Department of Neurobiology and Physiology (D.J.B., S.C.C., T.K.W.), Northwestern University, Evanston, Illinois 60208; Department of Medicine (T.K.W.), Northwestern University Medical School, Chicago, Illinois 60657
Address all correspondence and requests for reprints to: Teresa K. Woodruff, Ph.D., Department of Neurobiology and Physiology, Northwestern University, 2153 North Campus Drive, Evanston, Illinois 60208. E-mail: tkw{at}northwestern.edu
ABSTRACT
Betaglycan (the TGFß type III receptor) and InhBP/p120 (a membrane-tethered proteoglycan) were recently identified as putative inhibin receptors. Here, we review the current state of knowledge regarding these two proteins with respect to their potential roles in inhibin biology. Importantly, neither protein appears to satisfy all of the criteria required for classification as a bona fide inhibin receptor. Betaglycan does not appear to be expressed in pituitary gonadotropes, the primary target of circulating inhibins, and InhBP/p120 does not bind inhibins in conventional receptor binding assays. While both proteins appear capable of promoting inhibin-mediated antagonism of activin signaling, neither appears to generate inhibin-specific intracellular signals. Recently, additional inhibin binding proteins were identified in inhibin target tissues, including pituitary and Leydig cells. Characterization of these proteins, coupled with ongoing investigations of betaglycan and InhBP/p120, will lead to a clearer understanding of mechanisms of inhibin action.
Why Must There Be an Inhibin Receptor?
Ligands in the TGFß superfamily play diverse roles in a variety of physiological processes including growth, differentiation, and hormone secretion. Over the past decade, a general model of how these ligands affect target cells has emerged. The dimeric ligand binds a transmembrane serine/threonine kinase (the type II receptor), which then recruits and trans-phosphorylates a second serine/threonine kinase, the type I receptor. The activated type I receptor then phosphorylates intracellular signaling proteins of the Smad family. Activated Smads associate with a co-Smad and translocate to the nucleus where they interact with coactivators and corepressors to affect target gene transcription (1, 2, 3). While there are variations to this general signaling theme, the TGFßs, activins, bone morphogenetic proteins, and Müllerian-inhibiting substance all appear to use this dual receptor system to initiate intracellular signaling in target cells.
Inhibins are protein hormones that are produced primarily in the gonads
and that function to down-regulate pituitary FSH synthesis and
secretion. Inhibins are composed of an -subunit and one of two
ß-subunits (ßA or ßB), with
-ßA and
-ßB dimers forming
inhibin A and inhibin B, respectively. Dimers of the ß subunits form
the activins. Despite (or perhaps because of) the sharing of the ß
subunits, the activins and inhibins are functional antagonists in most
physiological contexts (4, 5). Although inhibins are part
of the larger TGFß superfamily, attempts to identify
inhibin-specific type I and type II receptors have been unsuccessful
(6). Inhibins can, however, bind activin type II receptors
through their ß-subunits. This binding does not lead to recruitment
or phosphorylation of the type I receptor and thereby provides a
mechanism for the inhibins to antagonize the actions of the activins
(Refs. 7, 8, 9, 10, 11).
Inhibin As affinity for the activin type II receptor isoforms (ActRIIA and ActRIIB) is 2- to 10-fold lower than that of activin A, suggesting that inhibins must be present in large excess of activins for antagonism to occur (7, 9, 10). This scenario is at odds with several systems in which inhibin A can antagonize activin A actions when at equimolar or even lower concentrations (12, 13, 14, 15). In addition, some activin-responsive cell lines are insensitive to inhibins, even at very high levels. This suggests that competitive binding to activin type II receptors alone cannot account for the actions of the inhibins and that additional inhibin binding proteins must be involved (8, 16). Consistent with this idea, nonoverlapping activin A and inhibin A binding sites have been observed in various tissues (17, 18, 19). These specific inhibin receptors or binding proteins may act to increase the affinity of the inhibins for the activin type II receptors or may propagate inhibin-specific signal transduction events.
Candidate Inhibin Receptor Proteins
Recently, two proteins were identified as candidate inhibin receptors: betaglycan and inhibin binding protein/p120 (InhBP/p120) (20, 21). Betaglycan was originally characterized as the TGFß type III receptor (22, 23). The amino acid sequence predicts a single transmembrane-spanning proteoglycan with a large ectodomain and a short cytoplasmic domain devoid of any signaling motifs. The protein was shown to bind TGFß1-3 with high affinity and to be necessary for high affinity association of TGFß2 with its type II receptor, TßRII (24, 25, 26). Surprisingly, betaglycan was also found to bind inhibin A with high affinity, suggesting a potential role for this protein in the inhibin receptor complex (20). InhBP/p120 was purified from bovine pituitary membrane extracts by its affinity for inhibin A (21). Like betaglycan, InhBP/p120 is a large transmembrane protein with a short, kinase-deficient cytoplasmic tail. Its large ectodomain contains 12 C-type Ig-like domains organized in groups of five and seven loops. Its high level of expression in the pituitary gland and its ability to bind inhibin A in an affinity column suggested a role for this protein in the inhibin receptor system.
We previously identified several criteria that need to be satisfied to
designate a candidate protein as a bona fide inhibin
receptor (6). The protein: must be expressed in inhibin
target tissues, particularly within the gonadotrope cells of the
anterior pituitary; must bind the inhibins with high affinity and with
high specificity; and must provide a mechanism for the inhibins to
antagonize the activins as well as provide a mechanism for independent
inhibin-mediated signaling. Here, we review data collected over the
past year and a half that enable us to assess the extents to which
betaglycan and InhBP/p120 satisfy these criteria (see Table 1).
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Given the potent inhibitory effects of the inhibins on FSH synthesis and secretion, FSH-producing gonadotropes are generally regarded as the canonical inhibin target cells. Therefore, one would predict that inhibin receptors must be expressed within this cell type. Northern blot analyses indicate that both betaglycan and InhBP/p120 are expressed in rat pituitary. In situ hybridization analyses further show that, at least for InhBP/p120, this expression is limited to the anterior pituitary (27). Preliminary immunohistochemical data indicated that InhBP/p120 was expressed in FSHß-producing gonadotropes (21), but the currently available antisera have been unreliable in producing consistent results in this regard (Bernard, D. J., and T. K. Woodruff, unpublished data). Nonetheless, the in situ hybridization data suggest that InhBP/p120 is likely expressed within multiple cell types of the anterior pituitary, including gonadotropes. In addition, pituitary InhBP/p120 expression is regulated across the rat estrous cycle and is inversely related to serum FSH levels (27).
Betaglycan is expressed highly within the intermediate lobe and is also expressed in some cells of the anterior pituitary. Importantly, however, we have never observed detectable expression of betaglycan in gonadotropes. This is true whether male or female tissue is analyzed or whether gonadotropes are labeled with FSHß or LHß polyclonal antisera (Ref. 27 and our unpublished results). However, it should be noted that two reports referring to unpublished data indicate that betaglycan is expressed in rat gonadotropes (20, 28). Also, others and we have detected betaglycan expression in the mouse gonadotrope cell line, LßT2 (20). The cause of these apparently discrepant results is not yet known, but clarification of this issue will be critical to understanding the role of betaglycan in inhibin-mediated FSH regulation.
High affinity inhibin A binding has been reported in rat and ovine pituitary cells in culture (29, 30), although the identity of the specific binding proteins has not yet been reported. In addition, murine adrenal AC cells and rat primary adrenal cells bind inhibin A with high affinity (30). We have observed high levels of expression of betaglycan as well as a novel splice variant of InhBP/p120 in rat adrenals; however, it is not yet known if either or both of these proteins contribute to inhibin A binding in this tissue (Ref. 27 and our unpublished results). Betaglycan immunoreactivity has also been reported throughout the rat ovary, including theca and granulosa cell layers and within the oocyte (20, 31). Previous in situ ligand binding data indicate that inhibin A binds specifically to antral granulosa cells of large preovulatory follicles, but not to other ovarian cell types (17). It is, therefore, unclear whether betaglycan functions as an inhibin binding protein in theca cells and oocytes. InhBP/p120 is expressed at low levels in the rat ovary, but the specific cell types expressing this protein have not yet been determined.
Inhibins and activins bind distinct proteins in a variety of tissues.
For example, iodinated inhibin A binds strongly to testicular Leydig
cells in rats, whereas iodinated activin A binds preferentially to germ
cells within the seminiferous tubules (18). Similarly,
tumors from inhibin knockout mice bind inhibin A strongly, but bind
activin A only weakly (19). Consistent with their presumed
roles as inhibin receptors, both betaglycan and InhBP/p120 are
expressed in rat Leydig cells (20, 21). Unlike the case
for rats, however, InhBP/p120 is not expressed in adult mouse testis
(but see Ref. 32), nor is it expressed at detectable
levels in gonadal tumors of inhibin
knockout mice (Bernard, D.
J., K. H. Burns, M. M. Matzuk, and T. K. Woodruff, unpublished
results). The mouse gonadal cell lines, TM3 and TM4, bind inhibin A
with high affinity, but do not express InhBP/p120 (32).
Betaglycan, on the other hand, is expressed in both TM3 and TM4 cells
and is part of a high affinity inhibin A binding complex in these
cells. While betaglycan is expressed in wild-type mouse testes and
ovaries, its expression in inhibin
knockout gonadal tumors does not
appear to be up-regulated, as one might predict given the increased
inhibin A binding observed in these tissues (19 ; Bernard,
D. J., K. H. Burns, M. M. Matzuk, and T. K. Woodruff,
unpublished results). Consistent with these observations, the predicted
sizes of the inhibin binding proteins in the inhibin
-subunit
knockout tumors (40, 54, 84, and 98 kDa) do not correspond to either
betaglycan (>110 kDa) or InhBP/p120 (
140 kDa)
(19).
Taken together, these data indicate that both betaglycan and InhBP/p120 are expressed in some but not all inhibin target tissues or cells. This suggests that these proteins may participate in the actions of the inhibins in some but not all contexts and that additional inhibin binding proteins or receptors must exist (see below).
Binding Affinity and Specificity
Although originally identified as a TGFß coreceptor protein, it is clear that betaglycan also binds inhibin A, and does so with high affinity (20). Cells transfected with betaglycan alone bind inhibin A with a Ki of 600 pM. This affinity is increased 3-fold in the presence of ActRIIA (20). The coexpression of betaglycan and ActRIIA or ActRIIB also increases total inhibin A or B binding in transfected cells (20, 33 ; Chapman, S. C., D. J. Bernard, and T. K. Woodruff, in preparation). Interestingly, inhibin A binding experiments in cultured ovine and rat pituitary cells indicate the presence of both high (280310 pM) and low affinity (3.95.3 nM) inhibin binding sites (29, 30). These binding affinities are similar to those reported for cells transfected with ActRIIA either alone (6.3 nM) or in conjunction with betaglycan (200 pM) (20). Whether these receptor combinations actually reflect the inhibin A binding sites in the pituitary remains to be determined, but it is worth noting that inhibin A can form a complex with ActRIIA and betaglycan expressed endogenously in the murine LßT2 gonadotrope cell line (20).
The extracellular region of betaglycan has domains that share sequence conservation with endoglin (the E-domain) and uromodulin (the U-domain). While TGFß ligands bind betaglycan in both the E- and U-domains, inhibin A appears to bind uniquely to the U-domain. In the absence of ActRIIA, this binding can be competed by TGFß2, and to a lesser extent by TGFß1 (33). However, in the presence of ActRIIA, inhibin A forms a strong ternary complex with betaglycan and the type II receptor, which is resistant to competition by activin A or TGFß1 (20). Thus, while inhibin A binding to betaglycan is not specific, it is of high affinity and, in the presence of ActRIIA, can prevent activin A binding to its type II receptor. Whether the inhibins can bind uromodulin has not yet been determined.
InhBP/p120 was initially identified by its ability to bind an inhibin A affinity column and by the fact that this binding was competed by preincubation with inhibin A (21). In addition, in in situ ligand binding studies, transfection of the full-length human InhBP/p120 conferred inhibin A and inhibin B binding to cells that under normal conditions do not bind either ligand (34). More recently, however, we have been unable to show specific binding of iodinated inhibin A or inhibin B to cells transfected with InhBP/p120 in standard competition and saturation binding studies (Chapman, S. C., D. J. Bernard, and T. K. Woodruff, in preparation). There were differences between the experimental paradigms employed in these studies, however, which may account for the discrepant results. For example, in the in situ ligand binding experiments, transfected CV-1 cells were incubated at room temperature overnight with iodinated inhibin A or inhibin B (34). In the competition binding studies, transfected COS-7 cells were incubated with the iodinated ligands for 4 h on ice. Both the time and temperature of incubation can affect iodinated inhibin binding (30). Moreover, it is possible that InhBP/p120 requires additional proteins to form a high affinity association with the inhibins, and these proteins may be more highly expressed in CV-1 than in COS-7 cells. Nevertheless, under the same binding conditions, we observed inhibin A and B binding to both betaglycan and ActRIIB in a manner consistent with previous reports (20, 33 ; Chapman, S. C., D. J. Bernard, and T. K. Woodruff, in preparation). Thus, the available data suggest that InhBP/p120 alone is not a high affinity inhibin binding moiety.
Mechanism for Activin Antagonism and for Independent Inhibin Signaling
Two general models have been proposed for how inhibins may affect target cells (6, 35). In the first model, inhibins compete for binding to the activin type II receptors. Because this interaction does not lead to recruitment and activation of the signaling type I receptor, the inhibins antagonize activin-dependent signaling. As such, all of the actions of the inhibins can be accounted for by abrogation of events mediated by the activins. In the second model, the inhibins may interact with an independent receptor or receptor complex that activates an intracellular signaling cascade. There is little or no published support for this latter model, and progress in this area has been severely impaired by a persistent failure to identify inhibin-responsive genes or to demonstrate inhibin actions that are activin independent. The observations that neither betaglycan nor InhBP/p120 contain any known signaling motifs in their intracellular domains further casts doubt on the existence of signal propagating inhibin receptors.
As described above, the inhibins have lower affinities than the
activins for the activin type II receptors but are often able to
antagonize activins actions when present at equimolar concentrations.
These seemingly contradictory observations led to the hypothesis that
the affinities of the inhibins for the type II receptor may be
increased by interaction with an additional binding protein
(8). As described above, betaglycan appears to function
well in this capacity. In the presence of betaglycan, inhibin A forms a
high affinity association with ActRIIA (200 pM), which
is similar to estimates of activin As affinity for the type II
receptor isoforms (100400 pM; 7, 9, 36, 37).
Importantly, molar excess quantities of activin A appear incapable of
disrupting the inhibin A/betaglycan/ActRIIA complex, providing a potent
mechanism for inhibin A antagonism of activin A binding
(20).
Betaglycan also appears to be sufficient to make cells sensitive to inhibin A, at least in terms of activin A antagonism. Activin A stimulation of the TGFß/activin promoter-reporter construct, p3TP-lux, is not antagonized by inhibin A in AtT20 corticotroph cells. These cells do not express betaglycan endogenously (23). When betaglycan is introduced to these cells by transient transfection, inhibin A potently antagonizes activin A-stimulated p3TP-lux activity (20). Activin A also suppresses ACTH secretion from this cell line and this effect is not suppressed by inhibin A (16). It will be important to demonstrate that in the presence of betaglycan, the inhibins can attenuate this "endogenous" activin-mediated event.
Unlike betaglycan, InhBP/p120 does not interact with ActRIIA or ActRIIB and therefore would not be expected to alter the binding of the inhibins to these receptors (34). However, InhBP/p120 forms a complex with the activin type IB receptor, ALK4, in a ligand-independent fashion when overexpressed in HeLa cells. This association may provide a means for the inhibins to interact with and presumably disrupt the activin signaling complex. In fact, we observe that when InhBP/p120 is expressed in human embryonic kidney TSA cells, activin A-induced p3TP-lux activity is inhibited, even in the absence of inhibins. When inhibin B, but not inhibin A, is added to this system, activin A-stimulated reporter gene activity is almost entirely abrogated (34). The nature of this inhibitory mechanism is not yet clear. In addition, given the apparent inability of inhibin A or B to bind InhBP/p120 alone or with ALK4 (Chapman, S. C., D. J. Bernard, and T. K. Woodruff, in preparation), the mechanism of the inhibin B specific response is also unclear.
As mentioned above, neither betaglycan nor InhBP/p120 contains any signaling motifs within their short intracellular domains. Thus, it appears that neither protein alone provides a means for inhibins to generate intracellular signals. Whether these proteins interface with other signaling receptors remains to be seen, but there is precedent within the TGFß superfamily for ligand binding to nonsignaling receptors that subsequently interface with signal propagating subunits (38, 39). In addition, recent data suggest that betaglycan may function as more than just a ligand presenting coreceptor and may play a role in directing activation of unique downstream signaling events (40, 41).
Are Betaglycan and InhBP/p120 Inhibin Receptors?
As summarized in Table 1, both betaglycan and InhBP/p120 meet
some, but not all, of the criteria for designation as an inhibin
receptor. Betaglycan is expressed in many, but not all inhibin target
tissues or cells (e.g. apparently not in gonadotropes) and
is expressed in some tissues that do not appear to bind inhibins
(e.g. ovarian theca cells and oocytes). On the other hand,
betaglycan binds inhibin A with high affinity, especially in the
presence of ActRIIA and provides a mechanism for inhibin As
antagonism of activin A. In addition, inhibin A forms a ternary
complex with endogenously expressed betaglycan and ActRIIA in several
immortalized cell lines (LßT2, KK-1, TM3, and TM4).
InhBP/p120 is highly expressed in the anterior pituitary gland and is dynamically regulated across the rat estrous cycle in a manner consistent with its role as an inhibin receptor. However, it does not bind inhibin A or B when expressed alone or with ALK4 or ActRIIB. In addition, while InhBP/p120 interacts with ALK4 and can antagonize activin A signaling in the presence or absence of inhibin B, its mechanism of action has not yet been thoroughly characterized. Moreover, its inability to bind either inhibin isoform suggests that these effects may be inhibin independent or that they may be dependent upon other accessory proteins. Lastly, neither betaglycan nor InhBP/p120 provides a mechanism for independent inhibin signaling. As such, the proteins are more accurately referred to as coreceptors. In the light of the available information, and the binding data in particular, betaglycan currently appears to satisfy the criteria for designation as an inhibin coreceptor to a greater extent than does InhBP/p120.
Are There More Inhibin Receptors on the Horizon?
While it is clear that betaglycan binds the inhibins, it is not
clear that it functions as the inhibin coreceptor mediating inhibins
effects on pituitary FSH secretion (given its apparent absence in
gonadotropes in vivo). This raises the possibility that
other inhibin binding proteins may be expressed in the pituitary and
other inhibin target tissues. In fact, the data from affinity labeling
experiments in inhibin knockout tumors (19), mouse
Leydig and Sertoli cells (32) and cultured rat pituitary
cells (30) indicate the existence of additional inhibin
binding proteins that do not correspond to activin type II receptors,
betaglycan, or InhBP/p120. Do these proteins share structural homology
with betaglycan (e.g. endoglin, uromodulin) or will they be
completely novel? Do any of these candidates provide a direct mechanism
for inhibin signaling or are all of the inhibins actions accounted
for by competitive binding to activin type II receptors? Are different
inhibin binding proteins expressed in different inhibin target tissues,
each providing a different mechanism of action? The answers to these
and other questions should quickly surface once these proteins are
characterized, and as a result, a clearer picture of the mechanisms of
inhibin action will emerge.
FOOTNOTES
The work was supported by NIH Grant HD-37096. D.J.B. is a Lalor Foundation Post-Doctoral Fellow. S.C.C. is a fellow of the Northwestern University Carcinogenesis Training Grant (T32-CA09560).
1 Present address: Center for Biomedical Research, Population Council,
1230 York Avenue, New York, New York 10021.
Abbreviation: InhBP/p120, Inhibin binding protein, a membrane-tethered proteoglycan.
Received for publication September 11, 2001. Accepted for publication November 7, 2001.
REFERENCES