH (1997) Inhibin interferes second is by interaction with specific and high affinity re- with activin signaling at the level of the activin receptor complex in Chinese hamster ovary cells Endocrinology 138 2928–2936 ceptors, which activate signal transducing pathways that Onichtchouk D, Chen Y-G, Dosch R, Gawantka V, Delius H, Massague J oppose activin action. Additional inhibin-binding proteins and Niehrs C (1999) Silencing of TGF-β signalling by the pseudoreceptor may be involved in either process. The strongest evidence for BAMBI Nature 401 480–485 inhibin receptors is the coincidence in the pituitary and gonads Soler RM, Dolcet X, Encinas M, Egea J, Bayascas JR and Comella JX (1999) of potent inhibin actions and tissue-specific and inhibin-specific Receptors of the glial cell line-derived neurotrophic factor family of neurotrophic factors signal cell survival through the phosphatidylinositol high-affinity binding proteins. Progress in this field is likely to 3-kinase pathway in spinal cord motoneurons Journal of Neuroscience 19 depend on the isolation and characterization of the key pro- 9160–9169 teins involved in the interaction of inhibin with the target cell, Wang QF, Tilly KI, Preffer F, Schneyer AL, Crowley WF, Jr and Sluss PM and the discrimination of receptors from ancillary binding (1996) Activin inhibits basal and androgen-stimulated proliferation and induces apoptosis in the human prostatic cancer cell line, LNCaP proteins. These advances will clarify the signalling pathway Endocrinology 137 5476–5483 of inhibin and whether its action is dependent on activin or Wang EY, Gitch J, Chong H, Draper LB, Shanmugum S, Pangas SA, is unique. Santiago J and Woodruff TK (1999) The tissue distribution of a

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membrane-bound protein which interacts with inhibin Proceedings of the *Xu J, McKeehan K, Matsuzaki K and McKeehan WL (1995) Inhibin 81st Annual Meeting the Endocrine Society Abstract P3–489 antagonizes inhibition of liver cell growth by activin by a dominant- Woodruff TK (1999) Editorial: hope, hypothesis, and the inhibin receptor. negative mechanism Journal of Biological Chemistry 270 6308–6313 Does specific inhibin binding suggest there is a specific inhibin receptor? Yamashita H, ten Dijke P, Huylebroeck D, Sampath TK, Andries M, Endocrinology 140 3–5 Smith JC, Heldin CH and Miyazono K (1995) Osteogenic protein-1 binds Woodruff TK and Mather JP (1995) Inhibin, activin and the female reproduc- to activin type II receptors and induces certain activin-like effects Journal tive axis Annual Review of Physiology 57 219–244 of Cell Biology 130 217–226

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