From the Departments of Medicine and Neurobiology and
Physiology, Northwestern University, Chicago, Illinois 60611, the
§ Departments of Pathology, Cell Biology, and Molecular and
Human Genetics, Baylor College of Medicine, Houston, Texas 77030, the
¶ Department of Reproductive Medicine, University of California,
San Diego, California 92093, and
Genentech, Inc., South San
Francisco, California 94080
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ABSTRACT |
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Inhibins and activins are dimeric proteins that
are functional antagonists and are structurally related to the
transforming growth factor- (TGF
) family of growth and
differentiation factors. Receptors for activin and TGF
have been
identified as dimers of serine-threonine kinase subunits that regulate
cytoplasmic proteins known as Smads. Despite major advances in our
understanding of activin and TGF
receptors and signaling pathways,
little is known about inhibin receptors or the mechanism by which this
molecule provides a functionally antagonistic signal to activin.
Studies described in this paper indicate that an independent inhibin
receptor exists. Numerous tissues were examined for inhibin-specific
binding sites, including the developing embryo, in which the spinal
ganglion and trigeminal ganglion-bound iodinated inhibin A. Sex cord
stromal tumors, derived from male and female inhibin
-subunit-deficient mice, were also identified as a source of inhibin
receptor. Abundant inhibin and few activin binding sites were
identified in tumor tissue sections by in situ ligand
binding using iodinated recombinant human inhibin A and
125I-labeled recombinant human inhibin A. Tumor cell
binding was specific for each ligand (competed by excess unlabeled
homologous ligand and not competed by heterologous ligand). Based on
these results and the relative abundance and homogeneity of tumor
tissues versus the embryonic ganglion, tumor tissues were
homogenized, membrane proteins were purified, and putative inhibin
receptors were isolated using an inhibin affinity column. Four proteins were eluted from the column that bind iodinated inhibin but not iodinated activin. These data suggest that inhibin-specific
membrane-associated proteins (receptors) exist.
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INTRODUCTION |
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In 1932, McCullagh (1) proposed that the gonads produce a
nonsteroidal factor that regulates pituitary function. The factor was
called inhibin and was finally isolated from bovine, ovine, and porcine
follicular fluid in 1985 (2). The mature inhibin protein core of 32 kDa
is composed of two disulfide-linked polypeptide chains of 18 (-subunit) and 14 (
-subunit) kDa, and its action is to inhibit
pituitary follicle-stimulating hormone
(FSH)1 secretion from
anterior pituitary gonadotrope cells (2-4). Concurrent purification of
FSH-stimulating factors from follicular fluid identified activin, a
dimeric, disulfide-linked protein composed of two inhibin
-subunits
(2-4). Five
-isoforms have been identified and are designated
A-,
B-,
C-,
D-, and
E-subunits (2, 5-7). Dimeric
inhibin A (
-
A), inhibin B (
-
B),
activin A (
A-
A), activin AB
(
A-
B), and activin B
(
B-
B) have been purified from follicular
fluid. Homo- or heterodimeric assembly of the
C-,
D-, and
E-subunits has not been
demonstrated to date.
The inhibin and activin -subunits are 30% homologous to TGF
based on alignment of conserved cysteine amino acids. Crystal structures of two TGF
family members (TGF
2 and osteogenic
protein-2) have been solved, and the overall structural similarity
between these proteins implies that a conserved topology exists between members of the superfamily (8, 9). Proteins within the TGF
family
span multiple species and appear to be central factors in bone
formation and morphogenesis of embryos, and the genes that encode them
may function as tumor suppressor genes (10).
Activin activity is inhibited by interaction with a bioneutralizing
binding protein called follistatin (11). Follistatin binds both inhibin
and activin through the -subunit; however, the ability of
follistatin to neutralize inhibin activity cannot be determined until a
cellular activity is described for inhibin that is not confounded by
activin. Follistatin is structurally complex and is highly conserved
between species with two conserved amino acid differences between rat
and human and 87% homology with the Xenopus follistatin
homologue (12, 13).
Cellular response to activin is transduced through two single
membrane-spanning serine-threonine kinase subunits (3). The ligand
binds a type II receptor (70-75 kDa) that transphosphorylates a type I
receptor (50-55 kDa). The holo-receptor complex is then competent to
initiate intracellular signaling cascades (3). Two ligand binding type
II receptor genes have been identified (type RII and type RIIB receptor
subunits), and four alternatively spliced variants of the type IIB
receptor have been cloned (14). The isoforms differ by changes in an
extracellular proline-rich region immediately preceding the
transmembrane domain and in a region between the transmembrane domain
and serine-threonine kinase domain. Inhibin is able to bind the type II
receptor but not recruit a type I receptor (15-17). It is therefore
likely that the ability of inhibin to inhibit activin action is based,
in part, upon this dominant negative interaction with receptor
subunits. Receptors for many of the individual ligands have been
identified, and they have structural characteristics similar to those
described for the activin receptor. Inhibin -subunit is distinct
within the TGF
superfamily because it is capable of heterodimeric
assembly (with activin
-subunit) and is not able to homodimerize.
The inhibin
-subunit is one of four proteins distantly related to the core ligands (activin/TGF
/bone morphogenic protein). Other ligands with distant homology include Mullerian inhibiting substance (the receptor of which is analogous to the TGF
structure), growth differentiation factor-9 (the receptor of which has not been
identified), and glial-derived nerve growth factor (the receptor of
which includes a glycosylphosphatidyl inositol-anchored binding protein
that presents the ligand to a tyrosine kinase receptor) (18-20).
An alternative hypothesis is that a separate inhibin receptor or inhibin accessory protein exists that mediates an inhibin-specific signal. Supporting the hypothesis that an independent inhibin receptor exists, inhibin-specific binding sites have been identified on ovarian granulosa cells and testicular Leydig cells (21-23). The most compelling evidence indicative of an inhibin receptor or cell surface ancillary binding protein is the identification of an inhibin-specific protein complex in a hematopoietic cell line (K562) (16). Taken together, these data indicate that inhibin activity involves an inhibin-specific receptor. Therefore, studies were initiated to isolate the inhibin receptor.
Mice that are genetically deficient in the inhibin -subunit are
deficient in inhibin A and inhibin B and overexpress activin A and
activin B (24, 25). These inhibin knockout mice are normal during
embryonic and early postnatal development. However, microscopic focal
gonadal tumors of the mixed or incompletely differentiated sex cord
stromal (granulosa/Sertoli cell) type develop in mice as early as 4 weeks of age. Eventually, 100% of these mice will have either
unilateral or bilateral tumors and die of a cancer cachexia-like
syndrome due to the activin secreted from the tumors (26). Upon
investigation, these tumors were found to bind inhibin specifically,
and data are presented identifying tumor-derived inhibin receptor
proteins.
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EXPERIMENTAL PROCEDURES |
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Recombinant Ligands-- Recombinant human (rh)-inhibin A and rh-activin A were obtained from Genentech, Inc. (South San Francisco, CA). The ligands were formulated in a buffer of 0.15 M NaCl and 0.05 M Tris, pH 7.4. Recombinant human follistatin (288aa) was obtained from the National Hormone and Pituitary Distribution Program (NIDDK, National Institutes of Health).
Iodination of Ligands--
rh-activin A and rh-inhibin A were
iodinated by a modified lactoperoxidase method. Briefly, 5 µg of
ligand was diluted in 0.4 M sodium acetate, pH 5.6, and 0.5 nmol of Na125I (0.5 nmol/mCi on calibration date), 0.5 IU
of lactoperoxidase, and 0.25 nmol of H2O2 were
added sequentially. The ligands were incubated at ambient temperature
with intermittent vortexing for 5 min. The reaction was quenched with
450 µl of phosphate-buffered saline + 0.05% Tween 20 + 0.5% bovine
serum albumin (Intergene, 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 Inc.). The specific activity of the ligands
was approximately 100 µCi/µg (range, 89-102 µCi/µg). To verify
that the iodinated inhibin and activin used in these studies were
active, ligands were assayed for bioactivity by perifusion of dispersed
rat pituitary cells and quantitation of FSH mRNA (27). Both
ligands retained full biological activity (data not shown). Moreover,
activin was able to bind type RII receptor expressed in a rat pituitary
cell line (
T3), indicating that this ligand would be able to bind its receptor in the eluate if it existed (data not shown).
In Situ Ligand Binding--
In situ ligand binding
was performed as described previously using rat embryos collected on
E13, E15, and E17 or control ovaries and gonadal tumors collected from
inhibin -subunit knockout mice (22, 28). Briefly, 12-µm cryocut
tissue sections were incubated for 3 h at room temperature in
blocking buffer: Dulbecco's modified Eagle's medium:F-12 (1:1), 20 mM HEPES, 0.05% cytochrome C, 0.3% bovine serum albumin,
0.01 mg/ml phenylmethylsulfonyl fluoride, 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, 40 pM
125I-rh-activin A or in the presence of 40 nM
excess homologous ligand (to define nonspecific background) or
heterologous ligand (to define low affinity binding to heterologous
ligands). The slides were washed in phosphate-buffered saline (two
times for 10 min each); fixed in 3.7% formalin, 2% glutaraldehyde (10 min); rinsed in water (four times for 1 s each); and allowed to
dry. Dry slides were exposed to autoradiographic film for 1-14
days.
Isolation of Membrane Proteins--
20-25 grams of gonadal
tumor tissue isolated from adult male and female inhibin -subunit
knockout mice were mechanically homogenized in 0.15 M NaCl
plus a mixture of protease inhibitors (aprotinin (10 µg/ml, serine
protease inhibitor); EDTA-Na2 (0.5 mg/ml, metalloprotease
inhibitor); leupeptin (0.5 µg/ml, serine and cysteine protease
inhibitor), and pepstatin (0.7 µg/ml, aspartate protease inhibitor)).
Insoluble material was removed by centrifugation at 10,000 × g. The resultant cleared extract was centrifuged at 100,000 × g, and the membrane pellet was resuspended
in 85 mM Tris, pH 7.8, 0.1% octyl
-glucoside, 30 mM NaCl, and protease inhibitors. The suspension was
centrifuged at 100,000 × g, and insoluble membranes
were discarded. Four separate isolations of protein were performed.
Construction of an Inhibin Affinity Column and Affinity
Purification--
An inhibin affinity column was prepared by coupling
5 mg of rh-inhibin A to AffiGel 10 (Bio-Rad) following the
manufacturer's protocol. Coupling efficiency was >98%. Soluble
membrane proteins were passed in series though a blank AffiGel-10
column (bed volume, 5 ml) and then over the rh-inhibin A-coupled column
(bed volume, 5 ml). The eluate was reprocessed twice through the
inhibin column. The column was washed extensively with 25 mM Tris, pH 7.5, 150 mM NaCl, 0.1% octyl
-glucoside. After a stable baseline was reached, the column-bound
proteins were eluted with 100 mM glycine, pH 3.0, 0.1%
octyl
-glucoside and immediately neutralized with 3 M
Tris, pH 8.5. The column fractions were analyzed by chemical cross-linking and mobility on SDS-PAGE (26). Briefly, 100,000 cpm of
biologically active iodinated inhibin or activin was added to
equivalent aliquots of column fractions and incubated at room temperature for 1 h. Disuccinimidyl suberate, dissolved in
Me2SO (Pierce), was added to a final concentration of 500 µM and allowed to react with the bound complexes for
1 h at room temperature. The products were analyzed by 12%
SDS-PAGE. The gels were dried and subjected to autoradiography.
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RESULTS |
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The successful identification of activin/TGF/BMP/MIS receptor
subunits has proceeded based on degenerate oligonucleotide priming from
the conserved serine-threonine kinase domain of the activin receptor
subunit. Studies in our laboratory were initiated to identify a subunit
receptor that bound inhibin and not activin using degenerate
oligonucleotides against the conserved serine-threonine kinase domain
in pituitary and inhibin
-subunit tumor tissue. No independent
receptor isoform was identified using this approach (data not shown).
Second, an expression library generated from rat ovaries was generated
and screened for inhibin-binding proteins. No inhibin-specific binding
proteins were identified using this methodology (data not shown).
Ligand binding studies in ovary (22), testis (23), and the developing embryo (Fig. 1) indicate that separate inhibin and activin binding sites exist in specific cellular groups. In the embryo, inhibin-specific binding sites are detected in the trigeminal ganglion and spinal ganglion. Because activin receptors and follistatin are also present near or coincident with the inhibin binding sites in ovary, testis, and the embryo, no attempt was made to purify the inhibin receptor from these source tissues (22, 23, 28). These studies indicate, however, that several tissues or cellular groups have inhibin binding sites that are distinct from activin binding sites.
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Based on our in situ ligand binding studies, we hypothesized
that gonadal tumor tissues arising from the genetic deletion of the
inhibin -subunit in mice would be a potential source of inhibin
receptor. Gonadal tumors (both ovarian and testicular) were collected
from adult inhibin
-subunit-deficient mice and embedded immediately
on dry ice. Tumor tissues were processed to examine inhibin or activin
binding sites using in situ ligand binding (Fig.
2). The tumors had a mixed sex cord stromal phenotype, and all cells bound labeled ligands (Fig. 2, B
(125I-inhibin A) and F (125I-activin
A)). Addition of 1000-fold molar excess unlabeled inhibin A competed
with the 125I-inhibin A (Fig. 2C), and excess
activin A competed with 125I-activin A (Fig.
2G). Inhibin and activin did not cross-compete with
heterologous labeled ligand, indicating that the tissues have distinct
inhibin and activin binding sites (Fig. 2, D and H). Sections of ovaries obtained from wild-type littermate
animals had low level inhibin binding to antral granulosa cells (Fig. 2A) and higher levels of activin A binding to ovarian
follicles (Fig. 2E). The pattern and intensity of inhibin
and activin binding in control mouse ovaries is identical to the
binding pattern of the two ligands previously described in normal rat
ovary (22).
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Because the tumor tissues appeared to be enriched for an inhibin binding moiety and had little activin binding (which we predicted would represent binding to follistatin, a cytoplasmic protein), ovarian and testicular tumor tissues were homogenized, and membrane proteins were isolated from multiple tumors. Protein was incubated with iodinated inhibin, cross-linked with disuccinimidyl suberate, and analyzed under nonreducing conditions on denaturing SDS-PAGE (Fig. 3). In addition, iodinated inhibin formed complexes with proteins in both ovarian and testicular tumor extracts. Incubation of membrane extracts with 100-fold excess unlabeled rh-inhibin A reduced the inhibin binding in ovarian tumor extracts and competed efficiently for the binding in testicular tumor homogenates. Ovarian and testicular tumor homogenates were passed over an inhibin affinity column, resulting in an enrichment of proteins that bind inhibin. Ovarian and testicular tumor homogenates were combined from this experiment forward.
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To examine whether the proteins that could be isolated would bind specifically to inhibin, solubilized membrane protein from ovarian and testicular tumors was passed over the inhibin affinity column twice, the column was washed, and the protein that bound the immobilized inhibin was eluted using a low pH buffer. Fractions representing the eluted protein peak were neutralized with Tris, and aliquots were incubated with iodinated inhibin and iodinated activin. The proteins were cross-linked with disuccinimidyl suberate and analyzed by SDS-PAGE (Fig. 4). When incubated with fractions representing the protein peak eluted from the inhibin affinity column, 125I-rh-inhibin A was specifically shifted upward in the chromatogram, whereas 125I-rh-activin A was not (Fig. 4, A and B, lanes 5-9). Four specific 125I-rh-inhibin A complexes corresponding to sizes of 130, 116, 86, and 72 kDa were identified (Fig. 4A, lane 7, a, b, c, and d, respectively). These proteins were identified in four independent experiments. However, attempts to microsequence the proteins were unsuccessful due to blocked N termini or lack of sufficient material. Internal sequence analysis was attempted in the cases of blocked N termini; however, no sequence data were generated due to the small amount of protein recovered in the purifications.
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To confirm that the proteins were receptors and did not include follistatin, two experiments were conducted. First, iodinated activin was used as a probe in a ligand binding blot to detect follistatin in the solubilized membrane protein fraction and in fractions isolated from the inhibin affinity column (Fig. 5A). Iodinated activin was used in this experiment because iodinated inhibin does not bind follistatin in this format (data not shown). Iodinated activin binds rh-follistatin in the positive control lane, yet it does not detect free follistatin in the total membrane protein fraction or in the column eluate fractions. This suggests that the homogenate and eluate are essentially devoid of free follistatin and indicates that the membrane preparation is likely free of cytoplasmic proteins. To determine whether membrane-bound proteoglycan-associated follistatin was present in the membrane or column fraction, an antibody against follistatin was used in an immunoblot analysis of isolated protein (Fig. 5B). The antibody detected a high molecular weight protein in the membrane fraction but not in the column eluate. These data suggest that follistatin is membrane-associated but is not in a form capable of binding activin and indicate that the proteins eluted from the inhibin affinity column do not include follistatin.
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DISCUSSION |
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Inhibin is an gonad-derived dimeric protein hormone. The principle
biological activity of inhibin is to suppress pituitary FSH secretion
in a classic endocrine fashion (2, 29, 30). It may also participate in
ovarian follicle development and oocyte maturation (31-33). Closely
related to inhibin (-
) is activin (
-
). Activin stimulates
pituitary FSH synthesis and secretion in a dose-dependent
manner and causes follicle atresia; however, it is synergistic with
inhibin in stimulating oocyte maturation (2, 21, 31-33). In addition
to its role in ovarian and pituitary function, activin is known to
regulate erythrodifferentiation (34), promote neuronal survival (35),
and regulate mesoderm development in Xenopus and mouse
embryos (36, 37). Activin regulates these functions through a family of
receptor kinases (38-40). One subunit, the type RII(B) receptor, binds
the ligand and then transphosphorylates a second type RI receptor. The
ligand-RII-RI complex is a functional serine threonine kinase, the
functional targets of which are the members of the Smad cytoplasmic
protein family (41).
Although specific activin receptor subunits have been identified and
cloned, efforts to isolate an inhibin receptor have not been
successful. Numerous studies were done to identify an inhibin receptor.
The inability to identify an inhibin receptor using oligonucleotides
directed against conserved regions of known activin receptors suggests
that the inhibin receptor may differ from the activin receptor
subunits. Numerous in situ ligand binding studies were done
using a wide variety of tissues to localize inhibin binding sites that
could be indicative of novel inhibin function and a source of potential
inhibin receptor. In the ovary, inhibin-specific binding sites are
associated with the granulosa cell (22). The ovary is capable of
producing inhibins, activins, and follistatins in response to pituitary
gonadotropins and to local growth regulatory factors (42). Inhibin A
and inhibin B are released from the ovary and regulate pituitary FSH in
a traditional endocrine feedback manner (43, 44). In addition to
endocrine regulations, the follicular granulosa cell, theca, cell and
ooctye are able to respond to inhibin and to activin, and these effects
can be modulated by the binding protein follistatin. Inhibin stimulates
granulosa cell proliferation, theca cell androgen production, and
ooctye maturation (21, 32-35, 45, 46). Each of these effects may be
coordinated through an independent inhibin receptor. In the testis,
inhibin-specific binding sites are present on interstitial cells that
are also positive for 3-hydroxysteroid dehydrogenase, suggesting
that these cells are the steroidally active Leydig cells (23).
In experiments described in this paper, we identified inhibin binding
sites in the trigeminal ganglion and spinal ganglion of the developing
rat embryo. The ability of inhibin to act on these sites requires that
the ligand be present. Inhibin -subunit mRNA is expressed in the
somites of the embryonic rat on E12, in the dorsal root ganglion from
E12 to E20 (47), and in the somites of the 10.5 day mouse embryo (48);
immunoreactive
-subunit protein is localized in the somites of chick
embryos (49). Moreover, both inhibin
- and
-subunit mRNAs are
detected in different stages of the early mouse embryo and in embryonic
stem and embryonic carcinoma cells that model early murine fetal
development (50, 51). Likewise, human pre-embryos have been shown to
secrete immunoreactive
-subunit protein (52). Therefore, the
developing embryo likely produces inhibin in restricted cellular sites,
and this inhibin may have effects specifically on cellular loci such as
the trigeminal ganglion and spinal ganglia, where inhibin-specific binding sites have been localized. Further analysis will be required to
delineate the specific effect(s) of inhibin (and activin) on these
cellular sites.
A powerful model system to delineate the physiological relevance of
inhibin and activins is the genetic deletion of subunit genes through
homologous recombination (the knockout mouse model). A series of
animals deficient in the - or
-subunits, in the receptors for
activin, and in the activin/inhibin-binding protein, follistatin, have
been generated (24, 53-57). Animals deficient in the inhibin
-subunit develop gonadal tumors (24-26), activin type RII receptor
is down-regulated (57), and, as shown herein, the tumors bound
iodinated inhibin A preferentially. Upon identification of the ovarian
tumors as a tissue source of a potential inhibin receptor, we initiated
studies to purify the protein using classical affinity chromatography
methods. Four inhibin-binding proteins were partially purified. The
receptor proteins were identified by cross-linking labeled ligand to
putative receptor and examining the retardation of the complex on
SDS-PAGE. Our inability to sequence the N terminus of the proteins
isolated by affinity purification means that we can only speculate on
the relationship between the proteins that bind iodinated inhibin. The
two smaller proteins (complexes of 86 and 72 kDa) may be proteolytic
cleavage products of the larger proteins. A broad spectrum mixture of
protease inhibitors was used throughout the purification procedure;
however, degradation of the larger proteins is a possibility.
Alternatively, the larger molecular weight shifts may represent
complexes of the smaller proteins with the iodinated inhibin. A third
possibility is that several classes of inhibin receptor proteins exist
in the tumor tissues. For example, inhibin may bind a yet-unidentified
type II receptor, may bind and activate a type RI receptor, may have a
structurally dissimilar receptor, or may use an adapter protein, such
as type RIII receptor, to act in concert (or competition) with activin
type RII or RI receptors. The existence of an inhibin-specific receptor
and competition with the activin receptor are not mutually exclusive
conclusions. Indeed, an inhibin-specific protein band has been
identified in the context of activin type RII and RI receptors in human
erythroid precursor cells (16). Whether the protein identified in the
K562 cell system is similar to one of the proteins identified in this
study remains to be clarified. Indeed, the complete elucidation of the
functional relationship between inhibin and activin receptors awaits
the cloning of the inhibin receptor.
The fact that the inhibin receptor can be clearly identified in the knockout mouse tumor tissue is significant. It is known that one of the phenotypes of these tumors is low expression of type RII receptor, likely due to down-regulation by activin (24-25, 57). Similarly, the inhibin receptor may be up-regulated by persistent activin or by the lack of negative feedback of inhibin. Clearly, additional studies are necessary to determine what role, if any, activin and inhibin have in tumorigenesis and whether an inhibin receptor is expressed in human epithelial ovarian tumors.
In summary, inhibin binding moieties were found to be abundant in
gonadal tumors that arise from the genetic elimination of the
-subunit of inhibin. Four inhibin-binding proteins were purified by
inhibin affinity chromatography from these tumors. Finally, the
proteins eluted from the inhibin column were found to be distinct receptor proteins and not follistatin. Further work will be required to
clone and characterize the inhibin receptor-generated proteins identified by affinity column purification; however, the results presented in this study represent an important first step toward the
elucidation of the inhibin receptor and signal transduction system.
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ACKNOWLEDGEMENTS |
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We thank Dr. Patrick Sluss (Massachusetts General Hospital) for the follistatin antibody and Mary Slikowski (Genentech, Inc.) for useful conversations. We also thank the Genentech Rare Reagents Program for inhibin and activin and NIDDK for follistatin.
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FOOTNOTES |
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* This study was supported by a Genentech Research Award (to T. K. W.), American Cancer Society, Illinois Division, Grant 96-17 (to T. K. W.), NCI, National Institutes of Health Grant CA60651 (to M. M. M.), and National Institutes of Health Grant HD 29464 (to V. J. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Northwestern University, Department of Medicine, Tarry Bldg I5-716, 303 Chicago Ave., Chicago, IL 60611. Tel.: 312-503-4229; Fax: 312-908-9032; E-mail: tkw{at}nwu.edu.
1 The abbreviations used are: FSH, follicle-stimulating hormone; TGF, transforming growth factor; rh, recombinant human; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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