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Prince Henry’s Institute of Medical Research, Clayton, 3168 Victoria, Australia
Address all correspondence and requests for reprints to: Dr. David Mark Robertson, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton 3168 Victoria, Australia.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit and a structurally related ß-subunit, either
ßA or ßB. Circulating inhibin is primarily
a product of the gonads and is involved in the closed loop feedback
inhibition of FSH synthesis and secretion by gonadotrophs of the
anterior pituitary gland (1, 2, 3). Disulfide-linked dimers of inhibin
ß-subunits form activins, which act as local growth and
differentiation factors in many tissues of the body. Within the
anterior pituitary gland, activins inhibit the functions of several
cell types, but stimulate FSH synthesis and release by the
gonadotrophs. In some circumstances, most notably the control of FSH,
inhibin has been shown to antagonize the actions of activin. Inhibin and activin are members of the transforming growth factor-ß (TGFß) superfamily of pleiotropic growth and differentiation factors that also includes bone morphogenetic proteins (BMP), Mullerian Inhibitory Substance (MIS), and glial cell line-derived neurotropic factor (GDNF) (1, 4, 5). Receptors for several members of this superfamily have now been identified (5, 6). Nearly all of these receptors share structural similarities in their extracellular, transmembrane, and intracellular regions, the last incorporating a serine/threonine phosphokinase domain. Transmembrane signaling by most TGFß superfamily members, including activin, characteristically involves ligand binding to a constitutively active type II receptor serine/threonine kinase and subsequent recruitment of a type I receptor, the serine/threonine kinase activity of which is then activated.
Inhibin signaling, in contrast, is poorly understood. Attempts to clone
the putative inhibin receptor(s) based on sequence homology with
receptors for activin/TGFß/BMP/MIS have been unsuccessful to date
(7), which raises the possibility that inhibin signals by a mechanism
that is atypical of the superfamily. Indeed, the recent elucidation of
signaling pathways for a distantly related subgroup of TGFß
superfamily members that includes GDNF, neurturin, and perhaps
persephin (8, 9, 10, 11) has established precedents for diversity of signaling
within the superfamily. Efforts to identify the inhibin receptor
through conventional ligand binding assays have been hampered by the
loss of biological activity that accompanies radiolabeling of inhibin
(12) (Hertan, R., P. G. Farnworth, K. L. Fitzsimmons, and D.
M. Robertson, personal observations) and by the binding of
inhibin to activin receptors (13, 14). Studies of inhibin binding using
radiolabeling (7, 15, 16, 17) and fluorescent labeling (18) methods have
identified binding sites in several tissues, including the pituitary
and gonads. Several inhibin-binding proteins were recently isolated
from gonadal tumors that arose in inhibin -subunit-deficient mice,
and the binding of [125I]inhibin was not displaced by
activin (7). However, there is limited information about the affinity,
specificity, and structure of such binding sites and no knowledge of
their intracellular signal transduction mechanisms.
The present report describes the iodination of inhibin by a modified lactoperoxidase procedure and the isolation and characterization of biologically active iodinated inhibin. Studies with [125I]inhibin provided evidence of binding sites on ovine anterior pituitary cells that show high affinity, saturability, reversibility, and specificity for inhibin, characteristics consistent with the presence of an inhibin receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Iodination of inhibin
Inhibin was iodinated by a lactoperoxidase procedure (19) with
modifications. Inhibin (2 µg, unless specified otherwise) was diluted
in 30 µl 0.5 M phosphate buffer (pH 7.4), after which
Na125I (0.5 mCi/5 µl) and lactoperoxidase (4 µg/20 µl
water) were added. The reaction mixture was then incubated for 2 min at
room temperature, in the absence of exogenous
H2O2, before the addition of PBS (20 µl; 10
mM phosphate buffer, pH 7.4, and 0.15 M NaCl),
then the reaction mixture was gel filtered on a NAP-5 column in PBS to
remove free 125I. The iodinated protein product collected
from the NAP-5 column was fractionated by gel filtration chromatography
on a Sephadex G-100 column (30 x 1.5 cm) in 0.1 M
HCl, and 1.5-ml fractions were collected. During the optimization
experiments, the masses of inhibin and 125I, the number and
concentration of H2O2 additions, and the
duration of incubation of the reaction were systematically varied in an
attempt to minimize aggregation and oxidative damage of the tracer and
to achieve a predictable, low level of 125I incorporation
per molecule of inhibin. These trials revealed an inverse relationship
between the mass of inhibin to be iodinated and the amount of earlier
eluting material (pools A and B in Fig. 1
) from 37% or more of the total counts
recovered from the column when 1 µg was used, to 33% for 2 µg and
22% for 5 µg. When the mass of inhibin was held constant at 1 µg,
variation in the other parameters had little effect on the extent of
aggregation, which generally accounted for 36–42% of the total
radioactivity eluted from the gel filtration column. The specific
activity of the inhibin tracer decreased when the mass of inhibin was
increased or when the concentration of either peroxide or radioactive
iodine was decreased. Neither the extent of aggregate formation nor the
specific activity of the tracer was affected by variation of the
reaction time between 1.5–15 min with a single addition of
peroxide.
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Inhibin in vitro bioassay
Primary cultures of ovine anterior pituitary cells were prepared
by trypsin/deoxyribonuclease digestion of the diced tissue from four to
eight sheep pituitary glands freshly obtained from a local abattoir
(22). Cells were suspended in DMEM-Ham’s F-12 medium buffered with
bicarbonate and containing antibiotics, nonessential amino acids for
MEM, glutamine, and 10% FBS. The cells were plated in 48-well culture
plates at a concentration of 7.5 x 104 viable
cells/0.30 ml·well and preincubated for 48 h at 37 C in a
humidified atmosphere of 5% CO2 in air. Fractions of
iodinated inhibin were bioassayed by the method of Tsonis et
al. (23) with modifications. Before addition of sample to the cell
culture, the cells were washed with serum-free DMEM-Ham’s F-12 medium
containing 0.1% BSA, and the wash medium was replaced with 0.30 ml
fresh medium containing serum and an estrogen antiserum (Y17, final
dilution of 1:200). Iodinated inhibin samples (0.10 ml; 70,000–100,000
cpm, highest dose) and the unlabeled inhibin as standard (0.10 ml, 1
ng/ml, highest dose, previously calibrated by bioassay against rh
inhibin First International Standard 91/624) were serially diluted in
DPBS-0.1% BSA for addition to triplicate wells in the bioassay. After
addition of inhibin samples, the cultures were incubated for a further
65–72 h, then the medium was collected and assayed for FSH by RIA
either immediately or after storage at -20 C (22), using second
antibody/polyethylene glycol precipitation with the following reagents:
ovine FSH-13-SIAFP RP-2 (lot AFP-4117A) as the standard, ovine FSH
(AFP-5679-C) for iodination by the Iodogen method, rabbit antiovine FSH
serum R 20 36/37 (final dilution, 1:40,000 in normal rabbit serum), and
goat antirabbit IgG serum GAR 11 (1:80 final dilution). Logit log(dose)
transformations and parallel line statistics were used to determine
biologically active masses of iodinated inhibin samples.
Binding of [125I]inhibin to cultured
ovine pituitary cells
Pooled [125I]inhibin fractions (see
Results) were gel filtered on a PD-10 column into DPBS-0.1%
BSA for cell binding studies. Ovine pituitary cells prepared as
described above were preincubated for 2 days at 37 C under 5%
CO2 in air at a concentration of 2 million cells/0.50 ml
DMEM-Ham’s F-12 medium·well in 24-well plates unless otherwise
specified in the text. Two cell preparations were fractionated on a
discontinuous Percoll gradient before culture (24), and cells retained
at the 26/40% and 40/44% Percoll interfaces were pooled and plated at
1 million cells/well for subsequent determination of inhibin binding.
After the preincubation, cell monolayers were washed, then incubated at
37 C for 60 min with medium (0.30 ml) and [125I]inhibin
(
40,000 cpm in 0.10 ml) in the presence or absence of unlabeled
inhibin or other hormones (0.5–200 ng in 0.10 ml medium). The cultures
were placed on ice to terminate the reaction, and the cells were washed
three times with ice-cold culture medium. Triton X-100 (0.1% (vol/vol)
in DPBS; 0.50 ml) was added and left for 60 min at room temperature,
then radioactivity in the recovered lysate was counted in a
-counter.
Binding affinity and binding site concentration were assessed by
incubating [125I]inhibin (40,000 cpm, 300–400
pg/well, corresponding to a final concentration of 19–26
pM) with 23 increasing doses of unlabeled inhibin (20 pg to
200 ng/well, corresponding to 1.3 pM to 13 nM).
Inclusion of more replicates was not possible because of plate to plate
variation in binding. Nondisplaceable binding, determined in the
presence of 200 ng unlabeled inhibin, was subtracted from all binding
data. Two approaches were employed to determine the binding parameters.
The first was the widely advocated nonlinear regression assessment of
saturation binding curves (25, 26) using Prism software (version 2.0
from GraphPad Software, Inc., San Diego, CA). Analysis of
the present binding data by this method gave evidence of saturability
and the presence of one type of binding site. The second approach was a
graphical (Scatchard) analysis that consistently showed evidence of two
sites. The high affinity binding sites formed a low proportion (<10%)
of the total, which seems to have precluded its resolution by nonlinear
regression assessment, and for this reason Scatchard analysis was
preferred. To obviate the major criticism associated with Scatchard
analysis (25, 26, 27), inhibin binding was assessed over a broad saturation
range (10–90%).
Binding of [125I]inhibin to cell
lines
Human embryonic kidney (293) and human acute T cell leukemia
(Jurkat) cell lines were assessed for [125I]inhibin
binding. The adherent 293 cells were maintained on 15-cm culture plates
in DMEM-Ham’s F-12 medium, and the suspension cultures of Jurkat cells
were grown in RPMI 1640 medium in flasks, each medium supplemented with
10% FBS, antibiotics, and glutamine as for the pituitary cell
cultures. Cells for binding studies were harvested from near-confluent
cultures. Binding of [125I]inhibin to 293 cells was
determined as described for ovine pituitary cells after incubation for
3.5 h at 4 C. For Jurkat cells, aliquots of 2 million cells in
0.10 ml medium were incubated in the presence of tracer (40,000 cpm
in 0.10 ml) with or without competing unlabeled ligand in 0.10 ml
medium (see Fig. 5 for details), with rotation of the cell suspensions
for 3 h at room temperature in 1.5-ml Eppendorf
tubes. The reaction was terminated on ice, and an aliquot of each cell
suspension was washed by centrifugation through dibutyl phthalate at
12,000 rpm for 30 min at 4 C (28). The tips of tubes containing the
cell pellets were cut off with scissors, and their radioactivity was
measured.
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Results |
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and Table 1). Each pool contained inhibin
immunoactivity, determined by RIA, and the material from all but the
first pool was also bioactive based on the dose-dependent inhibition of
FSH secretion from ovine pituitary cells in culture. However, most of
the inhibin bioactivity localized to the ascending and descending limbs
of the second region of radioactivity (Fig. 1B, pools C and D),
consistent with the presence of 31-kDa inhibin, as confirmed by
SDS-PAGE (data not shown). Tracer recovered in these pools represented
about 10% of the starting material, based on immunoactivity
determinations (Table 1). More significantly, the optimized iodination
procedure yielded [125I]inhibin in pools C and D, with an
iodine incorporation ratio of 1.7–2.0 mol 125I/mole
inhibin and a bioactivity/immunoactivity (B:I) ratio of 0.62–0.77
(Table 1) that proved suitable for studies of inhibin binding to cells
in culture. Tracer from pool D was used in all of the following binding
studies.
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Binding of [125I]inhibin to cultured
ovine pituitary cells
[125I]Inhibin bound to ovine pituitary cells in a
time- and temperature-dependent manner. At 37 C, specific binding
reached a maximum by 60 min and remained stable for at least another 60
min (Fig. 2). At 4 C, maximum binding was
reached by 14 h and remained stable for another 10 h (data
not shown). In other experiments, specific binding was proportional to
cell number up to 2 million cells/well during incubations for 60, 90,
and 180 min (data not shown). Standard assay conditions chosen were
60-min incubation at 37 C with 2 million cells/culture well. Under
these conditions, specific binding accounted for between 0.8–1.8%,
and nondisplaceable binding determined in the presence of 200 ng (13
nM) unlabeled inhibin ranged between 0.8–1.9% of the
radioactivity added. Attempts to increase the specific binding and/or
reduce nondisplaceable binding by purification of gonadotrophs on
continuous or discontinuous Percoll gradients (25–65%) gave little
improvement (20% only) in inhibin binding due to the wide range in
size and density of the gonadotrophs (results not shown).
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). Saturation binding isotherms provided
evidence of saturability (e.g. Fig. 3A). Scatchard plots of
[125I]inhibin binding data were curvilinear
(e.g. Fig. 3B). Scatchard analysis of the data obtained at
37 C revealed two binding sites with average dissociation constants
(Kd) of 0.28 nM (site 1) and 3.9 nM
(site 2), corresponding to approximately 250 and 3100 binding
sites/pituitary cell, respectively (Fig. 3B and Table 2). [125I]Inhibin binding
to ovine pituitary cells was specific, as assessed by the low
cross-reaction with activin A (<2%) and TGFß (<0.2%), and
negligible cross-reaction with follistatin (<<0.1%; Fig. 4).
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). No specific binding was detected with
Jurkat cells (Fig. 5). |
Discussion |
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The two identified inhibin binding sites differ in affinity for the ligand by a factor of 14. The presence of two classes of binding sites with a similar difference in Kd (0.13–0.4 vs. 1.5–3.5 nM) has previously been observed for activin binding to primary cultures of rat hepatocytes (30) and to several (but not all) leukemia/carcinoma cell lines (28, 31). Transfection of an endothelial cell line with the activin type II receptor also resulted in the emergence of two binding sites with Kd values of 0.25 and 6.6–16 nM, respectively (29). In the latter studies, the high affinity site was attributed to the dimerization of endogenous type I/III receptors with the transfected type II receptor, whereas the low affinity site was ascribed to activin binding to the excess transfected type II receptor. In the absence of the transfected type II receptor, no specific binding of activin was observed. On this basis, the two inhibin-binding sites on pituitary cells observed in the present study may represent an analogous inhibin receptor type I/II dimer and a putative inhibin receptor type II monomer, respectively.
The identified inhibin-binding sites may otherwise be ancillary binding
proteins, such as have been found for other TGFß superfamily members,
for example endoglin and ß-glycan for TGFß (36, 37), follistatin
for activin and osteogenic protein-1/BMP-7 (38, 39), GDNFR- for GDNF
(8, 40), and NTNR- for neurturin (9, 10). These binding proteins
exhibit high affinities for their respective ligands, but do not
generate an intracellular signal, although they may hold the ligand in
the juxtamembrane environment or "present" the appropriate ligand
to its matching receptor. For instance, GDNF, neurturin, and perhaps
persephin comprise a structurally distinct subgroup of TGFß
superfamily ligands. Within this subgroup, the ligand initially binds
with high affinity to its cognate ancillary protein (e.g.
GDNFR-) that is loosely associated with the cell membrane via a
glycophosphatidylinositol anchor, and each binary complex
subsequently recruits and activates a common membrane-spanning protein,
Ret, that includes an intracellular tyrosine phosphokinase domain
(8, 9, 10, 11, 40). The nature of the binding sites for the GDNF group of
ligands thus differs greatly from those discovered for activin, TGFß,
MIS, and BMP, but the pattern of ligand binding to one membrane-bound
site and recruitment of a second by the complex provides a common theme
that is consistent with the present findings for inhibin.
Nonlinear regression analysis of inhibin saturation binding isotherms,
the recommended method for determining binding site characteristics
(25, 26), did not account for the high affinity binding evident in the
Scatchard plots and resolved a single binding site with an average
Kd of 0.63 nM, corresponding to approximately
750 binding sites/pituitary cell. The failure of nonlinear regression
analysis to resolve the high affinity inhibin binding site on ovine
pituitary cells can be attributed to several factors, including
restriction of the number of observations per assay by technical
limitations in the cell culture and the finding that the higher
affinity sites represent a low proportion (7%) of the total.
Two cell lines were investigated for the presence of high affinity inhibin-binding sites. No response of Jurkat cells to inhibin has been documented, and no specific binding of inhibin to these cells was observed. The 293 cell line expresses activin receptors (Vale, W., personal communication) and thus was expected to show limited binding of [125I]inhibin due to the documented cross-reaction of activin type II receptors with inhibin (13, 14, 32). Activin and inhibin displaced bound [125I]inhibin from 293 cells with similar potency in accordance with this prediction, which suggests that 293 cells do not express the high affinity type of inhibin-binding sites identified in the ovine pituitary.
A major impediment to the identification and characterization of the
putative inhibin receptor has been the loss of bioactivity that
accompanies inhibin iodination. Others found that inhibin can be
radioiodinated by a lactoperoxidase procedure with retention of
bioactivity (12, 41), but studies employing such tracer have not
provided details of inhibin-binding site affinity and number. The
present iodination procedure, which employs mild oxidizing conditions
(i.e. relies on endogenous H2O2 in
the reaction solution) also damages inhibin, as evident by the presence
of inhibin aggregates after gel filtration and iodinated inhibin
fractions (pools A and B) with reduced B:I ratios. Each of the mature
subunits, C and ßA, of inhibin A has five
tyrosine residues (42, 43). However, fractions of iodinated inhibin
were identified that contained inhibin with high bioactivity and an
appropriate level of 125I incorporation of 1–2 mol/mol
inhibin. This level of incorporation is important because it means,
firstly, that the bioactivity more likely reflects the action of
iodinated inhibin molecules than of residual unlabeled inhibin that
would be present with an average 125I incorporation ratio
of less than 1 mol/mol inhibin. Secondly, excessive radioiodination of
ligand can lead to a significant difference between the binding
affinity of the labeled and the unlabeled form, with consequent
distortion of the binding data (26, 44). Finally, inhibin tracer with a
higher average incorporation ratio of 5 mol [125I]/mol
inhibin that was produced by the published lactoperoxidase procedure
gave a lower B:I ratio than the optimized tracer and proved unsuitable
for the cell binding studies.
In conclusion, the identification of saturable inhibin-binding sites with high affinity and specificity on ovine pituitary cells is consistent with the presence of specific inhibin receptors on these cells. The present findings support the proposition that inhibin signaling includes an inhibin-selective receptor, analogous to the cases for signaling by other TGFß superfamily members, including activin. It remains to be determined whether inhibin signaling operates through these binding proteins.
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Acknowledgments |
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Footnotes |
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Received August 3, 1998.
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References |
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-subunit knockout mice. J Biol
Chem 273:398–4033 is an orphan
member of the GDNF/neurturin/persephin receptor family. Proc Natl Acad
Sci USA 95:5801–5806-2 and GFR-3 are two
new receptors for ligands of the GDNF family. J Biol Chem 272:33111–33117-3,
a novel co-receptor belonging to the glial cell line-derived
neurotrophic receptor family. J Biol Chem 273:3502–3508This article has been cited by other articles:
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