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Endocrinology Vol. 141, No. 7 2281-2284
Copyright © 2000 by The Endocrine Society

ARTICLES

Editorial: In Search of Binding—Identification of Inhibin Receptors

Martin M. Matzuk, M.D., Ph.D.

Baylor College of Medicine Houston, Texas 77030

Address all correspondence and requests for reprints to: Martin M. Matzuk, M.D., Ph.D., Baylor College of Medicine, Department of Pathology, One Baylor Plaza, Houston, Texas 77030.


   Introduction
Top
 Introduction
References
 
"The future belongs to those that believe in the beauty of their dreams."— Eleanor Roosevelt

The cloning of the inhibin subunit genes in the mid-1980s sparked a passionate search for the inhibin receptors and the roles they play in inhibin action. Over this time frame, there have been major advances in understanding how activin signals, demonstration of inhibin binding sites in tissues, and isolation of inhibin receptor protein complexes (see Fig. 1Go). However, it was not until this year that two inhibin receptors were biochemically and molecularly identified.



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  		Figure 1. Model for signaling by inhibin and activin. Cells expressing p120 [inhibin receptor (IR)], betaglycan (BG), an activin type II receptor (II) such as ActRIIA or ActRIIB, and a TGF-ß superfamily type I receptor (I) such as ALK4 would have the full potential for inhibin and activin signaling. In the presence of an activin dimer (activin A, B, or AB), ActRIIA or ActRIIB will phosphorylate ALK4, which can subsequently phosphorylate Smad2 or Smad3, which together with Smad4 translocate to the nucleus to regulate target gene transcription. Additional transcription factors (TF) may be present in the complex to coregulate transcriptional responses. Smad6 and Smad7 are potential inhibitory Smads that could block the action of ALK4. Inhibin is hypothesized to play dual inhibitory roles in cells expressing BG and p120 to rapidly block activin signaling. One inhibin heterodimer (either inhibin A or inhibin B) would sequester all of the type II receptors through formation of a complex with betaglycan and ActRIIA or ActRIIB. This stable cell surface complex is suggested to be inert. At the same time, a second inhibin heterodimer would bind to p120 and ALK4 (or another type I receptor) to form a stable ternary complex that initiates its own unique signaling cascade. This second signaling pathway could function to block any residual activin signaling through activation of Smad6 or Smad7, via antagonism of transcription of activin-regulated genes, and/or through induction of its own distinct complement of inhibin-regulated genes.

 
The existence of a gonadal substance that played a negative regulatory role in pituitary function was demonstrated in the 1920s and 1930s (1, 2, 3). McCullagh (2) used the term "inhibin" to describe this inhibitory activity. With the advent of specific FSH immunoassays in the 1970s, gonadal fractions were identified which specifically inhibited (i.e. contained inhibins or follistatin) or stimulated (i.e. contained activins) FSH synthesis and secretion. These studies directly led to the identification and cloning of the cDNAs encoding the inhibin and activin subunits (4, 5, 6). At that time, it was shown that inhibins are heterodimers ({alpha}:ßA, {alpha}:ßB) that share a subunit with the activins (ßA:ßA, ßB:ßB, ßA:ßB). Surprisingly, these protein subunits had structural similarity to transforming growth factor-ß1 (TGFß1). The TGFß superfamily now contains over 35 members and is the largest family of secreted growth factors (7). The superfamily contains diverse members such as Müllerian-inhibiting substance (MIS), bone morphogenetic protein (BMP) 8a, BMP8b, and growth differentiation factor-9, which are all critical proteins in reproductive tract function (8, 9, 10, 11).

The next major breakthrough in the inhibin/activin field came in 1991 when Mathews and Vale cloned the first receptor that bound a TGFß superfamily protein (12). Using an expression cloning approach, they identified cDNAs encoding an activin type II receptor (also called ActRIIA). ActRIIA binds activin A and B with high affinity and inhibin A at a 10-fold lower affinity. It was called a type II receptor because it was larger (70–75 kDa) than type I receptors (50–55 kDa) based on nomenclature for TGFß binding studies. Eventually, type II receptors that bound other ligands in the superfamily were cloned as well as type I receptors. Interestingly, the majority of the TGF-ß superfamily ligands interact with these type I and type II receptors, which are transmembrane serine/threonine kinase receptors (13, 14). Activins bind either ActRIIA or ActRIIB, which subsequently recruit a type I receptor (activin-like kinase 4, ALK4) into the complex (see Fig. 1Go). Signal translation is initiated when the type II receptor phosphorylates the type I receptor in the GS box (a serine-rich region near the transmembrane region). The phosphorylated (activated) type I receptor subsequently phosphorylates downstream proteins including Smads (homologs of the C. elegans sma and D. melanogaster MAD proteins). Smad2 and Smad3 are phosphorylated by both activin and TGFß receptors and transduce the signal from the cytoplasm to the nucleus through an interaction with Smad4. Physiological confirmation that ActRIIA was a receptor for activin came through our demonstration that ActRIIA knockout mice have dramatically suppressed FSH, which causes a decrease in testis and ovary size and a block in ovarian folliculogenesis (15). These findings also demonstrated that ActRIIA is the major activin type II receptor for activin signal transduction in pituitary gonadotrophs. Although in vitro biochemical data have demonstrated convincingly that ALK4 functions as a type I receptor in activin signaling, in vivo data are lacking because absence of ALK4 in mice leads to early embryonic lethality, likely due to ALK4’s involvement in other TGF-ß superfamily signaling pathways (16).

In parallel with these activin signal transduction studies, Woodruff and colleagues used in situ ligand binding studies to show specific and distinct binding of iodinated recombinant inhibin A and activin A to ovarian and testicular sections (17, 18). At the same time, we demonstrated that knockout mice lacking the {alpha} subunit of inhibin had elevated serum FSH and developed granulosa/Sertoli cell tumors of the gonads (19) and adrenal tumors (20). The finding that inhibin could function both locally (19), and at a distance (21) as a tumor suppressor suggested that in the absence of inhibin ligand, these ovarian tumors (which could be as large as 1 g) could be an abundant source of inhibin receptors. In collaboration with the Woodruff laboratory (22), we confirmed this hypothesis. Ligand binding studies demonstrated specific binding of 125I-inhibin to tumor sections that could not be competed by activin. Although ActRIIA binds inhibin at 10-fold lower affinity than activin, the lack of competition by activin suggested that this receptor was not ActRIIA and instead was a unique inhibin receptor. Furthermore, a putative inhibin receptor could be purified from the tumor extracts using an inhibin affinity column. Unfortunately, the N-terminus of this receptor was blocked, and there was insufficient material from the tumors to obtain internal peptide sequence. Other groups also demonstrated inhibin binding sites in other tissues besides the gonads. Several studies confirmed that inhibin could directly antagonize activin through its binding to ActRIIA (23, 24, 25). More recently, high affinity binding sites for inhibin on ovine pituitary cells were detected (26). Two distinct binding sites with 10-fold different affinities were demonstrated. These observations further confirmed that at least one specific inhibin receptor exists in both the pituitary and the gonads.

If one wants to identify a cell surface receptor for a protein ligand, the four major approaches are: 1) Use an expression cloning strategy (similar to the cloning of the activin/TGFß type II receptors); 2) Use a candidate receptor approach (i.e. try binding to known receptors); 3) Attempt a biochemical (classical) approach using an abundant source of tissue, quite often from a large species such as the cow; or 4) Use a PCR-based or low stringency hybridization strategy to try to clone a receptor based on structural similarity to known receptors (as was performed for the MIS type II receptor). The second and third approaches turned out to be successful for the Vale and Woodruff groups, respectively. Based on their papers, it is obvious why approach 4 did not work (i.e. the inhibin receptors are structurally unrelated to the other receptor proteins in the family).

In the paper by Vale and colleagues (27), betaglycan, which was shown previously to function as an abundant type III receptor for TGFß, binds inhibin with high affinity. Furthermore, betaglycan and ActRIIA together, on the same cell, enhance the binding of inhibin. The fairly ubiquitous nature of betaglycan suggests that inhibin could theoretically bind to this receptor and block the effect of activins in multiple tissues.

The classical approach was taken by Woodruff and colleagues in this issue of Endocrinology (28) to isolate the inhibin receptor. Using 500 g of bovine pituitaries and an inhibin affinity chromatography column, over 20 bound proteins were eluted. One protein of approximately 120 kDa was specifically competed by inhibin for binding to the column, and 17 amino acids of peptide sequences were obtained for the protein, called p120. Interestingly, the bovine sequence exactly matched two human sequences coding for a 1336 amino acid immunoglobulin superfamily/cell adhesion protein. Northern blot and immunohistochemical analysis of rat tissues showed that the receptor is appropriately expressed in gonadotrophs of the pituitary and Leydig and Sertoli cells of the testes. Likewise, Leydig cells bound 125I-inhibin specifically, suggesting a tissue-restricted compartmentalization of inhibin and activin receptors. Although evidence for in vitro binding of inhibin to expressed p120 is not presented, the cellular localization, the structure of the protein, and the initial purification data are highly suggestive that a novel transmembrane inhibin receptor had been isolated.

An interesting finding about the p120 gene is that it maps to human chromosome Xq25. This chromosomal location suggests several putative in vivo functions of the p120 inhibin receptor. First, this region is associated with pan-hypopituitarism in humans. Second, X-chromosome deletion syndromes also implicate this same region in premature ovarian failure. Third, loss of heterozygosity of this region is frequently associated with ovarian epithelial cancer. Thus, absence and mutations of p120 by itself or in combination with other genes in this region might be associated with three major gynecological disorders.

Our previous studies of the {alpha}-inhibin knockout male mouse failed to demonstrate any primary defects in the Leydig cells (i.e. males were initially fertile and seminal vesicles were prominent suggestive of normal testosterone production), and the mice subsequently developed Sertoli cell tumors. How might one rationalize the high binding of inhibin to Leydig cells and the presence of p120 in these cells? One possibility is that the development of the Sertoli tumors is an indirect process. Inhibin may normally induce a Leydig cell factor that suppresses Sertoli cell proliferation; in the absence of inhibin, the suppressor is absent and Sertoli cell tumors develop. Interestingly, male mice lacking MIS or the MIS receptor develop Leydig cell hyperplasia and rare Leydig tumors in the late adult stage (8, 29). However, mice lacking both inhibin and MIS develop nonfunctional, multifocal Leydig cell tumors that arise as early as 1 week of age (30). Thus, both MIS and inhibin function synergistically to promote Leydig cell tumorigenesis. Although this process is still unclear, it is exciting to speculate on the interactions between MIS, inhibin, the type II MIS receptor, type I receptors, and p120 in a common negative regulatory pathway in Leydig cells.

Are betaglycan, p120, and type II activin receptors physiologic receptors for inhibin? One intriguing model in cells that express all of these receptors is presented in the figure. Activin binds ActRIIA and ALK4 to stimulate one signal transduction cascade. Alternatively, inhibin, by binding betaglycan and ActRIIA, could generate a nonfunctional complex and block the activin pathway. At the same time, another inhibin ligand could bind p120 and a type I receptor (e.g. ALK4) or another signal transducing transmembrane protein to stimulate its own pathway. In this way, inhibin could act quickly and precisely to antagonize the effect of activin and exert its own effects. This is an attractive model encompassing the roles of activin and inhibin in the gonadotrophs, testes, and ovaries where precise control is needed and where antagonist functions of inhibins and activins have been identified. In other tissues, single components of this regulatory system could be expressed where precise action of activin or inhibin are unnecessary or where only an on/off switch is required for activin signaling.

Several questions still need to be answered with regard to inhibin, p120, and betaglycan functions. For example, are p120 and betaglycan the only inhibin receptor in reproductive tissues? Are there independent receptors that can distinguish between the effects of inhibin A and inhibin B in the ovaries or testes? Do p120 knockout mice mimic any human X-chromosome-linked syndromes and develop gonadal tumors similar to inhibin {alpha} knockout mice? Do p120, betaglycan, type II receptors, and type I receptors interact physiologically in the same cells as suggested by the model? Clearly, the beauty of the dreams of the past decade will help to address these important questions in the years to come.

Received May 4, 2000.


   References
Top
 Introduction
 References
 

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  2. McCullagh DR 1932 Dual endocrine activity of the testes. Science 76:19–20[Free Full Text]
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  5. Mason AJ, Hayflick JS, Ling N, Esch F, Ueno N, Ying S-Y, Guillemin R, Niall H, Seeburg PH 1985 Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-b. Nature 318:659–663[CrossRef][Medline]
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  7. Dube JL, Wang P, Elvin J, Lyons KM, Celeste AJ, Matzuk MM 1998 The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes. Mol Endocrinol 12:1809–1817[Abstract/Free Full Text]
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  16. Gu Z, Nomura M, Simpson BB, Lei H, Feijen A, van den Eijnden-van Raaij J, Donahoe PK, Li E 1998 The type I activin receptor ActRIIB is required for egg cylinder organization and gastrulation in the mouse. Genes Dev 12:844–857[Abstract/Free Full Text]
  17. Krummen LA, Moore A, Woodruff TK, Covello R, Taylor R, Working P, Mather JP 1994 Localization of inhibin and activin binding sites in the testis during development by in situ ligand binding. Biol Reprod 50:734–744[Abstract]
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  19. Matzuk MM, Finegold MJ, Su J-GJ, Hsueh AJW, Bradley A 1992 {alpha}-Inhibin is a tumor-suppressor gene with gonadal specificity in mice. Nature 360:313–319[CrossRef][Medline]
  20. Matzuk MM, Finegold MJ, Mather JP, Krummen L, Lu H, Bradley A 1994 Development of cancer cachexia-like syndrome and adrenal tumors in inhibin-deficient mice. Proc Natl Acad Sci USA 91:8817–8821[Abstract/Free Full Text]
  21. Pierson MP, DeMayo FJ, Matzuk MM, Tsai SY, O’Malley BW Regulable expression of inhibin A in wild-type and inhibin a null mice. Mol Endocrinol, in press
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  23. Lebrun JJ, Vale WW 1997 Activin and inhibin have antagonistic effects on ligand-dependent heteromerization of the type I and type II activin receptors and human erythroid differentiation. Mol Cell Biol 17:1682–1691[Abstract]
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