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Department of Neurobiology and Physiology (H.C., S.A.P., D.J.B., E.W., J.G., L.B.D., T.K.W.), Department of Biochemistry, Molecular Biology, and Cell Biology (W.C.), Northwestern University, Evanston, Illinois 60208-2850; and Genentech, Inc. (E.T.C.), South San Francisco, California 94080-4990
Address all correspondence and requests for reprints to: Teresa K. Woodruff, Ph.D., Northwestern University, Department of Neurobiology and Physiology, O.T. Hogan 4–150, 2153 North Campus Drive, Evanston, Illinois 60208. E-mail: tkw{at}nwu.edu
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Abstract |
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Introduction |
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Inhibin is a disulfide-linked, dimeric glycoprotein composed of an
-subunit and one of two ß-subunits (2). Homodimers of the
ß-subunits form activin, a functional antagonist of inhibin. The
inhibin ß-subunit places that molecule into the larger transforming
growth factor-ß (TGFß) superfamily (3). Inhibin is distinct from
most TGFß-like ligands because its -subunit is capable of
heterodimeric assembly (with activin ß-subunits), but it is not able
to homodimerize with unpaired -subunits. Because inhibin is a member
of the TGFß superfamily and antagonizes activin action, we
hypothesized that some aspects of the cellular response pathways
activated by this ligand would be similar to those activated by TGFß.
Based on the unique structural nature of the inhibin -subunit and
the heterodimeric -ß ligand, we also predicted that novel
components of the inhibin signaling pathway exist.
While little is known concerning the mechanism of inhibin action, the activin signaling pathway has been described. The cellular response to activin is transduced through two membrane-bound, serine-threonine kinases: a ligand binding receptor subunit (ActRII) and a signal generating protein (ActRI) (4). ActRI subunits are phosphorylated by ligand-bound ActRII. Activated ActRI then phosphorylates cytoplasmic Smad proteins (5). Three classes of Smad proteins have been identified. These include the receptor Smads, which are phosphorylated by activated type I receptors, the inhibitory Smads, which inhibit receptor-mediated Smad phosphorylation, and the common activator Smad known as Smad4 (5).
Inhibin inhibits pituitary FSH synthesis and release and stimulates Leydig and theca cell androgen production (1, 2, 6, 7). One hypothesis regarding inhibin action is that inhibin binds and inactivates the ActRII subunit, thereby antagonizing activin action (8, 9). The association of inhibin with ActRII subunits blocks ActRI subunit phosphorylation and uncouples the activin signal transduction pathway (8, 9). Thus, when the inhibin concentration exceeds activin in a target cell, inhibin can act to disengage the activin signal pathway by competitive inhibition of the activin-binding receptor subunit. Because the affinity of inhibin for ActRII is approximately 10-fold lower than activin for ActRII, this mechanism of action is plausible when inhibin is in excess of the local activin signal.
In addition to the dominant-negative model of inhibin-mediated activin
antagonism, strong evidence exists to support an inhibin-specific
receptor or inhibin accessory protein(s) that mediate an
inhibin-specific signal. The most compelling evidence supporting an
independent inhibin receptor is the fact that inhibin is not able to
antagonize all of activin’s actions. For example, inhibin does not
antagonize activin-stimulated liver cell apoptosis (10) nor does it
block activin-induced animal cap development (M. Whitman, personal
communication). Further, inhibin and activin both promote oocyte
maturation (11) and Leydig cell P45017
messenger RNA (mRNA) accumulation (6). These experimental observations
suggest that a unique inhibin-specific signal transduction pathway may
exist. Alternatively, an inhibin-binding protein may act in concert
with activin receptor components to permit the antagonistic as well as
cooperative relationship between the two ligands. These two mechanisms
are not mutually exclusive, and cells may have the ability to use
inhibin in a number of different ways to modify an activin signal.
Identifying an inhibin-binding and signal-transducing molecule will
increase knowledge about the normal reproductive cycle and will lead to
a more complete understanding of diseases in which inhibin-signaling
cascades may be abnormally regulated. Toward this end, the present
manuscript describes efforts to isolate inhibin-binding proteins. The
bovine pituitary was used as a starting material to isolate and
characterize an inhibin- binding protein based on physiological data
(inhibin inhibits bovine pituitary FSH secretion), direct binding data,
and the availability of large amounts of tissue (12, 13, 14). The
purification strategy implemented in the present study was based on a
method developed to isolate inhibin-binding proteins from the gonadal
tumors of inhibin -subunit knockout mice (15). A protein that binds
inhibin and is expressed in inhibin target tissue, was isolated and
cloned, and its characteristics are described.
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Materials and Methods |
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Ligand affinity chromatography and microsequencing
Inhibin A was coupled to two support matrices. Inhibin was
coupled to cross-linked agarose (Affi Gel 10) through free amino groups
of inhibin according to the manufacturer’s instructions (Bio-Rad Laboratories, Inc., Richmond, CA). Inhibin was also coupled to
Affi Gel HZ through carbohydrate groups on the inhibin -subunit. A
blank column was prepared by immobilizing Tris to Affi Gel 10; 500
g of bovine pituitaries were suspended in 150 mM NaCl with
1 mM EDTA, 25 µg/ml aprotinin, 0.5 µg/ml leupeptin, and
0.7 µg/ml pepstatin and disrupted in a Waring blender. The pituitary
mixture was stirred at 5C for 1 h and then centrifuged at
10,000 x g to remove particulate matter. The resulting
supernatant was centrifuged at 105,000 x g for 3
h. The ultracentrifugation pellet was resuspended in 85
mM Tris, pH 7.8, 0.1% octyl ß glucoside
(OßG), 30 mM NaCl, 1 mM
EDTA, and 25 µg/ml aprotinin. The sample was treated with 1 U/µg
DNase (Roche Molecular Biochemicals, Indianapolis, IN)
overnight to reduce viscosity and improve sedimentation of particulate
matter during ultracentrifugation. DNase-treated samples were
sedimented at 105,000 x g for 3 h, and clear red
supernatants were removed and sedimented again at 105,000 x
g for 3 h. The affinity columns were washed extensively
with 25 mM Tris, pH 7.5, 150
mM NaCl, and 0.1% OßG. The membrane proteins
were eluted from the columns with 100 mM glycine,
pH 3, and 0.1% OßG. The eluents were neutralized with 3
M Tris, pH 8. Addition of excess inhibin A to the
membrane-solubilized proteins resulted in inhibition of one band of 120
kDa from the Affi Gel 10 column. The protein with molecular mass of 120
kDa, which will be referred to as p120, was enriched and purified to
homogeneity using reverse-phase HPLC (15 µm bead diameter C4
resin in a 2-cm column, 0–70% n-propanol/trifluoroacetic
acid gradient). The receptor-enriched reverse-phase HPLC
fractions were run on reduced 10% SDS-PAGE, transferred to
polyvinylidenefluoride (PVDF), and stained with Coomassie blue.
The single protein band at 120 kDa believed to be an inhibin-binding
protein was cut out of the transblot and sequenced. This protein was
purified only when inhibin was coupled through amines and not when the
carbohydrate moieties of the inhibin -subunit were used in the
coupling chemistry. These data suggest that the column purification was
specific for binding sites that were blocked when the space between the
-subunit (the only subunit that is glycosylated) and the
inhibin-binding protein was hindered.
p120 complementary DNA (cDNA) cloning
Internal sequence for the isolated protein was obtained from the
Genentech microsequencing facility (Genentech, Inc., South
San Francisco, CA). Two sequences were identical to the peptide
sequence and were similar to each other [accession numbers: Y10523
(IGDC1) and AF034198 (IGSF1)]. A cDNA was amplified by PCR from
human testes cDNA (CLONTECH Laboratories, Inc., Palo Alto,
CA) giving a full-length Y10523 cDNA. PCR errors, detected by sequence
analysis, were corrected to match Y10523.
Northern blot analysis
RNA was size separated on a formaldehyde-agarose gel and
transferred to a nylon filter by capillary action. The filter was
hybridized using a 32P-labeled rat cDNA probe
corresponding to part of the extracellular domain of the rat p120 cDNA.
After stringent washes, the blot was exposed to x-ray film for 2
days.
Tissue distribution analysis
In situ ligand binding was performed as described
previously (16). Briefly, 12-µm cryocut tissue sections were
incubated for 3 h at room temperature in blocking buffer: DMEM:F12
(1:1), 20 mM HEPES, 0.05% cytochrome C, 0.3%
BSA, 0.01 mg/ml phenylmethylsulfonylfluoride, 0.01% bacitracin, 0.4
µg/ml leupeptin. Slides were then incubated at room temperature
overnight in the same buffer containing 40 pM
125I-rh inhibin A, activin A, or in the presence
of 40 nM excess homologous ligand (to define
nonspecific background) or heterologous ligand. The ligands were
iodinated 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 U 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 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 in situ ligand-binding studies was approximately
100 µCi/µg. The slides were subsequently washed in PBS (two times
for 1 sec), PBS (two times for 10 min), and fixed in 3.7% formalin,
2% glutaraldehyde (10 min); rinsed in water (four times for 1 sec)];
and allowed to dry. Dry slides were exposed to x-ray film for 1–14
days and then dipped in NTB-3 emulsion (Eastman Kodak Co.,
Rochester, NY).
Immunohistochemical studies were done using an antibody directed against the protein sequence determined for the bovine p120 protein. The bovine and human epitopes are identical; however, 4 of 17 amino acids (aa) differ between the human and rat epitope. Paraffin-embedded testicular sections from adult rats were the gift of Dr. Erwin Goldberg, Northwestern University. The p120 polyclonal primary antibody was diluted in PBS (1:500), and the sections were incubated overnight at room temperature, washed, incubated with a biotinylated goat-antirabbit secondary antibody (Vector Laboratories, Inc., Burlingame, CA), washed, and incubated in ABC reagent (Vectastain ABC kit, Vector Laboratories, Inc.). The signal was detected using the DAB substrate kit (Vector Laboratories, Inc.). Sections were counterstained with hematoxylin. Control sections were not incubated in the primary antibody and did not have detectable brown stain (data not shown). The specificity of the antibody was confirmed by preabsorption of the antibody with recombinant p120 (100 µg of protein of a transient transfection extract) or oligopeptide (at a final concentration of 1 µg/ml). Tissue sections were analyzed using a Eclipse E600 microscope (Nikon, Melville, NY).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Twenty to 25 proteins were eluted
from the inhibin affinity column as determined by visual inspection of
SDS-PAGE (Fig. 1B). Two different membrane preparations revealed a
similar pattern of proteins eluted from the inhibin affinity column. To
eliminate false-positive results, inhibin was incubated with the
membrane-associated proteins for 30 min before affinity chromatography.
Two independent preparations of inhibin-competed membrane proteins
were analyzed by affinity chromatography. The abundance of one of the
eluted proteins was reduced when membrane proteins were preincubated
with inhibin (Fig. 1B, shown by arrow). This protein, with
an apparent molecular mass of 120 kDa under reducing conditions, was
analyzed further.
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).
cDNA cloning of p120
The oligopeptide sequence derived from microsequence analysis was
used to search NCBI-GenBank and was found to match a partial sequence
of a human cDNA isolated by two independent groups [accession numbers
Y10523 and AF034198 (IGSF1)] (Fig. 2A;
Refs. 17, 18). The human p120 protein has five predicted
extracellular N-linked glycosylation sites and a large putative
extracellular domain (1234 aa in length) followed by a single,
hydrophobic, potential transmembrane domain and a short cytoplasmic
tail (53 aa) that is 19% serine and threonine. The extracellular
domain contains a series of Ig-like domains. Using the GenBank match
(Y10523), primers were designed that contained convenient unique
internal restriction sites, and a cDNA was amplified from human testis
cDNA by PCR. The cDNAs were ligated together to form the full length
4.01-kb coding region encoding a predicted protein of 1336 aa.
Subsequently, partial sequence was obtained for a rat ortholog by
screening a testis cDNA library. Tissue expression and abundance of
p120 mRNA in male rat pituitary, testis, and liver were examined (Fig. 2B). Northern blot analysis revealed two major transcripts of
approximately 2.0 and 4.6 kb, corresponding to transcripts of similar
sizes detected in human testis (data not shown).
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, A and B).
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, A and B).
Activin specifically associated with testicular germ cells while
inhibin-specific binding sites were detected in Leydig cells
(Fig. 4, A–D). Low-level inhibin binding was detected in Sertoli
cells. p120 protein was detected primarily in the Leydig cell by
immunohistochemical analysis of thin tissue sections derived from adult
rat testis (Fig. 4, E and F). The positive signal was competed by
cellular extracts from p120 transfected cells (Fig. 4G) or with the
oligopeptide (data not shown), indicating that the antibody staining is
specific for p120. Long Sertoli cell cytoplasmic processes are positive
and can be detected throughout the interior portion of all seminiferous
tubules (Fig. 3H). Thus, p120 colocalizes with inhibin-binding sites in
the testis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p120 was not eluted from the inhibin column when the coupling chemistry
was through the carbohydrate moieties of the -subunit. This result
suggests that the interacting portion of inhibin for p120 is related to
the -subunit. The -subunit has limited sequence similarity to any
other TGFß ligand, including activin, and is distinct because it is
capable of heterodimeric assembly with activin ß-subunit but will
not homodimerize (20). Other ligand subunits with distant homology
to the TGFß core ligands include Müllerian- inhibiting
substance (whose receptor is similar to the TGFß receptor
structure), growth differentiation factor-9 (GDF-9, whose receptor has
not been identified), and glial-derived nerve growth factor (GDNF)
(whose receptor includes a glycosylphosphatidyl inositol-anchored
binding protein that presents the ligand to the Ret tyrosine kinase
receptor) (21, 22, 23). An antibody against the inhibin -subunit
N-terminal 1–30 aa bioneutralizes inhibin action in vivo
(24). Taken together, these data imply that the -subunit N-terminal
1–30 peptide may serve as a ligand-interacting domain.
Binding assays for inhibin are difficult due to the poor quality of the
iodinated ligand. Despite this well documented impediment (14), inhibin
iodinated using lactoperoxidase is capable of suppressing pituitary
FSH, suggesting that binding studies using modified (iodinated) ligand
can bind and act upon receptor proteins. A second challenge in the
binding studies is that inhibin may interact with numerous surface
receptors with different relative affinities. The activin type II or
type IIB receptor subunits, follistatin, or 2-macroglobulin all bind
inhibin, and the relative abundance of each of these proteins may
affect inhibin binding to its receptor (25, 26, 27). None of these
potential inhibin-binding proteins were purified from bovine pituitary
homogenates; however, p120 was efficiently purified. This result
suggests that the success of isolating p120 relative to the other
molecules is due primarily to the high-affinity interaction of p120 and
inhibin A. Thus, in tissues where p120 is expressed, interaction of
inhibin A with p120 is predicted to exceed interactions with the other
proteins. Previous binding studies using bovine pituitary cells
revealed inhibin-specific binding sites with an apparent dissociation
constant (Kd) of 1.2 nM (13) while
ovine pituitary cells had two inhibin-specific binding sites with
Kd values of 0.28 nM and 3.9
nM (14) consistent with our hypothesis. The p120 molecule
was purified from bovine pituitary membranes based on its ability to
bind inhibin A. Inhibin B may also bind p120, but insufficient purified
recombinant protein is available to determine the relative affinity of
this isoform for p120.
Importantly, nonoverlapping inhibin- and activin-binding sites are found in the testis. Activin binds germ cells while inhibin binds Leydig cells, and the predominant site of p120 production is the Leydig cell. This cellular localization suggests that inhibin and activin action are compartmentalized by various binding proteins such as p120 and ActRII. Further, the two binding compartments may permit differential action of inhibin and activin within the testis. Additional studies will be required to determine whether the inhibin- binding protein has a function and whether that function is restricted to inhibin alone or whether there is an interaction with other TGFß ligands.
The p120 Ig-like domains bear structural resemblance to other cytokine receptors (28) and some adhesion molecules (29). p120 bears no sequence or structural homology to the canonical TGFß-family type II or type I receptor subunits (4, 30). It is not surprising that p120 represents a departure from the previously described TGFß family of receptor subunits in light of the unsuccessful attempts to clone an inhibin receptor or inhibin-binding proteins based on homology to these proteins (15). p120 appears to be a unique and novel addition to the family of cell surface receptor molecules that associate with TGFß-like ligands.
The TGFß or activin type III receptor is also dissimilar from other cytokine receptors and bears no sequence or structural homology to the canonical type II or type I receptors (31, 32). The TGFß type III receptor and accessory protein endoglin are large proteins with no consensus signal transduction motif. There is no sequence similarity between these receptors and p120; however, p120 may act as a type III-like molecule in regulating inhibin action. For example, five TGFß isoforms are synthesized by various cells, and the predominant isoforms found in most tissues are TGFß1 and TGFß2 (33). TGFß1 binds directly to type II and activates type I; however, TGFß2 requires an additional ancillary protein, a single membrane-spanning, nonsignaling protein (type III), that presents TGFß2 to the type II-type I complex (34). An activin type III receptor has been identified in endothelial cells and can be assembled with activin A into a type III-type II-type I complex (35). Recent data in the K562 hematopoietic cell line are suggestive of an ancillary protein requirement for inhibin function (36). Ancillary proteins, such as p120, may assemble complexes of activin type II and type I receptors into a configuration that is not only antagonistic to an intrinsic activin signal, but also promotes inhibin signal transduction. Alternatively, there may exist inhibin-specific RII-like subunits, and the availability of p120 permits an investigation of an RII-like interaction.
The type III receptors assemble into disulfide-linked homodimers or may oligomerize into larger integral membrane glycoprotein structures (31, 32). Similarly, p120 can oligomerize into a higher ordered structure (S. Chapman and T. K. Woodruff, unpublished observation). Endoglin, a dimeric type III-like protein, has a RGD domain in the extracellular domain, making it a candidate for cell-cell interactions (32). p120 does not have an RGD domain; however, the Ig loops of the large extracellular domain may allow cell-cell interaction. The mechanism by which cellular contact might propagate an inhibin, activin or TGFß signal is not known and represents a new area of investigation for the field.
One additional interesting feature of p120 is a potential glycosyl
phosphatidylinositol (GPI)-linkage domain. GDNF, neurturin, and
artemin are members of a new family of factors within the TGFß
superfamily that use accessory receptor proteins for activity (37).
Ligand binds a glycosyl phosphatidylinositol (GPI)-linked protein
called GDNF family receptor s (GFR-1 and GFR-2) (38). These
proteins then associate with the Ret tyrosine kinase (39). To date, the
GDNF-like ligands are the only TGFß superfamily ligands that do not
use serine-threonine kinase-based receptors and therefore represent a
separate subclass of ligands within the larger family. Because p120
does not have intrinsic kinase activity, it is likely that this
ligand-binding protein associates with other signal-transducing
proteins. The interacting proteins may include ActRII, ActRI, a type
III-like molecule, or a tyrosine-kinase signaling molecule. Each of
these possibilities will be investigated and should provide new insight
into the mode of inhibin action.
p120 is produced by the pituitary and testis. It was predicted that
p120 would be expressed in the pituitary because this is the
compartment from which the protein was purified. Equally important, if
the protein is relevant to FSH regulation, then it must be expressed in
the gonadotrope cell. p120 and FSHß were detected in the same cell,
suggesting a colocalization of p120 with gonadotrophs. The testis has
the most distinct and nonoverlapping compartmentalization of inhibin
and activin binding of all tissues examined: inhibin binding is highest
in the Leydig cells, and activin binding is in specific germ cell
populations (present paper and Ref. 19). Inhibin binding can be
specifically competed by inhibin and not by activin. The Leydig cell
was positive for p120 as detected by the polyclonal p120 antibody.
Moreover, previous studies suggested that inhibin binds Sertoli cells,
although the resolution of in situ ligand binding was not
sufficient to identify clearly this compartment as inhibin binding.
p120 is positive in the cytoplasm of Sertoli cells, suggesting that the
low grain density detected over these cells represents true inhibin
binding. As was discussed earlier, this binding may predict activity.
Inhibin stimulates Leydig cell P45017 mRNA
accumulation while activin stimulates 3ß-hydroxysteroid
dehydrogenase mRNA (6). As predicted, inhibin can antagonize
activin-induced 3ß-hydroxysteroid dehydrogenase mRNA
accumulation; however, activin does not antagonize inhibin-stimulated
P45017. Much of the physiology of inhibin has
been directed at its role in pituitary FSH regulation and an apparent
antagonism of activin function. The identification of p120, together
with the differential compartmentalization of inhibin- and
activin-binding sites and functional actions of the two ligands,
suggests an additional level of complexity.
p120 had been previously identified as a large molecular weight expressed protein and then as a member of the a cell adhesion molecule family. In humans, the gene for the receptor is encoded by the Xq25 chromosome and is 20 kb with 19 exons (17, 18). Duplications in this region of the human chromosome are associated with X-linked pan-hypopituitarism or Pettigrew Syndrome, and loss of heterozygosity is related to advanced human ovarian carcinoma (40, 41). Both the gonads and the pituitary respond to inhibin, and these functional abnormalities identified in humans may imply an important role for the ligand (through p120) in pituitary development and in ovarian carcinogenesis. In concert with the later observation, a loss-of-inhibin mouse model has been developed and the animals develop, with 100% penetrance, gonadal tumors (42, 43).
In summary, inhibin plays critical paracrine and endocrine roles in regulating pituitary FSH, and absence of inhibin results in ovarian and testicular cancer in knockout mice. Until now, none of the downstream signaling proteins in the inhibin signal transduction cascade had been identified. An inhibin-binding protein (p120) was purified from several kilograms of bovine pituitaries using affinity chromatography. An oligopeptide sequence was determined for the isolated and purified protein and the cDNA cloned from human testis RNA. In addition, p120 is abundant in the testicular Leydig cell, a compartment enriched for inhibin-binding sites and devoid of activin-binding sites. The purification and cloning of an inhibin-binding protein provide a key reagent to our further understanding of the mammalian reproductive axis as well as the origin of reproductive oncogenesis.
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Acknowledgments |
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Footnotes |
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Received November 9, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit knockout mice. J Biol Chem 273:398–403-RET receptor
complex. Neuron 21:1291–1302[CrossRef][Medline]
-Inhibin is a tumour-suppressor gene with gonadal specificity
in mice. Nature 360:313–319[CrossRef][Medline]
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D. Gaddy-Kurten, J. K. Coker, E. Abe, R. L. Jilka, and S. C. Manolagas Inhibin Suppresses and Activin Stimulates Osteoblastogenesis and Osteoclastogenesis in Murine Bone Marrow Cultures Endocrinology, January 1, 2002; 143(1): 74 - 83. [Abstract] [Full Text] [PDF]
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T. M. Plant and G. R. Marshall The Functional Significance of FSH in Spermatogenesis and the Control of Its Secretion in Male Primates Endocr. Rev., December 1, 2001; 22(6): 764 - 786. [Abstract] [Full Text] [PDF]
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G. P. Risbridger, J. F. Schmitt, and D. M. Robertson Activins and Inhibins in Endocrine and Other Tumors Endocr. Rev., December 1, 2001; 22(6): 836 - 858. [Abstract] [Full Text] [PDF]
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A. J. Duleba, T. Pehlivan, R. Carbone, and R. Z. Spaczynski Activin |
