Modulation of Activin Signal Transduction by Inhibin B and Inhibin-Binding Protein (InhBP)
Stacey C. Chapman and
Teresa K. Woodruff
Department of Neurobiology and Physiology (S.C.C., T.K.W.)
Northwestern University and Department of Medicine
(T.K.W.) Northwestern University Medical School Evanston,
Illinois 60208-2850
 |
ABSTRACT
|
---|
An antagonistic relationship between
inhibin and activin is essential to the control of pituitary FSH
release and to normal gonadal function. Two inhibin ligands, inhibin A
and inhibin B, are made by the ovary in females, and each regulate
pituitary FSH at different times during the reproductive cycle. Inhibin
B, but not inhibin A, is produced by the testes and is therefore
responsible for all inhibin-dependent FSH regulation in males. Although
the activin signal transduction pathway has been well characterized,
little is known about the mechanism of inhibin signaling and its
relationship to activin antagonism. A recently cloned inhibin-binding
protein, InhBP (p120), associates strongly with the type IB activin
receptor (Alk4) in a ligand-responsive manner and interacts to a lesser
extent with other activin and bone morphogenetic protein (BMP)
type I and activin type II receptors. Activin stimulates the
association of InhBP and Alk4, and inhibin B, but not inhibin A,
interferes with InhBP-Alk4 complex formation. InhBP is necessary to
mediate a specific antagonistic effect of inhibin B on
activin-stimulated transcription. Appropriate stoichiometry between
InhBP and the activin type I receptor is crucial to InhBP function.
These findings suggest that InhBP is an inhibin B-specific receptor
that mediates antagonism of activin signal transduction through the
modulation of activin heteromeric receptor complex assembly.
 |
INTRODUCTION
|
---|
Hormones and growth factors produced by the hypothalamus,
pituitary, and gonads control normal reproductive function. The
pituitary hormones FSH and LH are produced by gonadotrope cells in the
pituitary (reviewed in Ref. 1), and release of these hormones is
facilitated by the hypothalamic releasing peptide, GnRH (reviewed in
Ref. 2). FSH and LH regulate ovarian follicle maturation and ovulation
in the female and maintain tonic testicular sperm and steroid
production in males. Hormones produced by the gonads feed back to the
pituitary gonadotrope and hypothalamus to regulate FSH and LH synthesis
and secretion. The gonadotropins share a common
-subunit, yet the
synthesis and release of each hormone is often discordant (3), and the
precise molecular mechanisms by which LH and FSH are differentially
regulated have not been determined. The gonadal hormone inhibin is one
of the ligands that mediate differential LH and FSH production. Inhibin
is an endocrine hormone that specifically inhibits FSH release in a
cycle-dependent manner in females and is critical to normal
testicular-pituitary function in males (4, 5). Despite the central
nature of gonadal inhibin to the regulation of the reproductive axis,
little is known regarding its molecular mechanism of action, largely
because a receptor for this ligand had not been identified until
recently.
Both inhibin and activin are dimeric hormones and members of
the transforming growth factor-ß (TGFß) superfamily of proteins
(reviewed in Ref. 6). Inhibin is assigned to the TGFß superfamily
because of its ß-subunit, yet it is unique among the ligands because
it is capable of heteromeric assembly (7). The inhibin heterodimer
consists of an
-subunit and one of two ß-subunits, ßA (inhibin
A) or ßB (inhibin B), and production of this hormone is largely
restricted to the ovary and testes (8). Activin is a dimer of
ß-subunits produced by many tissues throughout the body and is a
local regulator of pleiotropic cell homeostasis (9). In contrast,
inhibin is one of the only TGFß superfamily ligands that acts as an
endocrine hormone, and known inhibin activity is restricted to a
discrete population of cells in the pituitary and the gonads.
Although gonadal-derived inhibin has been primarily regarded
as an endocrine agent involved in the regulation of FSH release from
the pituitary, specific binding sites have been localized to the ovary
and testes of the rat (10). Injection of recombinant human (rh-)
inhibin A into the ovarian intrabursal space of female rats results in
the growth and accumulation of intermediate (350500 nm diameter)
recruited follicles (11). Furthermore, inhibin A treatment of cultured
gonadal cells has been shown to stimulate steroidogenesis in
vitro. Production and secretion of androstenedione and
dehydroepiandrosterone by cultured primary human thecal cells
increase significantly after treatment with rh- inhibin A (12, 13),
and rh-inhibin A stimulates expression of the steroidogenic enzyme
cytochrome p450-c17 in primary cultures of porcine Leydig cells (14, 15). These findings suggest that inhibin is an endocrine, paracrine,
and autocrine hormone of the reproductive axis.
Many, but not all, activin actions are opposed by inhibin (16). Inhibin
has been shown to antagonize activin in a variety of physiological
circumstances, the best characterized of which is its effect on
pituitary FSH production (4). Inhibin also abrogates local activin
actions in the gonads. For instance, inhibin has been shown to
antagonize activin inhibition of testosterone production in cultured
rat Leydig cells and activin stimulated 3ß-hydroxysteroid
dehydrogenase (3ß-HSD) expression in cultured porcine Leydig cells
(14, 15). The mechanism by which inhibin is able to antagonize activin
action is an important aspect of inhibin biology and is most likely
multifactorial. Molecular antagonism of activin by inhibin occurs, at
least in part, through competition for the inhibin ß-subunit. In the
ovary, levels of the
-subunit far exceed those of the ß-
subunit, thus favoring inhibin rather than activin dimer
assembly. Inhibin may also block activin action through an interaction
with the activin type II receptor (ActRII) (17). The affinity of
inhibin for ActRII is 10-fold lower than activin; therefore, an excess
of inhibin is required to block activin action through its receptor
(18, 19, 20).
In many physiological settings, molecular assembly and receptor
competition can not fully account for the antagonistic actions of
inhibin and activin. Indeed, many of activins actions are insensitive
to inhibin antagonism. For example, inhibin cannot antagonize
activin-stimulated hemoglobin synthesis in erythrocytes (18), and
inhibin does not affect activin-induced granulosa cell growth (21).
Thus, a distinct inhibin receptor or other inhibin-binding accessory
molecule is necessary to potentiate an inhibin response. Moreover, when
all pituitary activin is neutralized by an activin-specific antibody,
inhibin is still able to block FSH release, suggesting that not all of
inhibins actions are a result of activin antagonism (22). Further, an
independent inhibin-signaling pathway is predicted based on the ability
of inhibin to stimulate hCG-supported testosterone secretion in porcine
Leydig cells and steroidogenesis in gonadal tumor cells (12, 13, 15, 23).
Early efforts to purify an inhibin receptor focused on the
identification of candidate proteins based on homology to the known
receptors of the TGFß superfamily. The cellular response to activin
and most other TGFß superfamily members is transduced through a
heteromeric receptor complex comprised of two single membrane-spanning
serine-threonine kinase subunits (24). Ligand binds to a specific type
II receptor, which then phosphorylates and activates a type I receptor.
Together, type I and type II receptors form a complex that is then
capable of activating downstream signaling events (25, 26). When
inhibin binding to type II or type I-like receptors could not be
identified in tissues that were clearly sites of inhibin action,
affinity purification methodology was used to isolate inhibin-binding
proteins from gonadal tumors and bovine pituitaries (27, 28). These
efforts led to the cloning of a novel inhibin-binding protein, InhBP,
that is the focus of the current investigation.
InhBP, previously called p120, is expressed in inhibin target tissues
and is a single membrane-spanning protein that contains 12 Ig domain
repeats separated into 5 and 7 repeats by a short linker region (28).
InhBP has no discernible intracellular kinase domain, and this predicts
a mechanism of inhibin signal transduction that is distinct from the
heteromeric receptor model of other members of the superfamily. The
following studies describe the characterization of the biophysical and
signal-transducing properties of InhBP and address the critical role
InhBP plays in mediating inhibin action.
 |
RESULTS
|
---|
Localization and Inhibin Binding Properties of InhBP
InhBP must be expressed at the cell membrane and bind inhibin to
function as an inhibin receptor. The subcellular localization of InhBP
was determined by immunofluorescence analysis of CV-1 monkey kidney
cells transfected with Flag-tagged InhBP (Fig. 1A
). InhBP is excluded from the
nucleus and is expressed in a diffuse punctate pattern indicative of
membrane-localized proteins. Three-dimensional reconstruction of CV-1
cells transfected with InhBP confirmed membrane localization of the
expressed, fluorescently tagged construct (data not shown). Because
CV-1 cells do not express endogenous InhBP, the ability of InhBP to
confer inhibin binding to these cells was determined. CV-1 cells were
transiently transfected with Flag-tagged InhBP (Fig. 1
, C, E, G, and I)
and treated with either iodinated inhibin A (Fig. 1
, BE) or inhibin B
(Fig. 1
, FI). InhBP expression was colocalized with inhibin binding
by immunostaining with a Flag epitope-directed antibody (Fig. 1
, B, C,
F, and G). Neither iodinated inhibin A nor iodinated inhibin B binds
CV-1 cells that have not been transfected with InhBP (Fig. 1
, B, D, F,
and H). The expression of InhBP in CV-1 cells is necessary and
sufficient to confer inhibin binding ability to these cells (Fig. 1
, C,
E, G, and I). Thus, InhBP exhibits a subcellular localization pattern
and inhibin binding properties that support its role as an inhibin
receptor.

View larger version (41K):
[in this window]
[in a new window]
|
Figure 1. Transient Transfection of InhBP into CV-1 Cells
Confers Inhibin Binding Ability
A, The Flag-tagged full-length InhBP cDNA was transfected into CV-1
cells, and the expressed protein was localized to membranes by
immunofluorescence analysis using the anti-Flag antibody. BE, To
determine whether InhBP can confer inhibin binding to CV-1 cells,
Flag-tagged InhBP-transfected and nontransfected cells were incubated
with iodinated inhibin A (D and E, dark-field) or inhibin B (H and I,
dark-field) followed by immunolocalization of InhBP-transfected cells
with the anti-Flag antibody (B, C, F, and G, bright-field). No inhibin
binding was detected in nontransfected cells (B, D, F, and H).
Arrows in B and C and F and H denote same cell. Inhibin
binding was detected specifically in those cells that expressed InhBP
(C, E, G, and I).
|
|
Assembly of InhBP into Homooligomeric Complexes
Several groups have reported that both type I and type II TGFß
serine-threonine kinase receptors can assemble into homooligomeric
complexes in vivo and in vitro (29, 30, 31).
Therefore, the ability of InhBP to form homooligomers was
determined in transiently transfected HeLa cells. In addition, the role
of inhibin and activin in regulating assembly of higher order complexes
of InhBP was examined. InhBP was capable of forming ligand-independent
homooligomers (Fig. 2A
). The formation of
homooligomeric complexes was unaffected by treatment of the cells with
inhibin A, inhibin B, or activin A alone, and addition of inhibin A and
activin A together did not alter the complex. However, InhBP
homooligomer assembly was partially disrupted upon treatment with a
combination of inhibin B and activin A.

View larger version (40K):
[in this window]
[in a new window]
|
Figure 2. InhBP Assembles into Complexes with Activin
Receptor Subunits
A, InhBP forms homooligomeric complexes in a ligand-independent manner
(upper panel). HeLa cells were transiently transfected
with equal amounts of Flag-tagged InhBP and HA-tagged InhBP and treated
with 100 ng/ml of inhibin A, inhibin B, or activin A as indicated 30
min before lysis, and lysates were subjected to immunoprecipitation
with anti-HA antibody and immunoprecipitated complexes were analyzed by
SDS-PAGE, immunoblotting with anti-Flag antibody and detected by
chemiluminescence. Protein expression was confirmed by immunoblotting
total cell lysates with anti-HA or anti-Flag antibodies (lower
panels). Closed arrows indicate protein relative
molecular mass markers; open arrows indicate migration
of proteins. B, InhBP associates strongly with Alk4 and to a lesser
extent with Alk2, ActRII, and ActRIIB (upper panel).
HeLa cells were transiently transfected with equal amounts of
Flag-tagged InhBP and HA-tagged type I (Alk2), type IB (Alk4), type II,
or type IIB activin receptors. Cells were incubated with 100 ng/ml
inhibin A for 30 min before lysis. Lysates were subjected to
immunoprecipitation, immunoblotting, and analysis as in panel A.
Protein expression was confirmed by immunoblotting total cell lysates
with anti-HA or anti-Flag antibodies (lower panels).
Closed arrows indicate protein relative molecular mass
markers; open arrows indicate migration of proteins. C,
Truncation of the C-terminal serine-threonine kinase domain of Alk4
does not affect assembly with InhBP (upper panels). HeLa
cells were transiently transfected with equal amounts of Flag-tagged
InhBP and myc-tagged Alk4 C-terminal domain truncation mutant
(Alk4 CT). Lysates were subjected to immunoprecipitation with
anti-myc antibody and immunoprecipitated complexes were
analyzed as in panel A. Protein expression was confirmed by
immunoblotting total cell lysates with anti-myc or
anti-Flag antibodies (lower panels). Closed
arrows indicate protein relative molecular mass markers;
open arrows indicate migration of proteins. D,
Endogenous assembly of InhBP and Alk4 in human pituitary extracts.
Three samples of human pituitary tissue were prepared, and 2 mg of
total lysate were subjected to immunoprecipitation with anti-InhBP
antibody as indicated. Immunoprecipitated complexes were analyzed by
SDS-PAGE on a 412% bis-acrylamide Tris gradient gel
(Novex, San Diego, CA), immunoblotted with anti-ActRIB
antibody (R&D Biosystems), and detected by chemiluminescence
(upper panels). Protein expression was confirmed by
immunoblotting total cell lysates with anti-InhBP or anti-ActRIB
antibodies (lower panels). HeLa cells were transiently
transfected with Alk4 or InhBP, and protein expression was confirmed by
immunoblotting total cell lysate concurrently with the human pituitary
samples. HeLa cells express low levels of Alk4 and no detectable InhBP,
and complexes of endogenous HeLa Alk4 and expressed InhBP were not
observed (upper panel, lane 2). Closed
arrows indicate protein relative molecular mass markers;
open arrows indicate migration of proteins.
Asterisk denotes nonspecific protein-anti-Alk4 antibody
interaction that runs just below InhBP/Alk4 band
(arrowhead, upper panel).
|
|
Assembly of InhBP and Activin Receptor Subunit Complexes
Based on the fact that InhBP appears to have no kinase domain and
may rely on extrinsic serine-threonine kinase receptors to transduce a
signal, and because inhibin is known to bind with low affinity to the
activin type II receptor, functional assembly of InhBP with known
TGFß family member receptor subunits was examined. Flag-tagged InhBP
and various hemagglutinin (HA)-tagged or myc-tagged activin receptors
were coexpressed in HeLa cells, and the cells were treated with 100
ng/ml inhibin A for 30 min before lysis. InhBP-activin receptor
complexes were coimmunoprecipitated using antibodies directed against
the HA or myc epitope tags. A weak interaction between InhBP and
activin type II (ActRII) and type IIB (ActRIIB) receptors was detected
by immunoblotting with anti-Flag antibody (Fig. 2B
). A direct
association between InhBP and two signaling receptor subunits, Alk4
(activin type IB receptor) and Alk2 (activin type I receptor), was
readily observed by immunoprecipitation followed by Western detection
(Fig. 2B
). No interaction between InhBP and HA-tagged GH releasing
hormone receptor was detected (data not shown), indicating that the
interaction of InhBP with activin receptors is specific. To investigate
whether InhBP interacts with the conserved serine-threonine kinase
domain of Alk4, a myc-tagged Alk4 expression construct, which lacks the
kinase domain, was cotransfected with InhBP (32). A stable InhBP-Alk4
C-terminal deletion mutant (Alk4
CT) complex was detected by
immunoblot, suggesting that the interaction between these two cell
surface proteins is dependent on regions lying outside the C-terminal
serine-threonine kinase domain of Alk4 (Fig. 2C
).
In Vivo Association of InhBP and Activin Receptor Type
IB
To verify that InhBP can assemble with Alk4 in a physiological
context, we immunoprecipitated InhBP- containing protein complexes
from three samples of human pituitary lysate using the anti-InhBP
antibody. Interaction of endogenous InhBP with Alk4 was observed by
immunoblotting with a human anti-Alk4 antibody (Fig. 2D
, upper
panel). Identity of the complexed pituitary proteins was
established by comparison of proteins identified in human pituitary to
immunoblots of overexpressed InhBP and Alk4 in HeLa cell lysates run
concurrently with the pituitary samples (Fig. 2D
, lower
panels). Similarly, InhBP and Alk4 complexes were identified in
ovine pituitary lysates (data not shown).
Dynamic Regulation of InhBP and Activin Receptor Subunit
Complexes
Because activin A and inhibin B together interrupt the InhBP
homooligomer (Fig. 2A
), the role of these ligands on Alk4 and InhBP
complex assembly was investigated. Cells were transfected with
Flag-tagged InhBP and HA-tagged Alk4 and treated with 100 ng/ml inhibin
A, inhibin B, or activin A for 30 min before lysis (Fig. 3A
). InhBP and Alk4 can assemble in a
ligand-independent manner and activin A does not alter this stable
association (Fig. 3B
). A subtle but consistent decrease in complex
assembly was observed when cells were treated with inhibin B alone.

View larger version (43K):
[in this window]
[in a new window]
|
Figure 3. InhBP Associates with the Activin [Type IB]
Receptor, Alk4, in a Ligand- Dependent Manner
A, The assembly of InhBP/Alk4 complexes is antagonized in the presence
of activin and inhibin B. HeLa cells were transiently transfected with
equal amounts of Flag-tagged InhBP and HA-tagged Alk4. Cells were
incubated with 100 ng/ml activin A, inhibin A, or inhibin B as
indicated for 30 min before lysis. Lysates were subjected to
immunoprecipitation with anti-HA antibody as indicated.
Immunoprecipitated complexes were analyzed by SDS-PAGE on an 8%
acrylamide gel (Bio-Rad Laboratories, Inc.), immunoblotted
with anti-Flag antibody, and detected by chemiluminescence. Protein
expression was confirmed by immunoblotting total cell lysates with
anti-HA or anti-Flag antibodies (lower panels).
Closed arrows indicate protein relative molecular mass
markers; open arrows indicate migration of proteins. B,
Data from densitometric analysis of Fig. 3B are presented as percent of
ligand-independent (basal) InhBP/Alk4 complex assembly corrected for
protein expression levels in the lysate and reported in arbitrary
units.
|
|
To investigate whether the InhBP-Alk4 interaction was altered by
coincubation of the antagonistic ligands, cells were treated
simultaneously with 100 ng/ml inhibin A or B plus activin A, and
complex formation was evaluated (Fig. 3A
). Association of InhBP and
Alk4 was not altered by treatment with inhibin A and activin A together
(Fig. 3B
). However, when cells were treated with inhibin B and activin
A, the interaction between InhBP and Alk4 diminished to approximately
60% of the basal associated complex (Fig. 3B
). These results suggest
that a heteromeric cell surface receptor complex is formed that
includes InhBP and Alk4, and that formation of this complex is
regulated in a dynamic manner by activin and inhibin B.
Modulation of Activin-Regulated Transcriptional Responses by InhBP
and Inhibin
Because inhibins antagonize activin action in various
physiological settings, the role of InhBP in regulating
activin-dependent signal transduction events was investigated. The TSA
kidney epithelial cell line used in this study expresses type IB (Alk4)
and both type II activin receptors, but does not express InhBP (data
not shown), and therefore provides a model system in which to study
InhBP action and interaction with an endogenous activin signaling
pathway. Furthermore, as no inhibin-specific promoter has yet been
identified, a known activin/TGFß responsive reporter gene construct,
the plasminogen activator inhibitor-1 promoter ligated to a luciferase
reporter (p3TP-luc), was used to investigate the effects of InhBP and
inhibin on activin-stimulated gene transcription.
TSA cells were transiently transfected with p3TP-luc and the cells were
treated with the indicated concentrations of inhibin, activin, or with
inhibin plus activin for 24 h (Fig. 4
). In our TSA system, activin A was able
to stimulate luciferase activity 8- to 10-fold over basal. Treatment of
cells with equimolar concentrations of inhibin A and activin A resulted
in a 70% antagonism of activin-stimulated transcription of the
reporter construct (Fig. 4A
), while inhibin B was capable of abrogating
the activin A effect by approximately 40% (Fig. 4B
). This antagonism
may be attributed to competition between activin and inhibin for
binding to the endogenous type II activin receptor in TSA cells (17).
Interestingly, transfection of InhBP into this system resulted in
ligand-independent antagonism of activin-stimulated p3TP-luc
transcription, and the mechanism underlying this effect is currently
under investigation. Remarkably, in the presence of InhBP, inhibin B
treatment resulted in a nearly total loss (90%) of activin
A-stimulated p3TP-luc transcription (Fig. 4B
), while inhibin A
treatment of InhBP-transfected cells had no additional antagonistic
effect on the activin response (Fig. 4A
). The antagonistic effect of
inhibin B and InhBP on activin-stimulated p3TP-luc was approximately 2
times greater than that observed with inhibin B treatment alone, and so
could not be due to competition for activin receptor binding alone.
This result indicates a direct role for InhBP in a novel receptor-based
mechanism of antagonism of activin-stimulated p3TP-luc transcription by
inhibin B. These results also strongly support the view that there is a
fundamental difference in the molecular actions of inhibin A and
inhibin B.

View larger version (22K):
[in this window]
[in a new window]
|
Figure 4. InhBP Mediates Inhibin B-Mediated Antagonism of
Activin-Stimulated p3TP-luc Transcription in TSA Cells
A and B, TSA cells were transiently transfected with p3TP-luc and
either 50 ng pcDNA3.0 or InhBP expression plasmid. Cells were treated
with 100 ng/ml inhibin A, inhibin B, or activin A as indicated.
Expression of p3TP-luc is calculated as relative light units corrected
for total protein in the lysate and is plotted as the percent
stimulation of luciferase expression ± SE (s.e.m.) of
triplicates from a representative experiment. C and D, Alk4
overexpression opposes InhBP and inhibin B-mediated activin antagonism.
Cells were transiently transfected with p3TP-luc and either pcDNA3.0,
50 ng InhBP expression plasmid, or 25 ng of expression plasmid encoding
Alk4 as indicated. Cells were treated with 100 ng/ml inhibin A, inhibin
B, or activin A as indicated. Expression of p3TP-luc is calculated as
relative light units corrected for total protein in the lysate and is
plotted as the percent stimulation of luciferase expression ±
SE (s.e.m.) of triplicates from a representative
experiment. *, P < 0.05; **,
P < 0.01. E, Antagonism of p3TP-luc expression by
inhibin and InhBP is activin specific. TSA cells were transiently
transfected with p3TP-luc and either pcDNA3.0 or 25 ng InhBP expression
plasmid and treated with 10 ng/ml TGFß1 or 100 ng/ml inhibin B as
indicated. Expression of p3TP-luc is expressed as relative light units
normalized for total protein and is plotted as the mean ±
SEM of triplicates from a representative
experiment.
|
|
Role of Alk4 in the Modulation of Activin-Stimulated
Transcriptional Responses by Inhibin and InhBP
The biochemical data predict a strong, ligand-modulated
interaction between InhBP and Alk4. To investigate whether Alk4 is
involved in the InhBP/inhibin-mediated antagonism of activin-stimulated
p3TP-luc expression, TSA cells were cotransfected with the p3TP-luc
reporter construct and an Alk4 expression plasmid, and the cells were
treated with inhibin and activin. Overexpression of Alk4 partially
reversed the functional antagonism of p3TP-luc expression due to
competition between inhibin A and activin A for receptor binding (Fig. 4C
) and completely reversed antagonism of activin A action by inhibin B
(Fig. 4D
). Notably, Alk4 overexpression only partially restored
activin- stimulated p3TP-luc transcription in cells transfected with
InhBP and treated with inhibin B, from 90% (Fig. 4B
) to 56%
antagonism (Fig. 4D
). Thus, overexpression of Alk4 opposes inhibin B
and InhBP-mediated activin antagonism in this system.
Modulation of Transcription by Inhibin and InhBP Is Activin
Specific
It is imperative to establish the antagonistic effect of inhibin B
and InhBP as activin specific. Inhibin A was shown to antagonize
activin A-stimulated p3TP-luc expression while having no effect on
TGFß-stimulated p3TP-luc transcription in a CHO cell line (19).
Importantly and necessarily, the antagonistic effect of InhBP and
inhibin B on p3TP-luc transcription is activin specific (Fig. 4E
).
TGFß is capable of stimulating p3TP-luc transcription 8-fold
over basal in TSA cells (data not shown). As anticipated, neither
inhibin A (data not shown) nor inhibin B alone has a significant
antagonistic effect on TGFß-stimulated p3TP-luc transcription (Fig. 4E
). Unlike the ligand-independent antagonistic effect of InhBP on
antagonism of activin-stimulated p3TP-luc transcription,
TGFß-stimulated luciferase activity is unaffected by transfection of
InhBP into the system. Furthermore, cotransfection of InhBP did not
support antagonism of TGFß signal transduction by inhibin B
(Fig. 4E).
InhBP Interacts with Other Type I Receptors of the TGFß
Superfamily
Cross-talk within the TGFß family occurs frequently through
sharing of receptors by multiple ligands and the formation of
nontraditional receptor complexes. For example, bone morphogenetic
protein 2 (BMP2) and activin are able to signal through Alk2 in
some cell types (33), and it has been shown that inhibin can bind,
albeit weakly, to the activin type II receptor (17, 19). TGFß and
activin bound to their respective type II receptors can form complexes
with the type I TSR-1 (Alk1) (34), and BMP7 binds ActRII to form
functional complexes with the BMP type I receptors Alk3 and Alk6 (33).
Based on these observations, the interaction of InhBP with type I
receptor proteins of other TGFß family members, including Alk3 and
Alk6, as well as the dual specificity type I receptor Alk1, was
investigated. Flag-tagged InhBP and HA-tagged BMP type I receptors or
HA-tagged dual type I receptor were coexpressed in HeLa cells (Fig. 5
). Receptor complexes were
coimmunoprecipitated using antibodies directed against the HA epitope
tags. A very weak interaction between InhBP and Alk3 was detected by
immunoblot using antibody directed against the Flag epitope tag, while
InhBP readily formed complexes with Alk1 and Alk6.

View larger version (42K):
[in this window]
[in a new window]
|
Figure 5. InhBP Associates with Other Type I TGFß
Superfamily Receptors
InhBP interacts strongly with the dual-specificity type I receptor,
Alk1, as well as with the BMP type I receptor, Alk6, and to a lesser
extent with the BMP type I receptor, Alk3 (upper panel).
HeLa cells were transiently transfected with equal amounts of
Flag-tagged InhBP and HA-tagged dual specificity type I receptor
(Alk1), or the BMP type I receptors Alk3 or Alk6. Lysates were
subjected to immunoprecipitation with anti-HA antibody, and
immunoprecipitated complexes were analyzed by SDS-PAGE and
immunoblotting with anti-Flag antibody and detected by
chemiluminescence. Protein expression was confirmed by immunoblotting
total cell lysates with anti-HA or anti-Flag antibodies (lower
panels). Closed arrows indicate protein relative
molecular mass markers; open arrows indicate migration
of proteins.
|
|
 |
DISCUSSION
|
---|
Gonadal inhibin inhibits pituitary FSH synthesis, promotes ovarian
follicle maturation, and is important to the maintenance of normal
spermatogenesis. Although our knowledge of how activin acts on target
cells has expanded tremendously in the last several years, progress in
understanding the mechanism of inhibin action has been comparatively
slow. The means by which inhibin regulates distal or local cellular
events is gradually being revealed by the identification of cell
surface inhibin-binding proteins (27, 28, 35, 36). Biochemical
purification of InhBP and the recent characterization of betaglycan as
an inhibin-binding protein suggests that inhibin-binding cell surface
proteins act as docking molecules that permit assembly of specific
receptor complexes which then modulate ligand action (28, 36, 37, 38).
The Ig domain-rich nature of InhBP predicted a mechanism of inhibin
signal transduction differing significantly from other members of the
TGFß superfamily. Interestingly, Ig domains are found in a wide
variety of proteins, including growth factor receptors for epidermal
growth factor (EGF), platelet-derived growth factor (PDGF), and
hepatocyte growth factor (HGF), and in cell adhesion molecules
including neural cell adhesion molecule (N-CAM) and cadherin (39).
Furthermore, many members of the Ig superfamily are signal transducers;
for example, the Ig-domain containing PDGF receptor (PDGF-R) tyrosine
kinase initiates intracellular kinase cascades that lead to cell
division and proliferation (reviewed in Ref. 40), while activation of
N-CAM has been shown to result in changes in intracellular second
messenger levels and protein phosphorylation (reviewed in Ref. 41).
Further, the lack of an intracellular kinase domain suggested that
InhBP would require association with other proteins or extrinsic
kinases to transduce an inhibin signal. We observed that InhBP is
capable of interacting with the activin type IB receptor
serine-threonine kinase in a ligand-dependent manner. This observation
was unexpected because it was hypothesized that inhibin binds InhBP and
ActRII cooperatively through its
- and ß-subunits, respectively.
Accordingly, Lewis et al. (36) have reported that betaglycan
binds inhibin and disrupts activin signal transduction through
association with the activin type II receptor (Fig. 6B
). Similarly, interaction between
ligand-bound type II TGFß receptor and the accessory signaling
molecule endoglin interferes with TGFß signal transduction (Fig. 6C
)
(42).

View larger version (12K):
[in this window]
[in a new window]
|
Figure 6. InhBP and Inhibin B Antagonize Activin Signal
Transduction by Modulating Activin Receptor Complex Assembly
A, InhBP is assembled into the activin heteromeric receptor complex
through its ligand-independent association with Alk4, thus rendering
the complex inhibin B sensitive. Inhibin B binding to InhBP disrupts
the association of InhBP, Alk4, and ActRIIB and abolishes activin
signaling. B, Betaglycan binds inhibin A in cooperation with ActRII,
thereby disrupting heteromeric activin receptor complex formation and
activin signaling. C, Endoglin binds TGFß in the presence of the
TGFß type II receptor (TßRII), which disrupts complex formation
with TGFß type I receptor (TßRI) and interferes with TGFß signal
transduction.
|
|
However, it is the interaction between InhBP and an activin type I
receptor, Alk4, that is central to inhibin B action. InhBP associates
strongly with Alk4 in vivo and in vitro in the
absence of ligand, and this complex is stabilized in the presence of
activin. To our knowledge, interaction of Alk4 with InhBP is the first
example of in vivo assembly of any of the TGFß superfamily
receptors. Through this interaction with Alk4, we hypothesize that
InhBP is recruited to the Alk4/ActRII/B activin receptor complex.
Addition of inhibin to the system destabilizes the association between
InhBP and Alk4 and abrogates activin signal transduction through its
receptor complex (Fig. 6A
).
One of the unique features of inhibin is that it specifically
antagonizes some actions of activin but not those of other TGFß
superfamily ligands. One mechanism by which inhibin opposes activin
action is through binding interference of the inhibin ß-subunit to
the activin type II receptor (17). In our study, functional antagonism
of activin-stimulated p3TP-luc was observed when cells were treated
with equimolar amounts of activin A and inhibin A or inhibin B, which
resulted in approximately 50% inhibition of p3TP-luc expression.
LeBrun and Vale (18) have shown that inhibin abrogates
activin-stimulated p3TP-luc expression in K562 cells, but that in a
cell line which inducibly overexpresses the activin type IB and type II
receptors (KAR6), inhibin can no longer antagonize activin action even
at 8-fold molar excess. Thus, it was proposed that an additional
inhibin receptor or other accessory molecule was necessary to fully
antagonize activin action. Our results support this hypothesis. Cells
treated with inhibin A or inhibin B alone resulted in a partial
antagonism of activin-stimulated p3TP-luc, while inhibin B treatment in
the presence of cotransfected InhBP virtually abolished
activin-stimulated p3TP-luc expression. Molecular and functional models
of inhibin antagonism require that the concentration of inhibin
-subunit or inhibin dimer is such that it blocks the activin signal
transduction apparatus through abrogation of activin dimer assembly and
binding to its receptor. The data suggest that InhBP provides a means
through which low levels of inhibin B can antagonize activin signal
transduction, whereby the presence of InhBP in activin-stabilized
receptor complexes renders these complexes sensitive to antagonism by
inhibin B (Fig. 6A
).
Accordingly, overexpression of Alk4 in the system reverses
activin-stimulated p3TP-luc expression, perhaps by shifting the
stoichiometry of InhBP and Alk4 and favoring the assembly of activin
receptor complexes that do not contain InhBP. These complexes would be
inhibin B insensitive and remain intact upon inhibin B treatment. The
inability of excess inhibin to antagonize activin signaling in models
such as the activin receptor- overexpressing KAR6 cell line may be due
to a lack of endogenous InhBP in these cells or the assembly of a
greater number of activin receptor complexes that do not contain
InhBP.
It is apparent that InhBP may be an inhibin B receptor. Although InhBP
is capable of binding to both inhibin A and inhibin B, antagonism of
activin-stimulated p3TP-luc transcription by inhibin A was not enhanced
by the coexpression of InhBP, while inhibin B and InhBP were able to
abrogate activin signaling more than 90%. Once thought to be
interchangeable, inhibin A and inhibin B have very different synthesis
and secretion patterns during the female reproductive cycle (8, 43, 44). Furthermore, inhibin B, rather than inhibin A, predicts follicle
health and is an early indicator of ovarian aging (45, 46). In
addition, inhibin A and inhibin B are produced in a sexually dimorphic
manner: inhibin B, but not inhibin A, is produced by the testes and is
inversely correlated to FSH in several experimental models as well as
in disease states (8, 47, 48). Thus, the two species of inhibin exist
in discrete molecular, cellular, and endocrine niches. The observation
that InhBP mediates antagonism by inhibin B and not inhibin A suggests
that there is also a fundamental, functional difference between the two
species of inhibin. The basis for this difference remains unclear, but
may be partially attributed to the structural dissimilarity of the
inhibin ß-subunits, ßA and ßB, which share 63% identity.
Likewise, TGFß1 and TGFß2 exhibit approximately 80% amino acid
identity, yet they are regarded as functionally distinct proteins.
Comparison of these isotypes of TGFß in several studies revealed that
TGFß1 has a higher binding affinity for the type II TGFß receptor
and is a more potent inhibitor of hematopoietic progenitor cell and
endothelial cell proliferation than TGFß2 (49, 50, 51).
Furthermore, it is important that antagonism of p3TP-luc transcription
by InhBP and inhibin B is activin specific. The effects seen on
p3TP-luc expression upon treatment with inhibin B and activin are not
repeated when cells are treated with TGFß. Although InhBP interacted
with the TGFß dual specificity type I receptor Alk1, inhibin B and
InhBP had no effect on TGFß-stimulated p3TP-luc transcription. Thus,
antagonism of activin action by InhBP is a dynamic, ligand-dependent
process in which InhBP provides more than just a physical blockade to
signaling through the activin receptor.
These results, along with the fact that inhibin A and inhibin B are
differentially regulated throughout the human menstrual and rat estrous
cycles, support the existence of separate inhibin A and inhibin B
receptors within the female reproductive axis (8, 43, 52). In rat,
ovarian inhibin A subunit mRNA and protein levels remain low through
metestrus and diestrus, then rise to peak late in proestrus at the time
of the primary gonadotropin surges (8, 52). Conversely, a high
concentration of inhibin B is maintained through metestrus and
diestrus, then falls to its lowest levels after the primary FSH surge
and remains low through the secondary FSH surge (8). Activin A levels
throughout the cycle remain virtually undetectable until peaking
sharply just before the primary and secondary FSH surges. High levels
of inhibin B early in the cycle keep FSH low until the drop in inhibin
B levels coupled with GnRH and an activin peak in proestrus permit the
FSH surge and ovulation. Preliminary data from our laboratory suggest
that in the rat, pituitary InhBP expression closely follows
inhibin B expression, lending further support to the characterization
of InhBP as an inhibin B receptor (52A ).
Betaglycan was recently identified as an inhibin A-binding protein
(36). Several features of this cell surface molecule differ from InhBP
(53), indicating that different mechanisms of inhibin action may be
available to a variety of cell types. Unlike InhBP, which disrupts
receptor complex formation in the presence of inhibin B, betaglycan
binds and sequesters type II activin receptors in the presence of
inhibin A (Fig. 6B
). Cells that express both betaglycan and InhBP are
predicted to be exquisitely sensitive to the actions of inhibin. A
potential example of this type of cell is the pituitary gonadotrope.
Indeed, in humans, the gonadotrope may be able to respond to both early
follicular inhibin B via InhBP and luteal phase inhibin A via
betaglycan. Of course, this hypothesis will need to be tested in
vivo. Lastly, the testis secretes inhibin B and not inhibin A.
Thus, we predict that InhBP will be the dominant inhibin receptor in
the male pituitary.
Finally, it is interesting that both InhBP and endoglin share the
ability to assemble with various type I receptors of the TGFß
superfamily. However, in general, endoglin interacts with ligand
binding receptors within the superfamily (54), while InhBP can interact
with both activin and TGFß signaling type I receptors, such as Alk4
and Alk1, and with the BMP2 ligand binding type I receptor, Alk6.
Furthermore, while endoglin can only interact with various ligand
binding receptors in the presence of ligand, the role of ligand in
assembly of InhBP with type I receptors remains unclear.
In conclusion, the recently purified and cloned high-affinity
inhibin-binding protein, InhBP, is a crucial component of a heteromeric
receptor complex that can specifically modulate activin-stimulated
reporter gene expression in a model cell line. Despite its unique
properties, InhBP appears to be intricately involved in inhibin signal
transduction events through modulation of traditional activin receptor
complexes. Antagonism of activin action occurs through receptor complex
assembly-based mechanism whereby ligand-bound InhBP disrupts
formation of active activin receptor complexes. These data do not rule
out other means of activin antagonism mediated by InhBP and
inhibin. Future endeavors will focus on the identification of
other protein components of the InhBP signal transduction complex,
including the Smad proteins, as well as the identification of
inhibin-responsive genes. Taken together, these studies will help us to
better understand inhibin and InhBP signal transduction and its role in
normal reproductive function and gonadal oncogenesis.
 |
MATERIALS AND METHODS
|
---|
Recombinant Ligands
Recombinant human (rh) inhibin A, inhibin B, activin A, and
TGFß1 were purchased from R&D Biosystems (Minneapolis, MN),
Genentech, Inc. (South San Francisco, CA), or were
produced in the laboratory. The ligands were formulated in a buffer of
0.15 M NaCl and 0.05 M Tris, pH 7.5.
Immunofluorescence and Immunohistochemistry
The monkey kidney CV-1 cell line was transiently transfected
with the Flag-tagged InhBP and subjected to immunofluorescence analysis
using the mouse anti-Flag antibody (Sigma, St. Louis, MO)
followed by a rabbit-antimouse fluorescein isothiocyanate
(FITC)-conjugated secondary antibody (Pierce Chemical Co.,
Rockford, IL). Immunohistochemical studies were performed using the
rabbit anti-InhBP antisera or mouse anti-Flag antibody
(Sigma), followed by a biotinylated goat antimouse
secondary antibody or goat antirabbit secondary antibody (Vector Laboratories, Inc., Burlingame, CA), washed, and incubated with
ABC reagent (Vectastain ABC kit, Vector Laboratories, Inc.). Signal was detected using the DAB substrate
kit (Vector Laboratories, Inc.).
In Situ Ligand Binding
Inhibin was labeled using a modified lactoperoxidase method.
Briefly, 5 µg of ligand were diluted in 0.4 M sodium
acetate, pH 5.6, and 0.5 nmol Na125I (0.5
nmol/mCi on calibration date), 0.5 IU lactoperoxidase, and 0.25 nmol
H2O2 were added
sequentially. The ligand was incubated at ambient temperature with
intermittent vortexing for 5 min. The reaction was quenched with 450
µl PBS + 0.05% Tween 20 + 0.5% BSA (Intergen,
Purchase, NY). A 10 µl aliquot of the precolumn fraction was
removed for trichloroacetic acid (TCA) precipitation. Free iodine was
removed using Sephadex G-10 column chromatography (PD-10,
Pharmacia Biotech, Piscataway NJ). The specific activity
of the ligands used in the binding studies was approximately 100
µCi/µg. The biological activity of the iodinated ligand was
determined using male rat anterior pituitary cells. Iodinated inhibin
(40 pM) was added to cells for 12 h at room
temperature. Binding to InhBP-transfected CV-1 cells was detected by
exposure of emulsion and analyzed by dark-field microscopy.
Immunoprecipitation and Immunoblot Analysis
Cells were transfected with various receptor constructs using
the Vaccinia-T7 RNA polymerase hybrid expression system. HeLa cells
were infected with Vaccinia virus vTF7.3 expressing the bacteriophage
T7 RNA polymerase (obtained under license from Dr. Bernard Moss, NIH,
Bethesda, MD), for 1 h and the various plasmid DNA/liposome
mixtures were added and incubated for 14 h. Cells were treated
with ligand for 0.5 h before lysis. Cells were lysed in RIPA
buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 1
mM EDTA, 1% Triton-X-100, 0.5% deoxycholic acid, and
0.1% SDS plus protease inhibitors (Roche Molecular Biochemicals, Mannheim, Germany)]. The lysates were
clarified by centrifugation and incubated with mouse HA-specific
antibody 12CA5 (kindly provided by Dr. Robert A. Lamb, Northwestern
University) overnight at 4 C. Immunocomplexes were precipitated by
protein A-agarose beads (Vector Laboratories, Inc.) for
1.5 h and separated in a 8.5% SDS-PAGE gel (Bio-Rad Laboratories, Inc. Hercules, CA) or in a 412% gradient
bis-acrylamide-Tris NuPAGE gel
(Invitrogen/Novex, Carlsbad, CA). Proteins
were transferred to nitrocellulose membrane (Bio-Rad Laboratories, Inc.), blotted with mouse anti-Flag M2 antibody
(Sigma), mouse anti-HA antibody, mouse
anti-c-myc antibody (Sigma), or goat
anti-hActRIB (Alk4) antibody (R&D Biosystems), for 1 h at room
temperature, followed by 1 h incubation in horseradish peroxidase
(HRP)-conjugated goat antimouse antibody (Bio-Rad Laboratories, Inc.), HRP-conjugated donkey antirabbit antibody (Amersham Pharmacia Biotech, Buckinghamshire, UK), or HRP-conjugated
antigoat antibody (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA) at room temperature and detection by chemiluminescence
(Amersham Pharmacia Biotech).
DNA Constructs
The p3TP-luc reporter plasmid was provided by the J. Massague
laboratory. Activin receptor (type I, IB, II, and IIB) expression
plasmids were provided by the L. Mathews and K. Mayo laboratories and
modified by insertion of HA or Flag sequence at the 3'-end. HA-tagged
Alk1, Alk3, and Alk6 were a generous gift from the L. Attisano
laboratory and were modified by subcloning into the pcDNA3 vector
(Invitrogen). The myc-tagged Alk4 C-terminal deletion
mutant was donated by the W. Vale laboratory.
Transient Transfection and Luciferase Assays
TSA cells were maintained in DMEM high glucose media (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FCS
(Life Technologies, Inc.) and 1% antibiotic/antimycotic
(Life Technologies, Inc.) and incubated at 37 C, 5%
CO2. Cells were plated 1 day before transfection
in 24-well plates at 1.2 x 105 cells per
well and transiently transfected with p3TP-luc and various expression
plasmids encoding various activin, TGFß, and BMP receptors or empty
pcDNA3 vector (Invitrogen) using the calcium phosphate
transfection method (55). After an overnight recovery, cells were
treated with indicated ligands for 24 h in DMEM high-glucose
media, 1% antibiotic/antimycotic. Cells were lysed in Triton-X-100,
and luciferase activity was measured using standard techniques. Total
protein was measured using BCA reagent (Bio-Rad Laboratories, Inc.).
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Wei Chen, Huira Chong, and Kelly Mayo for
work on the initial cloning of InhBP. Many thanks to the Attisano/Wrana
Laboratory (University of Toronto, Toronto, Canada), the Vale
Laboratory (The Salk Institute for Biological Studies, La Jolla, CA),
the Mathews Laboratory (University of Michigan, Ann Arbor, MI), the
Massague Laboratory (Memorial Sloan-Kettering Cancer Center, New York,
NY), and the Mayo Laboratory (Northwestern University, Evanston, IL)
for their generous gifts of the various receptor constructs, as well as
Robert A. Lamb (Northwestern University) for kindly providing the mouse
anti-HA antibody. We would also like to thank Patrick Sluss
(Massachusetts General Hospital, Boston, MA) for human pituitary
tissue. Thanks to Huira Chong for cloning of plasmid constructs, InhBP
immunofluorescence, and in situ ligand binding studies. We
would also like to thank Brad Draper for preparation of plasmid
constructs, Stephanie Pangas for purified ligands, and Daniel J.
Bernard and Fernando Lopez-Casillas for helpful discussions.
This study was supported by NIH Grants HD-37096 and HD-28048 to
T.K.W. S.C.C. is a fellow of the Northwestern University Cellular
and Molecular Basis of Disease Training Grant (GM-08061).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Teresa K. Woodruff, Department of Neurobiology and Physiology, Northwestern University, O.T. Hogan 4150, 2153 North Campus Drive, Evanston, Illinois 60208. E-mail:
tkw{at}northwestern.edu
Received for publication October 6, 2000.
Accepted for publication December 13, 2000.
 |
REFERENCES
|
---|
-
de Kretser DM, Phillips DJ 1998 Mechanisms of protein
feedback on gonadotropin secretion. J Reprod Immunol 39:112[CrossRef][Medline]
-
Hayes FJ, Crowley Jr WF 1998 Gonadotropin pulsations across
development. Horm Res 49:163168[CrossRef][Medline]
-
Savoy-Moore RT, Schwartz NB 1980 Differential control of FSH
and LH secretion. Int Rev Physiol 22:203248[Medline]
-
Rivier C, Rivier J, Vale W 1986 Inhibin-mediated feedback
control of follicle-stimulating hormone secretion in the female rat.
Science 234:205208[Medline]
-
Woodruff TK, Krummen LA, Lyon R, Stocks DL, Mather JP 1993 Recombinant human inhibin A and recombinant human activin A regulate
pituitary and ovarian function in the adult female rat. Endocrinology 132:23322341[Abstract]
-
Vale W, Rivier C, Hsueh A, Campen C, Meunier H, Bicsak T 1988 Chemical and biological characterization of the inhibin family of
protein hormones. Recent Prog Horm Res 44:134[Medline]
-
Newfeld SJ, Wisotzkey RG, Kumar S 1999 Molecular evolution of
a developmental pathway: phylogenetic analyses of transforming growth
factor-ß family ligands, receptors, and smad signal transducers.
Genetics 152:783795[Abstract/Free Full Text]
-
Woodruff TK, Beseke LM, Groome N, Draper LB, Schwartz NB,
Weiss J 1996 Inhibin A and inhibin B are inversely correlated to
follicle-stimulating hormone, yet are discordant during the follicular
phase of the rat estrous cycle, and inhibin A is expressed in a
sexually dimorphic manner. Endocrinology 137:54635467[Abstract]
-
Meunier H, Rivier C, Evans RM, Vale W 1988 Gonadal and
extragonadal expression of inhibin
, ß A, and ß B subunits in
various tissues predict diverse functions. Proc Natl Acad Sci USA 85:247251[Abstract]
-
Woodruff TK, Krummen L, McCray G, Mather JP 1993 In
situ ligand binding of recombinant human
[125I]activin-A and recombinant human
[125I] inhibin-A to adult rat ovary.
Endocrinology 133:29983006[Abstract]
-
Woodruff TK, Lyon RJ, Hansen SE, Rice GC, Mather JP 1990 Inhibin and activin locally regulate rat ovarian folliculogenesis.
Endocrinology 127:31963205[Abstract]
-
Hillier SG, Yong EL, Illingworth PJ, Baird DT, Schwall RH,
Mason AJ 1991 Effect of recombinant inhibin on androgen synthesis in
cultured human thecal cells. Mol Cell Endocrinol 75:R1R6
-
Miro F, Hillier SG 1992 Relative effects of activin and
inhibin on steroid hormone synthesis in primate granulosa cells. J
Clin Endocrinol Metab 75:15561561[Abstract]
-
Lin T, Calkins JH, Morris PL, Vale W, Bardin CW 1984 Regulation of Leydig cell function in primary culture by inhibin and
activin. Endocrinology 125:21342140[Abstract]
-
LeJeune H, Chuzel F, Sanchez P, Durand P, Mather JP, Saez JM 1997 Stimulating effect of both human recombinant inhibin A and activin
A on immature porcine Leydig cell functions in vitro.
Endocrinology 138:47834791[Abstract/Free Full Text]
-
Ling N, Ying SY, Ueno N, Shimasaki S, Esch F, Hotta M,
Guillemin R 1986 Pituitary FSH is released by a heterodimer of the
ß-subunits from the two forms of inhibin. Nature 321:779782[Medline]
-
Mathews LS, Vale WW 1991 Expression cloning of an activin
receptor, a predicted transmembrane serine kinase. Cell 65:973982[Medline]
-
Lebrun JJ, Vale WW 1997 Activin and inhibin have antagonistic
effects on ligand-independent heteromerization of the type I and type
II activin receptors and human erythroid differentiation. Mol Cell Biol 17:16821691[Abstract]
-
Martens JWM, deWinter JP, Timmerman MA, McLuskey A, vanSchaik
RHN, Themmen APN, deJong FH 1997 Inhibin interferes with activin
signaling at the level of the activin receptor complex in Chinese
hamster ovary cells. Endocrinology 138:29282936[Abstract/Free Full Text]
-
Xu J, McKeehan K, Matsuzaki K, McKeehan WL 1995 Inhibin
antagonizes inhibition of liver cell growth by activin by a
dominant-negative mechanism. J Biol Chem 270:63086313[Abstract/Free Full Text]
-
Miro F, Hillier SG 1996 Modulation of granulosa cell
deoxyribonucleic acid synthesis and differentiation by activin.
Endocrinology 137:464468[Abstract]
-
Murata T, Saito S, Shiozaki M, Lu RZ, Eto Y, Funaba M,
Takahashi M, Torii K 1996 Anti-activin antibody (IgY) specifically
neutralizes various activin A activities. Proc Soc Exp Biol Med 221:100107
-
Sawetawan C, Carr BR, McGee E, Bird IM, Hong TL, Rainey WE 1996 Inhibin and activin differentially regulate androgen production
and 17ß-hydroxylase expression in human ovarian thecal-like
tumor cells. J Endocrinol 148:213221[Abstract]
-
Piek E, Heldin C-H, ten Dijke P 1999 Specificity, diversity,
and regulation in TGF-ß superfamily signaling. FASEB J 13:21052124[Abstract/Free Full Text]
-
Attisano L, Wrana JL, Montalvo E, Massague J 1996 Activation
of signaling by the activin receptor complex. Mol Cell Biol 16:10661073[Abstract]
-
Willis SA, Zimmerman CM, Li L, Mathews LS 1996 Formation and
activation by phosphorylation of activin receptor complexes. Mol
Endocrinol 10:367379[Abstract]
-
Draper LB, Matzuk MM, Roberts VJ, Cox E, Weiss J, Mather JP,
Woodruff TK 1998 Identification of an inhibin receptor in gonadal
tumors from inhibin a-subunit knockout mice. J Biol Chem 273:398403[Abstract/Free Full Text]
-
Chong H, Pangas SA, Bernard DJ, Wang E, Gitch J, Chen W,
Draper LB, Cox ET, Woodruff TK 2000 Structure and expression of a
membrane component of the inhibin receptor system. Endocrinology 141:26002607[Abstract/Free Full Text]
-
Chen R-H, Derynck R 1994 Homomeric interactions between type
II transforming growth factor-ß receptors. J Biol Chem 269:2286822874[Abstract/Free Full Text]
-
Henis YI, Moustakas A, Lin HY, Lodish HF 1994 The types II and
III transforming growth factor-ß receptors form homo-oligomers.
J Cell Biol 126:139154[Abstract]
-
Gilboa L, Wells RG, Lodish HF, Henis YI 1998 Oligomeric
structure of type I and type II transforming growth factor ß
receptors: homodimers form in the ER and persist in the plasma
membrane. J Cell Biol 140:767777[Abstract/Free Full Text]
-
Tsuchida K, Vaughan JM, Wiater E, Gaddy-Kurten D, Vale WW 1995 Inactivation of activin-dependent transcription by kinase-deficient
activin receptors. Endocrinology 136:54935503[Abstract]
-
Yamashida H, ten Dijke P, Huylebroeck D, Smapath TK, Andries
M, Smith JC, Heldin C-H, Miyazono K 1995 Osteogenic protein-1 binds to
activin type II receptors and induces certain activin-like effects.
J Cell Biol 130:217226[Abstract]
-
Attisano L, Carcamo J, Ventura F, Weis MB, Massague J, Wrana
JL 1993 Identification of human activin and TGF-ß type I
receptors that form complexes with type II receptors. Cell 75:671680[Medline]
-
Hertan R, Farnworth PG, Fitzsimmons KL, Robertson DM 1999 Identification of high affinity binding sites for inhibin on ovine
pituitary cells in culture. Endocrinology 140:612[Abstract/Free Full Text]
-
Lewis KA, Gray PC, Blount AL, MacConell LA, Wiater E,
Bilezikjian LM, Vale W 2000 Betaglycan binds inhibin and can mediate
functional antagonism of activin signaling. Nature 404:411414[CrossRef][Medline]
-
Woodruff TK 1999 Hope, hypothesis, and the inhibin receptor.
Does specific inhibin binding suggest there is a specific inhibin
receptor? Endocrinology 140:35[Free Full Text]
-
Matzuk MM 2000 Editorial: in search of binding-identification
of inhibin receptors. Endocrinology 141:22812284[Free Full Text]
-
Halaby DM, Mornon JPE 1998 The immunoglobulin superfamily: an
insight on its tissular, species, and functional diversity. J Mol Evol 46:389400[Medline]
-
Buck CA 1992 Immunoglobulin superfamily: structure, function
and relationship to other receptor molecules. Semin Cell Biol 3:179188[Medline]
-
Gordis C, Brunet J-F 1992 NCAM: structural diversity, function
and regulation of expression. Semin Cell Biol 3:189197[Medline]
-
Letamendia A, Lastres P, Botella LM, Raab U, Langa C, Velasco
B, Attisano L, Bernabeu C 1998 Role of endoglin in cellular responses
to transforming growth factor-ß. J Biol Chem 273:3301133019[Abstract/Free Full Text]
-
Groome NP, Illingworth PJ, OBrien M, Pai R, Rodger FE,
Mather JP, McNeilly AS 1996 Measurement of dimeric inhibin B throughout
the human menstrual cycle. J Clin Endocrinol Metab 81:14011405[Abstract]
-
Meunier H, Cajander SB, Roberts VJ, Rivier C, Sawchenko PE,
Hsueh AJ, Vale W 1988 Rapid changes in the expression of inhibin
-,
ßA-, and ßB-subunits in ovarian cell types during the rat estrous
cycle. Mol Endocrinol 2:13521363[Abstract]
-
Hall JE, Welt CK, Cramer DW 1999 Inhibin A and inhibin B
reflect ovarian function in assisted reproduction but are less useful
at predicting outcome. Hum Reprod 14:409415[Abstract/Free Full Text]
-
Seifer DB, Scott Jr RT, Bergh PA, Abrogast LK, Friedman CI,
Mack CK, Danforth DR 1999 Women with declining ovarian reserve may
demonstrate a decrease in day 3 serum inhibin B before a rise in day 3
follicle-stimulating hormone. Fertil Steril 72:6365[CrossRef][Medline]
-
Illingworth PJ, Groome NP, Byrd W, Rainey WE, McNeilly AS,
Mather JP, Bremner WJ 1996 Inhibin-B: a likely candidate for the
physiologically important form of inhibin in men. J Clin
Endocrinol Metab 81:13211325[Abstract]
-
Plant TM, Padmanabhan S, Ramaswamy S, McConnell DS, Winters
SJ, Groome N, Midgley Jr AR, McNeilly AS 1997 Circulating
concentrations of dimeric inhibin A and B in the male rhesus monkey
(Macaca mulatta). J Clin Endocrinol Metab 82:26172621[Abstract/Free Full Text]
-
Ohta M, Greenberger JS, Anklesaria P, Bassols A, Massague J 1987 Two forms of transforming growth factor-ß distinguished by
multipotential haematopoietic progenitor cells. Nature 329:539541[CrossRef][Medline]
-
Jennings JC, Mohan S, Linkhart TA, Widstrom R, Baylink DJ 1988 Comparison of the biological actions of the TGF ß-1 and TGF ß-2:
differential activity in endothelial cells. J Cell Physiol 137:167172[Medline]
-
Cheifetz S, Hernandez H, Laiho M, ten Dijke P, Iwata KK,
Massague J 1990 Distinct transforming growth factor-ß (TGF-ß)
receptor subsets as determinants of cellular responsiveness to three
TGF-ß isoforms. J Biol Chem 265:2053320538[Abstract/Free Full Text]
-
Woodruff TK, DAgostino J, Schwartz NB, Mayo KE 1988 Dynamic
changes in inhibin messenger RNAs in rat ovarian follicles during the
reproductive cycle. Science 239:12961299[Medline]
-
Bernard DJ, Woodruff TK 2001 Inhibin binding protein in rats:
alternative transcripts and regulation in the pituitary across the
estrous cycle. Mol Endocrinol 15:654667[Abstract/Free Full Text]
-
Lopez-Casillas F, Cheifetz S, Doody J, Andres JL, Lane WS,
Massague J 1991 Structure and expression of the membrane proteoglycan
betaglycan, a component of the TGF-ß receptor system. Cell 67:785795[Medline]
-
Barbara NP, Wrana JL, Letarte M 1999 Endoglin is an accessory
protein that interacts with the signaling receptor complex of multiple
members of the transforming growth factor-ß superfamily. J
Biol Chem 274:584594[Abstract/Free Full Text]
-
Chen C, Okayama H 1987 High-efficiency transformation of
mammalian cells by plasmid DNA. Mol Cell Biol 7:27452752[Medline]