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Northwestern University
Address all correspondence and requests for reprints to: Teresa K. Woodruff, Ph.D., Northwestern University, Department of Neurobiology and Physiology, 2153 North Campus Drive, Hogan Building 4–150, Evanston, Illinois 60608.
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Introduction |
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 Top Introduction References |
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Inhibin is a gonadal-derived hormone that inhibits pituitary FSH in a
cycle-dependent manner in females and maintains tonic FSH production in
males (2). Structurally, inhibin is a disulfide-linked dimeric
glycoprotein composed of an 
-subunit and a ß-subunit. One gene
encodes the -subunit. The ß-subunit is encoded by at least four
different genes in mammals. The four mammalian ß-subunits
(ßA, ßB, ßc,
ßE) are expressed in restricted cellular sites. The
ßA and ßB-subunits are coexpressed with
-subunits in the ovary leading to the biosynthesis of inhibin A
(-ßA) and inhibin B (-ßB). In the
testis, the ßB-subunit is coexpressed with the
-subunit, leading to the exclusive production of inhibin B in the
male. The inverse relationship between ovarian inhibins and FSH forms a
classic endocrine negative feedback system that is established in the
prepubertal years leading to the first ovulation, is maintained in a
cycle-dependent manner during the reproductive lifespan, and is lost
following follicular exhaustion in the postmenopausal years.
Inappropriate inhibin signals in the primate result in an imbalance
between LH and FSH, a delay in the follicular phase, and luteal phase
deficiency (3, 4). Absence of inhibin in animals genetically deficient
in the ligand and results in unopposed FSH secretion and abnormal
follicle growth leading to tumor formation (5).
Inhibin is a ligand within the transforming growth factor-ß (TGFß)
superfamily of proteins (6). The TGFß superfamily is a large and
complex group of dimeric ligands that regulates neuronal growth, bone
morphogenesis, oocyte growth, limb formation, pattern formation, and
many other cellular metabolic and secretory functions. TGFß homologs
are distributed from C. elegans through human with equally
conserved cell surface receptors and signaling coregulators. Many of
the advances in the identification of receptor and signaling molecules
for the TGFß superfamily have come from conserved functions,
particularly in early development, and easily manipulated genetics of
lower organisms. To date, no ortholog for inhibin -subunit has been
identified outside of mammals, suggesting that this subunit and the
dimeric inhibin protein may have recently emerged to fulfill the
demands of an endocrine-driven reproductive axis.
Activin is a homodimer of the inhibin ß-subunits (7). It was originally identified as a functional antagonist to inhibin; however, the cellular systems that respond to activin are more diverse than the systems that are regulated by inhibin. Activin regulates hormonogenesis (FSH, GH, ACTH, insulin), cellular homeostasis (divide or die pathways), and differentiation programs (during development and in adult cells). Cellular response to activin is transduced through two single membrane spanning serine-threonine kinase subunits (7). Each ligand in the TGFß superfamily binds to a receptor in a ligand discriminate way. Generally speaking, ligand binds a type II receptor (70–75 kDa) that transphosphorylates a type I receptor (50–55 kDa). The holo-receptor complex is then competent to initiate intracellular signaling cascades (6, 7). Inhibin is able to bind the activin type II receptor with low affinity; however, the activin type I receptor is not recruited into a binary complex (8, 9, 10).
Although our knowledge regarding activin action has expanded tremendously in the last several years, progress in understanding inhibin actions has been comparatively slow. The slower pace of advance in the elucidation of inhibin cellular mechanisms is due to: 1) mammalian restricted molecules; 2) the difficulty in purification of native inhibin and expression and purification of recombinant inhibin; 3) the restricted set of inhibin target tissues including highly differentiated cells of the reproductive system; 4) lack of a known inhibin receptor; and 5) target genes, whose expression is modulated by inhibin, have not yet been identified. Within the past year, several of these obstacles have been addressed and tools are becoming available to illuminate the inhibin signal transduction pathway. Specifically, rh-inhibin A has been produced, is biologically active, and is currently available through the NHPP; independent inhibin cellular binding sites have been identified in the gonads, pituitary, and in tumors derived from inhibin knockout mice; and, inhibin receptor proteins have been identified in tumor tissue membrane extracts. These studies prepare the way for a detailed analysis of inhibin signal transduction pathways.
Several criteria must be met to clearly prove that an inhibin receptor exists. First, it must be specific for inhibin and not activin or other members of the TGF superfamily. In addition, it must be expressed in pituitary gonadotropes. Sites outside the gonadotrope are likely; however, at a minimum, the gonadotrope must be receptor bearing. Third, the receptor must be of high affinity. The amount of inhibin that reaches the pituitary is in the pg/ml range, indicating that the receptor must have high affinity for ligand. Last, the inhibin receptor or its signaling targets must provide a mechanism for functional antagonism with activin but allow for independent activity; in other words, it should connect to a signaling pathway.
In the current issue, Hertan et al. (1) provide evidence that inhibin-selective binding sites are present on sheep pituitary cells. Two binding sites with binding constants of 280 and 3900 pM have been identified, satisfying the affinity requirement mandated by the physiology of inhibin in the pituitary. Moreover, the binding sites are saturable and specific for inhibin A. These data are strong evidence that an inhibin receptor exists. However, until the receptor is examined biochemically and its complementary DNA cloned, we can only speculate about the molecular characteristics of the inhibin receptor. Before this study, many laboratories, including our own, began the search for an inhibin receptor through molecular cloning of complementary DNAs related to the activin/TGFß family of receptors. Degenerate oligonucleotides directed against the serine-threonine kinase domain, the transmembrane domain, and the extracellular ligand binding domain were used in a variety of experimental paradigms. However, these efforts were not successful in identifying novel receptors. We therefore turned to an alternative approach that did not rely on assumptions that the inhibin receptor would be related to the activin receptor and began a biochemical characterization of inhibin-binding proteins. A breakthrough came when we identified the ovarian and testicular tumors derived from inhibin-deficient mice as a tissue source replete in inhibin binding and deficient in activin binding sites (11). This observation led to a biochemical purification strategy and identification of a separable inhibin-binding moiety in ovarian tumors and, more recently, in bovine pituitaries. The important study by Hertan et al. and the previous purification studies support the hypothesis that inhibin activity involves an inhibin specific receptor.
How might an inhibin-specific receptor regulate cellular function? It is possible that an independent, membrane-bound inhibin protein exists that binds inhibin and transduces a signal. This hypothesis is supported by two pieces of evidence: 1) inhibin-specific binding sites exist in normal and tumor tissue (11, 12, 13); and, 2) inhibin binding proteins can be purified from tissues exhibiting inhibin-specific binding sites (11). The signaling component of the inhibin receptor may be a serine-threonine kinase, similar to the signaling domains of the activin/TGFß receptors. However, based on the inability to clone additional members of the receptor serine-threonine kinase family, it appears unlikely that the inhibin receptor will be a serine-threonine kinase. A negative result cannot be used as compelling evidence that inhibin receptors are not serine-threonine kinase proteins; however, it stimulates us to think more broadly about the molecular characteristics of the inhibin receptor.
In some systems, no independent inhibin receptor may be present. Inhibin, in this case, could act by dominant negative regulation of activin signal transduction pathways. Evidence that inhibin can act in this manner is provided by in vitro studies using activin type II and type I receptors (8, 9, 10). Inhibin A, through its ßA-subunit, is able to bind to activin type II receptor with low affinity and binding blocks activin interaction with its receptor, thus uncoupling downstream signal transduction pathways. This pathway is valid only if inhibin is in excess of activin to overcome the affinity differences between the two ligands, and if the action of activin on an inhibin-regulated event is constitutively active. And, perhaps, if the cell lacks an inhibin receptor-like protein. We view this prediction as compelling for in vitro constructed test systems but less likely in vivo for three reasons. First, it is unlikely that inhibin is in excess of activin throughout the ovulatory cycle (particularly in early estrus or follicular phase when FSH is inhibited by a small rise in ovarian inhibin). Second, inhibin B binds the activin A receptor with a much lower affinity than inhibin A. Therefore, the ability of inhibin B to modulate activin A receptors in a biologically relevant manner is unlikely. Does inhibin B have a separate receptor? This issue is important and can only be addressed after the identification of an inhibin A receptor. Third, dominant negative repression of gene activation suggests that inhibin is a "brake" in persistent activin cellular action. While plausible in the pituitary, activin is not persistently active in the ovary; therefore, additional models of inhibin action much be generated. Last, neither the inhibin binding sites demonstrated in the current study nor the proteins that were isolated from the inhibin knockout tumors bind activin A, indicating that they differ from the type II subunit (1, 11).
A third hypothesis is that the inhibin receptor is an ancillary docking protein that causes activin receptor to become responsive to the antagonist. This hypothesis is true for two isoforms of TGFß. Similar to activin, TGFß signals through a binding TGFß-specific type II receptor and a signaling type I receptor. Five TGFß isoforms are synthesized by various cells, and TGFß1 and TGFß2 are the major forms studied. TGFß1 binds directly to type II and activates type I (6, 7). However, TGFß2 requires an additional ancillary protein, a single membrane spanning, nonsignaling protein (type III), that presents TGFß2 to the type II-type I complex (12). Is an analogous ancillary protein necessary for inhibin activation of cellular mechanisms? An activin type III receptor has been identified in endothelial cells and can be assembled with activin A into a type III-type II-type I complex (13). Recent data in the hematopoietic cell line, K562, is suggestive of a requirement for an ancillary protein that permits an inhibin function in this cell type (14). The ancillary activin-receptor docking protein as determinant for inhibin action is feasible and testable. Ancillary proteins may assemble complexes of activin type II and type I receptors into a configuration that is not only antagonistic to an intrinsic activin signal but also inhibin signal transducing.
The last, and most provocative hypothesis is that inhibin receptor is fundamentally different from activin or TGFß receptor complexes and is, instead, analogous to the glial-derived nerve growth factor (GDNF) receptor. GDNF is the most distant ligand of the TGFß superfamily, and it binds a nonsignaling GPI coupled protein that then presents the ligand to the ret-tyrosine kinase coupled receptor (15). The evidence that inhibin might signal through a completely different signal transduction pathway is incomplete because we lack the molecular tools to precisely probe this problem.
Hertan et al. have contributed an important study that lends substantial validity to the hypothesis that a true inhibin receptor exists. It appears that the field of inhibin biology is on the edge of finally gaining an elusive member of its molecular family, a bona fide receptor.
Received October 29, 1998.
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References |
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TopIntroductionReferences |
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- subunit knockout mice. J Biol Chem 273:398–403
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D. J. Bernard and T. K. Woodruff Inhibin Binding Protein in Rats: Alternative Transcripts and Regulation in the Pituitary across the Estrous Cycle Mol. Endocrinol., April 1, 2001; 15(4): 654 - 667. [Abstract] [Full Text]
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S. C. Chapman and T. K. Woodruff Modulation of Activin Signal Transduction by Inhibin B and Inhibin-Binding Protein (InhBP) Mol. Endocrinol., April 1, 2001; 15(4): 668 - 679. [Abstract] [Full Text]
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 R.L. Jones, L.A. Salamonsen, H.O.D. Critchley, P.A.W. Rogers, B. Affandi, and J.K. Findlay Inhibin and activin subunits are differentially expressed in endometrial cells and leukocytes during the menstrual cycle, in early pregnancy and in women using progestin-only contraception Mol. Hum. Reprod., December 1, 2000; 6(12): 1107 - 1117. [Abstract] [Full Text] [PDF]
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