The activins and inhibins are dimeric cytokines with important
roles in development and physiology(1) . Although first
discovered as gonadal peptides that regulate spermatogenesis and
oogenesis, both cytokines are widely expressed and cause diverse
responses in cell types from both intragonadal and extragonadal
tissues(1, 2) . Dependent on cell, tissue, and
biological activity, activin or inhibin can act as a positive or
negative effector, but both are generally antagonists of the
other(1, 2, 3) . Activins are
:
homodimers while inhibins are
:
heterodimers that share the
subunit. Both are members of the transforming growth factor-
(TGF-
) (
)superfamily of ligands whose activities are
mediated by transmembrane serine/threonine kinase
receptors(4, 5, 6, 7, 8) .
Similar to TGF-
1(9) , activin has recently been
demonstrated to be an inhibitor of hepatocyte DNA synthesis (10) and to reduce liver mass by induction of hepatocyte
apoptosis when administered in vivo(11) . In contrast
to TGF-
1, which is expressed in nonparenchymal cells after partial
hepatectomy and is a candidate paracrine regulator(9) ,
expression of the activin
A subunit increases in parenchymal
hepatocytes; therefore, activin appears to be a candidate for an
autocrine regulator of hepatocyte proliferation(10) . The
active receptor complex for homodimeric ligands of the TGF-
family
including activin appears to obligatorily involve a heterodimeric
complex of two genetically distinct kinases which have been designated
type I and type II according to sequence homology and
size(4, 5, 6, 7, 8) . Our
laboratory cloned two type I serine/threonine kinase receptors, SKR1 (12) and SKR2(13) , from a well differentiated human
hepatoma cell, HepG2, which exhibits many properties of parenchymal
hepatocytes. We have shown that four SKR2 variants, SKR2-1,
SKR2-2, SKR2-3, and SKR2-4, arise by alternative
splicing and poly(A) addition and vary in carboxyl-terminal domains,
potential phosphorylation sites, and kinase activities(13) .
Subsequent to our work, SKR1 was also cloned from different species and
named as R1(14) , TsK7L(15) , ALK2(16) , or
ActXIR(17) . The homolog of SKR2-1 has been designated as
R2(14) , ActR-IB(18) , or ALK4 (16, 19) by others. Although inactive alone, SKR1 and
SKR2 were subsequently shown to bind TGF-
1 or activin in concert
with the TGF-
1 or activin type II receptor,
respectively(6, 7, 8) . (
)However,
in transfected mammalian cell types examined so far, both SKR1 and SKR2
appear to elicit biological effects only in concert with the activin
type II receptor (ActRII) and
activin(7, 18, 19) .
Artificially
constructed monomers with defects in catalytic activity or substrate
binding that heterodimerize with a native subunit have been widely
employed as dominant-negative inhibitors to demonstrate the requirement
for dimeric transmembrane receptors in signal transduction. A
dominant-negative effect of an artificial mutant subunit of the
homodimeric ligand, platelet-derived growth factor, has been reported (20) . However, reports of naturally occurring
dominant-negative mechanisms of regulation of ligand-activated
transmembrane receptors are rare(21, 22) . We report
here that the inhibin heterodimer has properties of a natural
dominant-negative inhibitor of the heterodimeric type II-type I
receptor kinase complex promoted by homodimeric activin in liver cells.
EXPERIMENTAL PROCEDURES
Overexpression of Recombinant ActRII and Ectodomain of
SKR2 in Baculoviral-infected Insect Cells
The full-length coding
sequence of mouse ActRII cDNA (4) was subcloned into the NotI/BamHI sites of baculoviral transfer plasmid
pVL-1392 (Invitrogen). A human SKR2 cDNA was amplified from the cloned
SKR2 cDNA template (13) in the polymerase chain reaction using
5` (5`-TATGAATTCGGTTACTATGGCGGAGTCGGC-3`) and 3` primers
(5`-TAAGGATCCCTCTTGTAAAACGATGGTTCG-3`). The resultant cDNA spanned the
sequence from 7 base pairs upstream of the translational initiation
site through coding sequence for 62 residues of the intracellular
juxtamembrane domain. To facilitate immunochemical analysis, a portion
of human fibroblast growth factor receptor cDNA coding for the M1C4
monoclonal antibody epitope (23) was ligated in-frame with the
SKR2 cDNA at its 3`-end at a BamHI site. The tagged SKR2 cDNA
was cloned into the EcoRI sites of pVL-1392. Recombinant
baculoviruses bearing ActRII and SKR2 cDNAs were prepared as described
elsewhere(23) . Except where indicated, the infected Sf9 cells
were harvested within 65 h. The epitope-tagged chimeric protein
containing SKR2 extracellular, transmembrane, and intracellular
juxtamembrane domains from cell lysates was analyzed by immunoblot and
immunoprecipitation methods by using monoclonal antibody M1C4 as
described previously(23) .
Ligand Binding and Covalent Affinity
Cross-linking
Recombinant human activin A and inhibin A were
radiolabeled with
I-iodine as described
elsewhere(12) . Unless otherwise indicated, baculoviral
infected Sf9 cells (7.5
10
) were suspended in 0.5
ml of binding buffer (50 mM HEPES (pH 7.5), 128 mM NaCl, 5 mM KCl, 5 mM MgSO
, 1.2
mM CaCl
, 2 mg/ml bovine serum albumin) and mixed
with 0.5 pmol of
I-labeled activin (2.38
10
cpm/pmol) or inhibin (3.84
10
cpm/pmol) for
90 min at room temperature in the absence or presence of an excess
amount of unlabeled ligand. The bound ligand was covalently
cross-linked to receptor sites with 0.5 mM disuccinimidyl
suberate in binding buffer without bovine serum albumin. The cells were
extracted with ice-cold cell lysis buffer (10 mM Tris-HCl (pH
7.0), 125 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10
µg/ml leupeptin, 50 µg/ml aprotinin, 0.3 mM phenylmethylsulfonyl fluoride). The affinity-labeled complexes
from 7.5
10
cells were analyzed by
immunoprecipitation, SDS-PAGE, and autoradiography(23) .
Monolayers of HepG2 cells were affinity-labeled with
I-labeled activin or inhibin by using the same procedure.
Immunoprecipitation
Lysates of Sf9 cells (3
10
) from binding assays were absorbed with normal
rabbit IgG immobilized to protein A-Sepharose CL-4B beads (Pharmacia
Biotech Inc.) before reaction with the M1C4 monoclonal antibody
immobilized to protein A-Sepharose as previously
described(23) . Lysates (0.25 ml) of HepG2 cells (2
10
) prepared as described above were adjusted to 0.3% SDS,
boiled for 4 min, diluted 2-fold, and clarified by centrifugation at
16,000
g for 5 min at 4 °C. The supernatant was
then mixed with 35 µl of protein A beads and 10 µl of anti-SKR2
serum N19S or serum from the same rabbit obtained prior to
immunization. Antiserum was prepared against a synthetic polypeptide
sequence N19S (NRIDLRVPSGHLKEPEHPS) in the extracellular juxtamembrane
of SKR2 (13) by previously described methods(23) .
After incubation for 3 h at 4 °C, the beads were extracted and
analyzed by SDS-PAGE and autoradiography(23) .Untreated and
SDS- and heat-treated lysates of HepG2 cells described above were also
subjected to analysis by immunoprecipitation with anti-ActRII
monoclonal antibody, mAb149/1, as described elsewhere(24) . The
conditioned medium of hybridoma mAb149/1 was a generous gift from Dr.
R. Bicknell (Imperial Cancer Research Fund, Oxford, UK). For
immunoprecipitations, 0.30 ml of cell lysate was mixed with 0.15 ml of
the mAb149/1-conditioned medium and 35 µl of goat anti-mouse
IgG-agarose beads (Sigma).
DNA Synthesis
Acid-insoluble
[
H]thymidine incorporation was determined in 1.5
10
HepG2 cells in 24-well plates containing 1 ml of
minimum Eagle's medium and 1% fetal bovine serum after 3 days of
exposure to activin, inhibin, or both at the indicated concentrations.
Cells were incubated at 37 °C for 4 h with 0.5 µCi added to
each well in 10 µl of phosphate-buffered saline. After a wash with
1 ml of phosphate-buffered saline, cells were soaked in 1 ml of 10%
trichloroacetic acid at 4 °C for 30 min, then solubilized in 0.25
ml of 0.5 N NaOH, and the solution was counted by liquid
scintillation.
RESULTS AND DISCUSSION
Activin A Homodimer Binds to Recombinant ActRII and the
Ectodomain of SKR2
To study the binding of activin and inhibin
to ActRII and SKR2, full-length recombinant ActRII protein and an
epitope-tagged SKR2 chimeric protein exhibiting the extracellular,
transmembrane, and intracellular juxtamembrane domains of SKR2 and a
specific monoclonal antibody epitope M1C4 at its COOH terminus (23) were expressed in baculoviral infected Sf9 insect cells.
Western blot analyses with ActRII-specific monoclonal antibody mAb149/1 (24) and SKR2 chimeric protein-specific monoclonal antibody
M1C4 (23) indicated that the apparent molecular masses of the
insect cell-expressed recombinant ActRII and the tagged SKR2 ectodomain
on SDS-PAGE were 72 and 43 kDa, respectively. Table 1lists the
possible complexes that can arise and their relative abundance observed
experimentally from interaction of activin A, inhibin A, ActRII, and
SKR2 after incubation and subsequent exposure to a covalent affinity
cross-linking reagent followed by analysis of stable complexes by
SDS-PAGE and autoradiography. When expressed alone, the recombinant
ectodomain of SKR2 failed to bind
I-labeled activin A (Fig. 1A). Immunoassay with the M1C4 antibody confirmed
the presence of the 43-kDa antigen in the infected cell lysates
(results not shown). Cells expressing the 72-kDa ActRII product
exhibited
I-labeled bands of 98 and 85 kDa which
correlated with the size of the ActRII product cross-linked to a 28-kDa
:
homodimer and a single 14-kDa
chain of activin,
respectively (Fig. 1A). The results suggest that
:
activin binds to ActRII through one of its subunits. The
significant band at 28 kDa above the dye front containing the 14-kDa
chain monomer of activin indicated that the two disulfide-linked
chains of the homodimer can be cross-linked when bound to ActRII
or in free nonspecifically bound form (Fig. 1A).
Significant amounts of higher molecular mass ligand-labeled material
were apparent at the top of the gels; however, these analyses could
neither confirm the presence of the RII
:
RII
species (Table 1) nor shed light on the stoichiometry of the
binding of ActRII to each
chain within the activin homodimer.
Cells which were co-infected with both viruses bearing epitope-tagged
chimeric SKR2 and ActRII cDNAs exhibited an additional band at 57 kDa
which is the size predicted for the 43-kDa epitope-tagged SKR2 product
linked to one 14-kDa
chain (Fig. 1A). A 70-kDa
band indicative of SKR2 linked to a 28-kDa
:
activin
homodimer was notably absent. Immunoprecipitation with monoclonal
antibody M1C4 confirmed that the 57-kDa band contained the SKR2 product (Fig. 1B). In addition, the 85-kDa complex of an
I-labeled activin
chain and ActRII coprecipitated
with the anti-SKR2 M1C4 antibody (Fig. 1B). In contrast
to cell lysates, the 98-kDa band of ActRII bearing the activin
:
dimer was barely detectable in the immunoprecipitates. In
contrast to the lysates, the relative intensity of the 57- and 85-kDa
bands in the immunoprecipitates was near 1:1 at all three ratios of
co-infection. The results confirmed that the binding of
:
activin to ActRII facilitates binding of SKR2 to the ActRII-activin
complex in stoichiometric amounts. Although we cannot eliminate the
possibility that an ActRII and SKR2 complex is bound to each
chain of the activin
:
dimer, the absence of a significant
band at 128-kDa indicative of a ActRII

SKR2 complex (Fig. 1B and results not shown) suggests an
ActRII
:
SKR2 complex in which ActRII and SKR2 are
bound respectively to each chain of the
:
dimer. The absence
of the SKR2
:
species in both lysates and
immunoprecipitates and the ActRII
:
species in the
immunoprecipitates may indicate a spatial relationship between the two
chains of activin when bound to both ActRII and SKR2 that reduces
the efficiency of interchain cross-linking.
Figure 1:
Binding of
I-labeled
activin to recombinant ActRII and ectodomain of SKR2 in
baculoviral-infected Sf9 insect cells. A, radiolabeled bands
from cell lysates. B, radiolabeled bands precipitated with an
SKR2 antibody. Cells were infected with virus-bearing ActRII and SKR2
cDNA as indicated. Antisense indicates cells infected with
virus bearing SKR2 cDNA in the antisense orientation. Different
infection ratios of ActRII and SKR2 viral titers at a constant titer of
ActRII virus are indicated at the bottom. Where indicated,
unlabeled activin A was present in 100-fold excess of the
I-labeled activin. Affinity-labeled complexes and
immunoprecipitation samples were analyzed by SDS-PAGE and
autoradiography. Molecular mass standards are indicated in
kilodaltons.
Inhibin A
:
Heterodimer Binds to ActRII but Not
SKR2
I-Labeled inhibin covalently cross-linked to
bands of 105 and 85 kDa on Sf9 cells which were infected with virus
bearing the ActRII cDNA (Fig. 2A). The major 105-kDa
band was the predicted size of 72-kDa ActRII bearing the 32-kDa
:
inhibin heterodimer while the 85-kDa band correlated with
that of ActRII cross-linked to the 14-kDa
subunit of inhibin (Table 1). No distinct 90-kDa band indicative of ActRII linked to
the unique monomeric 18-kDa
chain of inhibin could be detected.
In addition, high molecular material at the top of the gels indicative
of oligomers of ActRII and the labeled
:
inhibin ligand was
much less than the same analyses employing activin as ligand (Fig. 1A). The intense band at 32 kDa as well as the
high yield of the ActRII
:
complex (105 kDa) indicated
that, similar to activin, disuccinimidyl suberate efficiently
cross-linked the
and
subunits of inhibin to each other when
bound both to ActRII or nonspecifically as the free ligand (Fig. 2A, Table 1). The high intensity of the
ActRII
:
species relative to the ActRII
complex probably indicates a higher specific activity of the
I-labeled
chain relative to the
chain.
Neither cells infected with single virus bearing the SKR2 cDNA nor
those co-infected with viruses bearing SKR2 and ActRII cDNAs yielded
SKR2 bands labeled with inhibin in the total cell lysates (Fig. 2A) or in immunoprecipitates with the M1C4
antibody (Fig. 2B). The results suggest that inhibin
binds to ActRII through its
subunit which is shared with activin,
but inhibin cannot promote formation of the ActRII
SKR2
heterodimer because of inability of SKR2 to bind to the inhibin
chain. Although steric interference by presence of the unique
chain cannot be eliminated, these results further suggest that it is
unlikely that ActRII and SKR2 assemble on a single
chain of the
activin
:
homodimer.
Figure 2:
Binding of
I-labeled inhibin
to recombinant ActRII and ectodomain of SKR2. A, radiolabeled
bands from cell lysates. B, radiolabeled bands precipitated
with M1C4 antibody. Conditions were the same as in Fig. 1except
that
I-labeled and unlabeled inhibin A was substituted
for activin A.
Activin A and Inhibin A Compete for Binding to
Recombinant ActRII
High levels of nonspecific binding of labeled
activin and inhibin impaired a direct comparative Scatchard analysis of
the affinity of the two ligands for ActRII. As an alternative, the
ability of unlabeled activin and inhibin to reduce the amount of
labeled activin that was covalently cross-linked to ActRII was
performed. Both activin-labeled ActRII bands at 98 and 85 kDa in
baculoviral infected Sf9 cells (Fig. 1A and Fig. 3, lane 2) were reduced by an excess of unlabeled
activin (Fig. 3, lanes 3-6) and inhibin (Fig. 3, lanes 7-10). In experiments not shown
here, the same results were achieved when Sf9 cells were co-infected
with baculoviruses bearing ActRII and SKR2 cDNAs. Activin-labeled
ActRII and SKR2 bands were proportionally reduced by unlabeled activin
or inhibin. Quantitative analysis of the results by scanning
densitometry revealed that on a molar basis 2-4 times more
inhibin than activin was required to reduce the activin-labeled ActRII
complexes to the same level (Fig. 3). Currently, it is unclear
whether this reflects an intrinsically lower affinity of the inhibin
chain for ActRII due to influence of the
chain or that the
molar ratio of binding of ActRII to the
:
activin homodimer
is greater than one.
Figure 3:
Competition of inhibin and activin for
binding of activin to recombinant ActRII. Sf9 cells (5
10
) were infected with baculoviruses bearing antisense (lane 1) or ActRII (lanes 2-10) cDNAs and
incubated for 90 min at room temperature in binding buffer containing
0.30 pmol of
I-labeled human recombinant activin and the
indicated amounts in picomoles of unlabeled activin (lanes
3-6) or inhibin (lanes 7-10) in a total
volume of 0.25 ml. After addition of DSS, the
I-activin-labeled complexes were analyzed by SDS-PAGE and
autoradiography. The relative intensities of the radiolabeled 98- and
85-kDa ActRII bands (bottom) was determined by scanning
densitometry across the area of the film containing the two ActRII
bands.
Activin and Inhibin Receptors in HepG2 Cells
To
test whether native activin and inhibin receptors in liver cells
exhibited properties similar to recombinant ActRII and SKR2, analyses
were carried out in human hepatoma cells (HepG2) which express ActRII
and from where SKR2 was originally cloned(12, 13) .
Both labeled activin and inhibin yielded a labeled 89-kDa band, activin
labeled specific bands at 72 and 165 kDa, and inhibin labeled a
specific band at 110 kDa. All four bands were eliminated by addition of
unlabeled activin or inhibin to the binding reaction (Fig. 4A). The common 89-kDa band labeled by both
ligands had the predicted molecular mass of a 75-kDa mammalian ActRII
linked to the common 14-kDa
subunit of activin and inhibin. The
72-kDa band that was specifically labeled with
I-labeled
activin was characteristic of a 59-kDa type I activin receptor linked
to a single 14-kDa
chain. Since the 165-kDa weak band that was
uniquely labeled with activin is sensitive to unlabeled inhibin and
near the sum of the 89- and 72-kDa bands, it is likely the heterodimer
of type II and type I activin receptors bearing the homodimeric
:
activin ligand. The 110-kDa band that labeled uniquely with
I-labeled inhibin correlates with the size of a complex
of the 75-kDa ActRII monomer and the 32-kDa
:
inhibin
heterodimer. All three activin-labeled bands precipitated with an
antiserum (N19S) against the extracellular juxtamembrane of SKR2 (Fig. 4B). No inhibin-labeled bands were precipitated
with the antiserum. In contrast to results in Fig. 1B and Fig. 2B using the M1C4 monoclonal antibody,
efficient reactivity of ligand-labeled complexes with the N19S
anti-serum required denaturation of extracts prior to
immunoprecipitation. This resulted in loss during the
immunoprecipitation of the fraction of the cross-linked 89-kDa
ActRII
species from ActRII
:
SKR2
complexes in which the adjacent
chain was not concurrently
cross-linked to SKR2 (Fig. 4B). This was reflected in a
lower yield of the 89-kDa ActRII
complex relative to the
SKR2
band at 72 kDa. The low yield of the 165-kDa
ActRII
:
SKR2 complexes that survive the
immunoprecipitation and SDS-PAGE analysis may reflect (a) the
lower probability of concurrent covalent cross-linking events occurring
between ActRII
, SKR2
, and the two
chains of
homodimeric activin within the same complex or (b) a low
efficiency of inter-
chain cross-linking within the
:
homodimer when it is bound to both ActRII and SKR2 (discussed earlier).
Figure 4:
Characterization of activin and inhibin
receptors in human liver cells (HepG2). A, radiolabeled
receptor sites from cell lysates. Monolayers of HepG2 cells (1
10
) were incubated with
I-labeled activin or
inhibin in the absence or presence of a 100-fold excess of unlabeled
ligand as indicated and radiolabeled binding sites were analyzed as
described in Fig. 1. B, immunoprecipitation of
ligand-labeled sites with SKR2 antiserum. The denatured lysates of
HepG2 cells (2
10
) from binding assays were
prepared and subjected to immunoprecipitation by using SKR2-specific
antiserum N19S or preimmune serum from the same rabbit (negative
control). C, immunoprecipitation of ligand-labeled binding
sites with anti-ActRII monoclonal antibody. The anti-ActRII mAb149/1
was used to precipitate both
I-activin and
inhibin-labeled bands from HepG2 cell lysates. An equal amount of
normal mouse IgG was used as negative control. The dried gel was
exposed to a storage phosphor screen and visualized by screening on a
Molecular Dynamics PhosphorImager.
In contrast to the anti-SKR2 antibody (N19S), a monoclonal antibody
against ActRII (mAb149/1) precipitated the three activin-labeled bands
at 72, 89, and 165 kDa and the inhibin-labeled bands at 89 and 110 kDa
from the HepG2 cell lysates (Fig. 4C). In addition, the
anti-ActRII antibody revealed a weak activin-labeled band at 101 kDa
that varied in intensity among assays (Fig. 4C). The
101-kDa band is characteristic of a complex of ActRII and the
:
activin dimer that was apparent in whole lysates of the
baculoviral infected Sf9 cells (Fig. 1). mAb149/1 precipitated
labeled ActRII (89 kDa) and activin type I receptor (ActRI) complexes
(72 kDa) in a 1:1 ratio from undenatured cell lysates, but yielded
predominately the labeled ActRII
complex from lysates that
were denatured prior to immunoprecipitation for reasons discussed above
in the analysis with the anti-SKR2 antibody N19S (Fig. 4C).
I-Activin-labeled 72-kDa
(type I), 89-kDa (type II), and 165-kDa (types I and II complex) bands
from the HepG2 cells were proportionally reduced by both unlabeled
activin and inhibin in a dose-dependent manner (Fig. 5). Similar
to the results with recombinant ActRII and SKR2 expressed in the Sf9
insect cells, quantitation of relative band intensities indicated that
inhibin was 3-4 times less effective than activin in the ability
to compete with labeled activin for binding to ActRII (Fig. 5).
Figure 5:
Competition of the binding of activin to
HepG2 cells with inhibin. Monolayers of HepG2 cells (1
10
) in 60-mm dishes were incubated with 0.4 pmol of
I-labeled activin in the presence of the indicated
amounts in picomoles of unlabeled activin (lanes 2-5) or
inhibin (lanes 6-9) in a total volume of 0.20 ml for 90
min at room temperature. Affinity-labeled complexes from cell lysates
were analyzed by a PhosphorImager as described in Fig. 4.
Relative intensities (below) of the 89/72-kDa receptor bands at each
level of competitor was determined from the image by
densitometry.
These results confirm that activin and inhibin share the same native
type II receptor in liver cells; inhibin blocks the binding of activin
to type II receptors and thereby prevents heterodimerization of the
type II and type I receptors. HepG2 cells exhibit neither a
ligand-specific type II receptor nor a type I receptor for inhibin that
can be detected by covalent affinity cross-linking.
Antagonistic Effects of Activin and Inhibin on HepG2 Cell
Growth
Activin inhibited DNA synthesis in the HepG2 cells in a
dose-dependent fashion (Fig. 6A). Inhibition was
half-maximal at about 100 pM activin and reached a maximum at
300 pM (Fig. 6A). In contrast, inhibin had no
effect on DNA synthesis at concentrations up to 2.5 nM (Fig. 6B). However, at 300 pM activin in
which DNA synthesis was maximally inhibited, inhibin antagonized the
effect of activin (Fig. 6B). About a 2 times molar
ratio of inhibin to activin was required to antagonize the inhibitory
effect of activin by 50% (Fig. 6B). This result is
consistent with the ability of inhibin to compete with activin for
binding to ActRII and inhibit formation of the
ActRII.
:
ActRI receptor complex in both baculoviral
infected insect cells and the HepG2 cells.
Figure 6:
Antagonistic effect of inhibin on
inhibition of HepG2 cell DNA synthesis by activin. A, HepG2
cells (1.5
10
each) were treated with activin at
the indicated concentrations of 37.5, 75, 150, 300, and 600 pM for 3 days and [
H]thymidine incorporation
was determined as described under ``Experimental
Procedures.'' Data points are the mean of duplicates and presented
as a percent of DNA synthesis in controls containing no activin (100%). B, inhibin at 150, 300, 600, 1200, and 2400 pM was
added to HepG2 cell cultures in the absence of activin (circles) or in the presence of 300 pM activin (triangles) for 3 days, and then the incorporation of
[
H]thymidine was
determined.
Conclusions
We suggest that some, if not all,
biological activities of inhibin may be mediated by its role as an
inhibitor of formation of the active heterodimeric activin receptor
complex of type II and type I serine/threonine kinases (Fig. 7).
We have shown that SKR2, a widely expressed activin type I
serine/threonine kinase(13, 18) , is recruited into an
ActRII
:
SKR2 complex by association with a
chain of the activin
:
homodimer (Fig. 7). Recently,
SKR2 (also called ActRIB) in conjunction with ActRII has been shown to
be a mediator of the growth-inhibitory and extracellular matrix
transcriptional responses of cells to
activin(18, 25) . Through its
chain subunit
which is shared with activin, the inhibin
:
heterodimer
competes with activin for binding to activin type II receptors,
inhibits formation of the ActRII
:
ActRI complex
and thereby inhibits the activin-dependent response.
Figure 7:
Hypothetical scheme of inhibition of
assembly of the heterodimeric activin receptor complex by inhibin.
Activin
:
homodimer binds to the type II receptor through one
of the
subunits which induces capacity of the unoccupied
subunit to bind the type I receptor (SKR2 in the current study). The
subunit of inhibin is incapable of binding type I receptors and
results in a dominant-negative inhibition of assembly of the
heterodimeric activin receptor.
In addition to
significant differences in size and sequence, the unique
chain of
inhibin differs from the common
chain of activin and inhibin by
absence of the two half-cystines which form the disulfide which anchors
the NH
-terminal
1 helix to the rest of the monomer
structure(26) . Homodimeric ligands, TGF-
1 and TGF-
2,
which also bind to SKR2 when anchored to the TGF-
type II
receptor,
exhibit a similar structure in this domain. The
conformation of the NH
-terminal domain may be important in
the binding of one chain within the homodimeric ligands within the
TGF-
family to type I receptors, while the other chain is anchored
to the appropriate type II receptor. The domain may be a target for
alteration and design of mutant:wild-type heterodimeric antagonists of
TGF-
ligands.