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Prince Henry’s Institute of Medical Research, Clayton 3168, Victoria, Australia
Address all correspondence to: Dr. David M. Robertson, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, 3168 Victoria, Australia. E-mail: david.robertson{at}med.monash.edu.au
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Introduction |
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- subunit and one of
two ß-subunits (ßA or
ßB) (3) and, based on sequence
homology, are members of the transforming growth factor-ß (TGFß)
family of growth and differentiation factors (4). Homo- or
heterodimers of the ßA- and
ßB-subunits form activins (A, AB, and B) that
are functional antagonists of inhibins (3, 5). Within the
anterior pituitary gland, activins stimulate FSH synthesis and release
by the gonadotropes (3). The mechanism of inhibin action is unclear, although two models have been proposed (6); in one, inhibin competes with the structurally related molecule, activin, for access to the activin receptor, and in the second, inhibin binds specifically to its own receptor and transduces an independent signal. The cellular response to activin, in contrast, has been well characterized and is transduced through two types of membrane-bound, serine-threonine kinases, one a ligand-binding receptor (ActRII) and the other a signal-generating receptor (ActRI) (5). Both receptor types have an extracellular ligand-binding domain, a single transmembrane domain, and a cytoplasmic portion that has serine/threonine kinase activity. ActRI subunits are phosphorylated and thus activated by ligand-bound ActRII (5). Activated ActRI then phosphorylates cytoplasmic Smad proteins, which form part of the postreceptor signal transduction system (7). Inhibin is able to bind ActRII with 10-fold lower affinity than activin, but does not recruit ActRI (8, 9). It is therefore likely that the ability of inhibin to inhibit activin action is based in part upon the dominant negative interaction with receptor types. However, there are cells and tissues in which inhibin is unable to attenuate the activin response (10, 11), suggesting that additional components are required for inhibin action. In addition, specific, high affinity binding sites for inhibin have been detected on ovine pituitary cells (12), and in situ ligand binding studies show specific binding of iodinated recombinant inhibin A and activin A to ovarian and testicular tissue sections (13, 14).
Recently, significant progress has been made in the elucidation of the additional components required for inhibin action. Lewis et al. (15) showed that the type III TGFß receptor, betaglycan, is an inhibin-binding protein that can associate with ActRII. Betaglycan is a heparan sulfate proteoglycan that binds inhibin with high affinity (Kd = 600 pM) and enhances binding of inhibin to ActRII in cells coexpressing ActRII and betaglycan. The resulting complex is resistant to activin competition. Moreover, betaglycan confers inhibin A sensitivity to cell lines that normally respond poorly to this hormone, thereby facilitating inhibin’s antagonism of activin action. Another membrane-anchored proteoglycan with affinity for inhibin, p120, has been identified in bovine pituitary cells (16). p120 interacts strongly with ActRIB in a ligand-responsive manner, and both inhibin A and B partially block the assembly of a p120-ActRIB complex (17). p120 immunostaining is intense in gonadotropes of the rat pituitary and in testicular Leydig cells, both of which are targets of inhibin action (16). However, p120, like betaglycan, does not appear to have an intrinsic signaling function and therefore is unlikely to mediate an inhibin-specific signal alone. Thus, there may be additional binding proteins representing the inhibin receptor present in inhibin target tissues.
We have screened a number of gonadal cell lines as putative inhibin target cells. The present study describes the characterization of inhibin A-specific binding proteins in mouse Leydig (TM3) and Sertoli (TM4) cell lines. A high affinity inhibin A binding complex consisting of five components was identified on the surface of TM3 and TM4 cells, and its characteristics are described.
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Materials and Methods |
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-subunit of
inhibin (R1) and the ßA-subunit of inhibin (E4)
were provided by Biotech Australia (Sydney, Australia). The p120
antibody was a gift from T. Woodruff. Lactoperoxidase (from bovine
milk, 80–120 U/mg protein), BSA, EDTA, phenylmethylsulfonylfluoride
(PMSF), Triton X-100, Nonidet P-40, sodium deoxycholate, and
n-octyl-ß-D-glucopyranoside were
purchased from Sigma (St. Louis, MO). The cross-linker
bis-[sulfosuccinimidyl]suberate (BS3) and
Pansorbin (protein A) were products of Pierce Chemical Co.
(Rockford, IL). Protein G agarose was obtained from Roche Molecular Biochemicals (Mannheim, Germany).
Na125I was a product of Amersham Pharmacia Biotech (Aylesbury, UK). DMEM, Ham’s F-12, glutamine, and FBS
were obtained from Trace Biosciences (Castle Hill, Australia).
Dulbecco’s PBS was obtained from Life Technologies, Inc.
(Gaithersburg, MD). Antibiotics for cell culture were obtained from CSL
(Parkville, Australia), and disposable plastic cluster plates for cell
culture were purchased from Costar (Cambridge, MA).
Recombinant inhibin A and B and activin A
Recombinant human (rh-) inhibin A (obtained from Biotech
Australia was stored at -70 C in 0.1% trifluoroacetic
acid/acetonitrile after purification. The physicochemical and
biological characterization of rh-inhibin A has been described
previously (18). Rh-activin A was purified to homogeneity
from culture medium obtained from mammalian cells stably transfected
with the human inhibin and ßA complementary
DNAs (18). Purity was confirmed by SDS-PAGE under
nonreducing and reducing conditions, and by in vitro
bioassay and activin A enzyme-linked immunosorbent assay. Rh-inhibin B
(lot 1070051, R & D Systems) was determined in this
laboratory to be 1.7-fold more immunoactive on a wt/wt basis than the
inhibin B standard in the inhibin B enzyme-linked immunosorbent assay
(inhibin B detection antibodies and inhibin B standard provided by
Oxford Bioinnovations, Oxfordshire, UK). This preparation possessed
0.28-fold the bioactivity of rh-inhibin A standard (National Institute
of Biological Standards and Control 96/784) in an inhibin in
vitro bioassay using FSH suppression by rat pituitary cells in
culture as an end point with an ID50 of 0.25
ng/ml.
Cell lines
Mouse Leydig (TM3) and Sertoli (TM4) cell lines derived from the
testis of immature BALB/c mice were originally characterized based on
their morphology, hormone responsiveness, and metabolism of steroids
(19). Cell lines were maintained in DMEM/F-12 medium
supplemented with glutamine, antibiotics, and 10% FBS at 37 C. Cells
were cultured as follows, unless otherwise specified: 75,000 cells/1.0
ml medium/well were seeded in 24-well cluster dishes for binding and
competition assays; 1 x 106 cells/6 ml/well
were plated in 6-well cluster dishes for binding and affinity
cross-linking assays. After culture for 1 day, serum-containing medium
was changed to serum-free medium containing insulin (10 µg/ml),
transferrin (1 µg/ml), BSA (6%, wt/vol), and antibiotics
(penicillin, streptomycin, and fungizone) for 24 h.
Binding of [125I]inhibin to TM3 and TM4
cell lines
[125I]Inhibin A and
[125I]inhibin B (
120 µCi/µg) were
prepared as previously described (12). Cell cultures were
washed, then incubated in 50 mM HEPES-buffered DMEM/F-12
medium containing 0.1% BSA and protease inhibitors (0.4 mM
EDTA and 50 ng/ml PMSF). Binding affinity and binding site
concentration were assessed by incubating cells in duplicate for 4
h at 23 C on a rotary mixer (50 cycles/min) with
[125I]inhibin (40,000 cpm/0.4 ml·well,
corresponding to a final concentration of 20–30 pM) in the
absence or presence of increasing doses (5 pM to 20
nM) of unlabeled inhibin A, inhibin B, or activin A.
Nonspecific binding, identified as binding that was not displaceable at
higher concentrations of unlabeled inhibin A (20 nM), was
subtracted from all binding data. The binding reaction was terminated
by placing the culture plates on ice and washing the cell monolayers
three times with 0.2 ml ice-cold PBS (10 mM
NaH2PO4/Na2HPO4
and 154 mM NaCl, pH 7.4). Cells were lysed in 0.30 ml 0.5%
Triton X-100 in PBS for 15 min at room temperature, and radioactivity
in the recovered lysate and a single rinse were pooled and counted in a
-counter. The binding parameters were determined by nonlinear
regression assessment of saturation binding curves using Prism software
(version 2.0, GraphPad Software, Inc., San Diego, CA) and
by Scatchard analysis.
Affinity cross-linking
TM3 or TM4 cells (1 x 106) were
incubated for 4 h at 23 C in 0.6 ml binding medium containing 300
pM [125I]inhibin A or
[125I]inhibin B (400,000 cpm) with or
without increasing concentrations (5 pM to 20
nM) of unlabeled inhibin A, inhibin B, or activin A. Cell
monolayers were washed three times with ice-cold cross-linking buffer
(50 mM HEPES, 125 mM NaCl, 5 mM
KCl, 5 mM MgSO4, and 1 mM
CaCl2, pH 7.4), and bound hormone was
cross-linked to cell surface proteins with BS3
(0.25 mM in cross-linking buffer) for 30 min at 4 C. The
cells were washed twice with ice-cold quench buffer (85 mM
Tris and 30 mM NaCl, pH 7.8), lysed with 0.50 ml
octyl-ß-D-glucopyranoside (1% in quench buffer
containing 0.4 mM EDTA and 50 ng/ml PMSF), and the
recovered lysates were centrifuged (10,000 x g for 10
min). The supernatant was concentrated (Speed-Vac, Savant
Instruments, Farmingdale, NY), combined with Tricine sample buffer, and
binding proteins were separated by nonreducing 6.5% SDS-PAGE
(20). Gels were dried and analyzed by autoradiography
using BioMax film (Eastman Kodak Co., Rochester, NY).
Immunoprecipitation of inhibin cross-linked complexes
To immunoprecipitate [125I]inhibin A
cross-linked complexes, 2 µg antibodies directed against the
C-subunit of inhibin (R1), betaglycan, p120, ActRIIA, or ActRIIB
were added to lysates (1 x 106 cells/ml) of
cross-linked samples and incubated for 16 h at room temperature.
Immune complexes were obtained by the addition of 20 µl Pansorbin
(protein A) or protein G agarose and incubation for an additional
1 h at room temperature. The resulting immobilized immune
complexes were pelleted by centrifugation, washed twice with 1 ml RIPA
buffer [50 mM Tris (pH 8), 150 mM NaCl, 10
mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, and
0.1% SDS], and bound protein was eluted by boiling in 40 µl Tricine
sample buffer. Immunoprecipitated, cross-linked complexes were analyzed
by SDS-PAGE and autoradiography.
HPLC
All liquid chromatographic separations were performed using a
Beckman Coulter, Inc., Gold HPLC system (Palo Alto, CA),
and UV absorbance was monitored at 214 nm with a Beckman Coulter, Inc., 166 UV/visible detector. Samples were analyzed by reverse
phase HPLC (RP-HPLC) using a Brownlee Aquapore RP-300 RP column
(100 x 2.1 mm, 7-µm particle size) on a gradient of 25–70%
acetonitrile in 0.1% trifluoroacetic acid at 0.5 ml/min over 30
min.
Betaglycan, ActRIIA, ActRIIB, and p120 messenger RNA (mRNA)
expression in TM3 and TM4 cell lines
Total RNA was extracted from TM3 and TM4 cells using the RNeasy
Mini Kit (QIAGEN, Hilden, Germany) or CsCl centrifugation.
RNA (0.5 µg) was amplified by RT-PCR in a 50-µl reaction
volume with avian myeloblastosis virus (AMV) reverse
transcriptase and Taq polymerase (Roche Molecular Biochemicals, Castle Hill, Sydney, Australia) using the
following mouse-specific primer pairs: ActRIIA sense, 5'-
CTAATGTGGTCTCTTGGAATGAAC-3'; ActRIIA antisense,
5'-TCATAGACTAGATTCTTTGGGAGG-3'; ActRIIB sense,
5'-CCTCCTGGGGATACCCATGGA-3'; ActRIIB antisense,
5'-TTAGATGCTGGACTCTTTAGGGA-3'; p120 sense,
5'-GGAAGGGAAAGGCATTGGAAAC-3'; p120 antisense,
5'-TGGTTACACTCTTCAAGGACTACGG-3'; betaglycan sense,
5'-AAAAGATGGTCGTGGCTGTAG-3'; and betaglycan antisense, 5'-
GGAGTATGGAGAGAGAAAGCAGG-3'. For betaglycan and p120, RT-PCR was
performed at 50 C for 30 min (RT) and at 95 C for 5 min (denaturation),
followed by 35 cycles of 95 C for 1 min, 55 C for 1 min, and 72 C for 1
min. The elongation step at the last cycle was at 72 C for 5 min to
ensure complete extension of DNA fragments. For ActRIIA and ActRIIB,
real-time PCR was performed for 40 cycles using the FastStart DNA
Master SYBR Green 1 kit (Lightcycler, Roche Molecular Biochemicals) with a final Mg2+
concentration of 3.5 mM, annealing conditions of
64 C for 5 sec and extension at 72 C for 25 sec, and product
quantitation at 79 C. An aliquot of the amplified samples (8 µl) was
electrophoresed on a 1.2% agarose-1 x TBE gel and visualized by
ethidium bromide staining. For ActRIIA and ActRIIB, PCR products were
also analyzed by their melting curve profiles by real-time PCR
(Roche Molecular Biochemicals).
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Results |
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, A and E). When the binding reaction
was performed at 37 C, [125I]inhibin A bound to
cells more rapidly, but the level of binding was 80% of that observed
at 23 C, possibly reflecting internalization and degradation of the
ligand (data not shown). Standard assay conditions of 4-h incubation at
23 C with 1 million cells/culture well were used. Under these
conditions, nonspecific binding of
[125I]inhibin A to TM3 and TM4 cells was low
(1%), and total binding ranged between 8–10% of the added tracer.
Biphasic Scatchard plots with similar Kd values
were obtained with 4- and 16-h incubations (data not shown).
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(B and F).
Scatchard plots of data obtained for
[125I]inhibin A binding to these cell lines
(Fig. 1, C and G) resolved both high and low affinity binding
components (TM3: Kd(1) = 85 ± 35
pM and 4,160 ± 1030 sites/cell;
Kd(2) = 520 ± 200 pM and
12,500 ± 2540 sites/cell; TM4: Kd(1) =
61 ± 5 pM and 2,620 ± 550 sites/cell;
Kd(2) = 520 ± 90 pM and
10,400 ± 3030 sites/cell; Table 1).
Competition curves for binding of [125I]inhibin
A to TM3 and TM4 cells (Fig. 1, D and H) indicated high affinity
inhibin A binding (ED50: TM3 = 97 ± 32
pM; TM4 = 75 ± 28 pM) that was
specific, as assessed by the low cross-reaction with activin A
(ED50 = >100 nM). Interestingly,
inhibin B cross-reacted weakly with
[125I]inhibin A-binding sites
(ED50 = 6 nM with both TM3 and TM4
cells).
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) and TM4 (Fig. 2B) cells,
[125I]inhibin A was incubated with cells for
4 h at 23 C, covalently linked to cell proteins with the
bifunctional cross-linker BS3, then products were
extracted with 1% octyl-ß-D-glucopyranoside,
immunoprecipitated with an antibody directed against the inhibin
-subunit (R1), and analyzed by nonreducing SDS-PAGE and
autoradiography. Affinity-labeled protein bands of relative masses more
than 200, 155, 145, 95, and 70 kDa, corresponding to proteins of more
than 200, 125, 115, 65, and 40 kDa after subtraction of inhibin (Table 2) were evident in both cell lines (Fig. 2A, lanes 2 and 6), and such binding was displaced by excess unlabeled
inhibin A (5 nM; Fig. 2A, lanes 4 and 8), but not by
unlabeled activin A (5 nM; Fig. 2A, lanes 3 and 7). Other
bands present represented free [125I]inhibin A
tracer and a tracer-related species (Fig. 2A, lanes 1 and 5).
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, lane 4)
interacted with the same group of binding proteins as
[125I]inhibin A (Fig. 2B, lane 1). Compared
with inhibin A (Fig. 2B, lane 2), inhibin B (Fig. 2B, lane 3) was a
poor competitor of [125I]inhibin A binding to
TM4 cells. In contrast, inhibin A (Fig. 2B, lane 5) was more effective
in displacing [125I]inhibin B from all affinity
cross-linked complexes than were 10-fold higher concentrations of
unlabeled inhibin B (Fig. 2B, lane 6). Competition curves for binding
of [125I]inhibin B to TM4 cells (Fig. 2C)
confirmed that inhibin A (ED50 = 50
pM) was more effective than inhibin B
(ED50 = 400 pM) at displacing
cell-bound [125I]inhibin B. Similar results
were seen with TM3 cells (data not shown).
To resolve cell-derived [125I]inhibin A-binding
proteins from [125I]inhibin A tracer-related
species, cross-linked TM3 (Fig. 3B)
and TM4 (Fig. 3C) extracts were fractionated by RP-HPLC, analyzed by
SDS-PAGE, and compared with [125I]inhibin A
tracer treated with BS3 (Fig. 3A). Free 30-kDa
[125I]inhibin A eluted predominantly in
fractions 19–25, with some tailing across the chromatogram. Other high
molecular mass [125I]inhibin A tracer-related
components coeluted with [125I]inhibin A in
this region. [125I]Inhibin A cross-linked
complexes in TM3 and TM4 cells were identified in fractions 27–33
(54–60% CH3CN), in which the 145- to 155-kDa
complexes eluted first, followed by the 95- and 70-kDa complexes. The
70-kDa protein complex was more evident in TM4 than TM3 cells (compare
lanes 29–30 of Fig. 4C and Fig. 4B, respectively). The more than 200-kDa cross-linked complex was poorly
resolved by RP-HPLC and was spread across fractions 26–33, suggesting
heterogeneity of this species.
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) and TM4 (Fig. 4C) cells were incubated with
[125I]inhibin A and increasing concentrations
of unlabeled inhibin A (10 pM to 2.5 nM),
cross-linked, immunoprecipitated with an antibody directed against the
inhibin -subunit (R1), and separated by 6.5% SDS-PAGE. Inhibin A
dose-response curves (Fig. 4, B and D), generated after densitometric
analyses of the resulting autoradiograms, indicated that displacement
of [125I]inhibin A from each cross-linked
species (ED50 = 60–110 pM) closely
resembled inhibin displacement from intact TM3
(ED50 = 97 ± 32 pM) and TM4
(ED50 = 75 ± 28 pM) cells
(Table 2). Low affinity inhibin-binding sites in TM3 and TM4 cells were
not resolved by affinity cross-linking. An affinity cross-linking time
course of [125I]inhibin A binding to TM3 and
TM4 cells (Fig. 5, A and C) suggested
that inhibin associates with each of its binding proteins within 15
min. Densitometric analyses of the autoradiograms indicated that the
time courses of binding of [125I]inhibin A to
all of its binding partners on TM3 cells were similar (Fig. 5B),
whereas on TM4 cells the larger molecular mass species may bind first
(Fig. 5D).
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To establish whether the inhibin A-binding proteins include betaglycan,
ActRII, and/or p120 in TM3 and TM4 cells, cultures were labeled with
[125I]inhibin A, cross-linked, and subjected to
immunoprecipitation using antibodies directed against inhibin
-subunit (R1), inhibin ßA-subunit (E4),
ActRIIA, ActRIIB, p120, or betaglycan. All of the antibodies tested
reacted with their target antigens in immunoprecipitation studies,
although the activin receptor antibodies were not particularly
effective (data not shown). In TM4 cells the R1 antibody recognized all
of the cross-linked species (i.e. 70, 95, 145, 155, and
>200 kDa; Fig. 6, lane 2, and Table 2).
The E4 and betaglycan antibodies immunoprecipitated
[125I]inhibin A cross-linked complexes with
relative molecular masses of 95, 145, and more than 200 kDa (Fig. 6, lanes 1 and 3). The 70- and 155-kDa species were not immunoprecipitated
with either of these antibodies. Anti-ActRIIA (Fig. 6, lane 3),
anti-ActRIIB (not shown), and anti-p120 (Fig. 6, lane 4) antibodies did
not immunoprecipitate any [125I]inhibin
cross-linked complexes. Similar results were obtained with TM3 cells.
An excess of unlabeled inhibin A blocked the immunoprecipitation of
[125I]inhibin A cross-linked complexes by
inhibin or betaglycan antibodies, but similar concentrations of
unlabeled inhibin B or activin A competed poorly (data not shown).
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, lanes 2 and 3) despite positive
expression in rat pituitary (Fig. 7A, lane 4) and rat (Fig. 7A, lane 5)
and mouse (Fig. 7A, lane 6) testicular samples. In contrast, betaglycan
(Fig. 7A, lanes 2 and 3), ActRIIA (Fig. 7B, lanes 2 and 4), and ActRIIB
(Fig. 7B, lanes 3 and 5) mRNA expressions were detected in TM3 and TM4
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Affinity cross-linking and immunoprecipitation studies using an inhibin
-subunit-directed antibody identified five binding proteins for
inhibin A with deduced molecular masses, after subtraction of inhibin,
of more than 200, 125, 115, 65, and 40 kDa. Interestingly, similar
studies using an inhibin ßA-subunit antibody
failed to identify the 125- and 40-kDa proteins, possibly reflecting
the involvement of the ß-subunit in binding to these two proteins.
Inhibin A displacement of [125I]inhibin A from
each of the cross-linked species (ED50 = 60–110
pM) closely resembled inhibin displacement from intact TM3
(ED50 = 97 ± 32 pM) and TM4
(ED50 = 75 ± 28 pM) cells,
suggesting that these proteins are all part of a high affinity inhibin
A-binding complex. The components of the low affinity inhibin A-binding
site were not resolved by these studies. An affinity cross-linking time
course of [125I]inhibin A binding to TM3 and
TM4 cells suggests that inhibin A does not initially bind to any single
protein that mediates complex formation but, rather, binds to a
preformed complex.
Precedents exist within the TGFß superfamily for ligand binding to preformed heterooligomeric receptor complexes. Experiments using a yeast two-hybrid interaction assay, double immunoprecipitation analyses, and antibody-mediated immunofluorescence copatching (24, 25, 26) have shown that TGFß receptor type I (TßRI) and TGFß receptor type II (TßRII) can form heteromeric complexes in the absence of TGFß. These studies indicated that the TßRI-TßRII heteromeric receptor complexes preexist in latent forms and are activated by TGFß (27). The TGFß receptor type III, beta-glycan (28), also interacts in a ligand-independent and -dependent manner with TßRI and TßRII, respectively, to form heterooligomeric complexes that mediate TGFß actions (29). In addition to these three receptors, several other high molecular mass (150–180 kDa) cell surface proteins, including endoglin (30) and glycosyl phosphatidylinositol-anchored proteins (31, 32), have been shown to interact with the TGFß signaling receptor complex in different cell types. There is also some evidence that activin A may bind to a heterotrimeric receptor complex, consisting of ActRI, ActRII, and an unidentified 160-kDa protein, on vascular endothelial cells (21). Endoglin can also associate with activin A via association with ActRII (33).
Of the known inhibin-binding proteins only glycosylated betaglycan (>200 kDa) and betaglycan core protein (115 kDa) were confirmed as components of the inhibin-binding complex in TM3 and TM4 cells. Betaglycan is a membrane- anchored proteoglycan with an 853-amino acid core protein that carries heparan sulfate and chondroitin sulfate glycosaminoglycan chains attached to Ser535 and Ser546 (7). The cytoplasmic region of betaglycan is short (43 amino acids) and lacks any discernible signaling motif. Recently, Lewis et al. (15) showed that betaglycan can associate with inhibin (but not activin), and that the resulting inhibin/betaglycan complex competes with activin for access to ActRII, thereby preventing formation of the activin/ActRII complex required for recruitment of ActRI and initiation of the activin-signaling cascade.
Both ActRIIA and ActRIIB are expressed in TM3 and TM4 cell lines and, based on previous studies (15), may correspond to the 65-kDa protein detected by affinity cross-linking. This species was coprecipitated with betaglycan; however, our immunoprecipitation studies using antibodies directed against both ActRIIA and ActRIIB could not confirm that either of the ActRII isoforms is a component of the inhibin A-binding complex in these cells. This may reflect the low level of expression of the 65-kDa protein or the affinities of the ActRII antibodies used. It is worth noting that in [125I]activin cross-linking studies a band consistent in size with the 65-kDa inhibin-binding protein was observed, and this species was displaceable with unlabeled activin (Farnworth, P., unpublished observations). The other recently identified cell surface inhibin-binding protein, p120 (16), is not involved in the inhibin-binding complex in TM3 or TM4 cells. Although a 155-kDa cross-linked complex (125 kDa plus inhibin) of a size consistent with p120 was identified on these cell lines, it was not precipitated using an antibody directed against p120. This is consistent with the failure of RT-PCR studies to detect p120 mRNA in either of these cell lines. Interestingly, Chong et al. (16) detected p120 protein in Leydig cells and in Sertoli cell cytoplasmic processes by immunohistochemical analysis of tissue sections derived from adult rat testis.
Thus, there are two and possibly three unidentified proteins (125, 65?, and 40 kDa) on the surface of TM3 and TM4 cells that bind inhibin A with high affinity and may represent inhibin A-specific receptors. Activin A did not block inhibin A cross-linking to any of the proteins identified, suggesting specificity of the interactions. Interestingly, inhibin B also displayed low cross-reactivity (5%) for [125I]inhibin A binding to TM3 and TM4 cells. This suggests a potential difference in the molecular actions of inhibin A and B and supports the concept that a different class of binding proteins may mediate inhibin B’s actions. However, binding and cross-linking studies showed that inhibin B interacts with the same binding proteins as inhibin A, albeit with lower affinity. Thus, inhibin B may function as a weak agonist of inhibin A in TM3 and TM4 cell lines. The significance of this observation in vivo is unknown, although it would clearly have ramifications in both the male, where inhibin B is the only detectable form of inhibin (34), and the female, where the levels of the two inhibin forms fluctuate throughout the menstrual cycle (35).
The identification of a high affinity inhibin A-binding complex on the surface of Leydig (TM3) and Sertoli (TM4) cell lines provides additional evidence that inhibin acts as a paracrine/autocrine regulator in the testis. Previous studies have indicated that gonadal inhibin A acts directly on interstitial (Leydig) cells, although low level inhibin binding was also detected in Sertoli cells (14, 16, 36). In contrast, activin may exert its activity through interactions with Sertoli and spermatogonial cells (14, 36). As is the case in the pituitary, inhibin appears to antagonize activin action in the testis. Activin acts as an inhibitor of LH/hCG action on androgen biosynthesis at all stages of Leydig cell differentiation, whereas inhibin facilitates LH action on immature Leydig cells (37, 38). Activin also stimulates the proliferation of spermatogonial cells in vitro (39), and inhibin decreases spermatogonial proliferation when injected locally into hamster testis (40).
Two models for inhibin signaling have been proposed (6, 15). In one, inhibin competes with the structurally related molecule activin for access to the activin receptor, and thus blocks activin action. In the second, inhibin binds specifically to its own receptor and transduces an independent signal. The data presented here suggest that inhibin may operate via both mechanisms in TM3 and TM4 cells. By binding beta-glycan, and possibly ActRII, inhibin A could block the activin pathway. At the same time, other components identified in this study as part of the inhibin A-binding complex may initiate downstream signal transduction events. In this way, inhibin A could act to antagonize the effect of activin and exert its own specific actions.
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Acknowledgments |
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Footnotes |
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Received November 15, 2000.
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 D. J. Bernard, K. H. Burns, B. Haupt, M. M. Matzuk, and T. K. Woodruff Normal Reproductive Function in InhBP/p120-Deficient Mice Mol. Cell. Biol., July 15, 2003; 23(14): 4882 - 4891. [Abstract] [Full Text] [PDF]
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L. M. Bilezikjian, A. M. O. Leal, A. L. Blount, A. Z. Corrigan, A. V. Turnbull, and W. W. Vale Rat Anterior Pituitary Folliculostellate Cells Are Targets of Interleukin-1{beta} and a Major Source of Intrapituitary Follistatin Endocrinology, February 1, 2003; 144(2): 732 - 740. [Abstract] [Full Text] [PDF]
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 J.-F. Ethier, P. G. Farnworth, J. K. Findlay, and G. T. Ooi Transforming Growth Factor-{beta} Modulates Inhibin A Bioactivity in the L{beta}T2 Gonadotrope Cell Line by Competing for Binding to Betaglycan Mol. Endocrinol., December 1, 2002; 16(12): 2754 - 2763. [Abstract] [Full Text] [PDF]
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 C. Welt, Y. Sidis, H. Keutmann, and A. Schneyer Activins, Inhibins, and Follistatins: From Endocrinology to Signaling. A Paradigm for the New Millennium Experimental Biology and Medicine, October 1, 2002; 227(9): 724 - 752. [Abstract] [Full Text] [PDF]
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 S. A. Pangas, A. W. Rademaker, D. A. Fishman, and T. K. Woodruff Localization of the Activin Signal Transduction Components in Normal Human Ovarian Follicles: Implications for Autocrine and Paracrine Signaling in the Ovary J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2644 - 2657. [Abstract] [Full Text] [PDF]
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 R. L. Jones, L. A. Salamonsen, Y. C. Zhao, J.-F. Ethier, A. E. Drummond, and J. K. Findlay Expression of activin receptors, follistatin and betaglycan by human endometrial stromal cells; consistent with a role for activins during decidualization Mol. Hum. Reprod., April 1, 2002; 8(4): 363 - 374. [Abstract] [Full Text] [PDF]
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L. A. MacConell, A. M. O. Leal, and W. W. Vale The Distribution of Betaglycan Protein and mRNA in Rat Brain, Pituitary, and Gonads: Implications for a Role for Betaglycan in Inhibin-Mediated Reproductive Functions Endocrinology, March 1, 2002; 143(3): 1066 - 1075. [Abstract] [Full Text] [PDF]
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D. J. Bernard, S. C. Chapman, and T. K. Woodruff Minireview: Inhibin Binding Protein (InhBP/p120), Betaglycan, and the Continuing Search for the Inhibin Receptor Mol. Endocrinol., February 1, 2002; 16(2): 207 - 212. [Abstract] [Full Text] [PDF]
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, nondisplaceable binding)
of an excess of unlabeled inhibin A (final concentration, 10
nM) was performed as described in Materials and
Methods, except that incubations were terminated at the times
indicated. Specific binding (
) was calculated as the difference
between total and nondisplaceable binding at each time point.
Saturation binding curves (B and F) and Scatchard plots (C and G) of
[125I]inhibin A binding to TM3 and TM4 cells. Binding was
performed at 23 C on 1 million cells/well, and nondisplaceable binding
was determined in the presence of 13 nM (200 ng) unlabeled
inhibin A. Data are from one representative experiment (see Table 1
), inhibin B
(
). Data at each point represent the average
result for binding obtained from duplicate wells.
), and 95-kDa (
) cross-linked species
were generated by densitometric analysis of cross-linked bands and were
compared with [125I]inhibin A displacement from TM3 (B,
•) and TM4 (D, •) cells. Data are representative of three separate
experiments. A dose-response curve was not generated for the 70-kDa
cross-linked species because of the faintness of the band in these
experiments. The sizes of molecular mass standards and inhibin
cross-linked bands are shown in kilodaltons.
