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INTRODUCTION |
The TGF-
1 family
consists of a large group of structurally related, but functionally
diverse polypeptides that control the growth and differentiation of
many cell types in vitro and in vivo (1-4).
TGF-
s, activins, and bone morphogenetic proteins (BMPs) exert their
biological effects through binding to two types of serine/threonine
kinase receptors, termed type I (± 53 kDa) and type II (± 70 kDa)
receptors (1, 5, 6). Type I and II receptors can form high affinity
receptor complexes at the cell surface and this is necessary for signal
transduction (7-11). Overexpressed type II receptors can bind ligand
in the absence of type I receptor with moderate affinity, while it is
generally accepted that type I receptors require type II receptors to
bind ligand in the high affinity receptor complex. The type II receptor phosphorylates the type I receptor after ligand binding, and the latter
propagates the signal to downstream effectors, the Smad proteins (see
Ref. 12).
TGF-
members are biologically active as dimers. Like other members
of the TGF-
family, the activins are synthesized as large precursor
proteins consisting of a signal peptide, a glycosylated prodomain and a
mature domain. The maturation of activin requires intracellular
cleavage by protein convertases, such as furin, at the basic cleavage
site which separates the mature chain from the prodomain (13, 14).
Removal of the prodomains from the precursor dimer is necessary for
biological activity of the mature 25-kDa dimer, since unprocessed high
molecular weight forms of activin A display no biological activity (13,
15, 16).
Thus far, TGF-
2, TGF-
3, and BMP-7 (also called osteogenic
protein-1 (OP-1)) have been crystallized, and the three-dimensional structures of the mature, dimeric molecules have been elucidated (17-19). These proteins share a common three-dimensional polypeptide folding pattern, although their amino acid sequence identity is limited
to 36% (BMP-7 compared with TGF-
2). Hence, it is likely that this
structure is the prototype for the whole family and might be
extrapolated to activins as well. The common fold of the monomer is
defined by seven cysteines that are conserved throughout the family.
Six of these form intrachain disulfide bonds and make up the cystine
knot, while the seventh cysteine forms an interchain disulfide bond
that stabilizes the dimer (17-19). By analogy with a left hand, the
monomeric structure consists of the N-terminal thumb region, two
antiparallel pairs of
-strands that build up four fingers, two loops
that connect the fingers (loop 1 connects finger 1 and 2; loop 2 connects finger 3 and 4), and a long
-helix at the heel of the hand
(see Fig. 1). This prototype structure defines four solvent-accessible,
flexible and divergent regions, which may contain putative
receptor-binding sites, i.e. the N terminus, loop 1, loop 2, and the C-terminal end of the long
-helix (17-19).
Amino acids important for biological activity have been defined by
limited structure-function analysis of TGF-
members and by molecular
characterization of naturally occurring mutations that cause drastic
phenotypes in different organisms. Mutation analysis revealed that the
nine cysteines, including the seven conserved cysteines in the family,
of mature activin A are essential for either the biosynthesis or the
(full) biological activity of activin A (20), and that a phenylalanine
to isoleucine substitution at position 21 of activin B creates a
dominant-interfering protein (16). In mature TGF-
1, the C-terminal
portion (amino acids 83-112) has recently been defined as necessary
for high affinity binding to the TGF-
receptor type II (T
RII)
(21). In addition, frameshift and/or point mutations (i.e.
replacement of the first conserved cysteine by tyrosine) in hCDMP-1
(human cartilage-derived morphogenetic protein), and growth and
differentiation factor (GDF)-8, have been identified in human
chondrodysplasia and double muscling in cattle, respectively
(22-24).
However, no specific receptor binding determinants are known for any
TGF-
member. Detailed mutagenesis studies of TGF-
family members
would provide insight into how such mutations affect their biological
activities, and this may facilitate the development of therapeutic
agents that can be used in TGF-
-related diseases. In order to
identify amino acids important for receptor binding and biological
activity, we started structure-function analysis of activin A by
introducing single amino acid substitutions in the mature domain, in
regions that are thought to be involved in receptor interaction (18).
In this way, we identified two amino acids in activin A which are
important for its biological activity and its interaction with the type
II receptor: Asp-27 and Lys-102, located in loop 1 and 2, respectively.
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EXPERIMENTAL PROCEDURES |
Mutagenesis--
Oligonucleotide-directed mutagenesis was
performed using plasmid PTZ18R, which contains a mouse activin A
cDNA cloned in the sense orientation with respect to the T7
promoter, and the Muta-Gene phagemid in vitro mutagenesis
kit (Bio-Rad). Mutations were introduced using the single-stranded
sense oligonucleotides listed in Table I.
Mutant TGF-
2L1 was constructed using two complementary
oligonucleotides representing the DNA sequence of loop 1 of TGF-
2: 5'-CGATTTCAAGAGAGATCTAGGGTGGAAATGGATACACGAACCCT-3' and
5'-CCGGAGGGTTCGTGTATCCATTTCCACCCTAGATCTCTCTTGAAAT-3' This
sequence was cloned into an activin mutant construct in which a
ClaI site and a MroI site had been introduced
upstream and downstream, respectively, of the loop 1 (Val-18 to Pro-32) sequence.
Mutant activin V18I/S19D/N26I/D27G was generated by partial double
annealing via the nine 3'-nucleotides of
5'-GAAACAGTTCTTTATCGATTTCAAGGACATTGGCTGGATTGGCTGG-3'. This
generated two extra mutations (N26I and D27G) in addition to those
generated by oligonucleotide V18I/S19D.
All mutations, listed in Fig. 1B, were confirmed by DNA sequencing.
Vaccinia Virus T7 Expression System--
This expression system
has been described previously (13), but it was applied in a slightly
modified manner. Subconfluent HeLa or PK15 cells (10-cm2
dishes) were infected with a recombinant vaccinia virus expressing phage T7 RNA polymerase (multiplicity of infection: 5) for 1 h at
24 °C. These cells were then transfected with T7 promoter-containing plasmids encoding the wild type and mutant activins A, using DOTAP (Boehringer Mannheim). For the heterodimerization assay, cells were
cotransfected with T7 plasmids encoding zebrafish activin B (a gift
from F. Rosa, U368 INSERM, Ecole Normale Supérieure, Paris,
France). Cells were incubated with this DNA/DOTAP mix for 6 h at
37 °C in an atmosphere containing 5% CO2. Cells were
then washed with methionine-free minimum essential medium before
starvation in this medium for 1 h at 37 °C. The cells were next
pulse-labeled for 1 h by addition of 1 ml of the same medium
containing 50 µCi of [35S]methionine and
[35S]cysteine (ICN). The cells were chased by addition of
1 ml of Dulbecco's modified Eagle's medium supplemented with 20 µg/ml bovine serum albumin and a 10-fold higher concentration of cold methionine than is normally present in this medium. After 14 h at
37 °C, the medium was collected and centrifuged, and the supernatant was frozen. Samples were prepared for electrophoresis after
precipitation of the proteins with trichloroacetic acid, as described
previously (13).
FSH Assay in Rat Pituitary Cells--
The follicle-stimulating
hormone (FSH) assay was performed as described (25). Briefly, primary
rat pituitary cells were cultured for 2 days in serum-free medium (as
specified in Ref. 25) containing dilutions of the activin A mutant
proteins. The medium was then collected, and the FSH concentration was
determined by radioimmunoassay (RIA). Each mutant activin was added to
three wells, and the RIA for FSH was performed in duplicate using
the FSH-RIA kit (NIDDKD, National Institutes of Health,
Rockville, MD) according to Denef et al. (26).
Mesoderm Induction Assays in Xenopus--
Xenopus
embryos were obtained by in vitro fertilization (27). They
were maintained in 10% Normal Amphibian Medium (28) and staged
according to Nieuwkoop and Faber (29). Animal pole regions were
dissected from mid-blastula (stage 8) embryos (30) and cultured in 75%
Normal Amphibian Medium containing 0.1% (w/v) bovine serum albumin and
wild type or mutant activin (2.5 ng/ml). A preliminary assessment of
mesoderm induction was based on the elongation of the animal caps.
Animal pole regions were then frozen on dry ice, and expression of the
mesoderm-specific gene Brachyury (Xbra) (31) was
assayed by RNase protection analysis as described by Jones et
al. (32).
Radiolabeling of Activins and Follistatin--
Wild type and
mutant activins, and follistatin were iodinated using a modified
chloramine-T method (33). Two µg of protein (in 10 µl of 30%
acetonitrile, 0.1% trifluoroacetic acid) were diluted with 10 µl of
600 mM sodium phosphate (pH 7.5) and 5 µl of
Na125I (0.25 mCi; Amersham Pharmacia Biotech) and 5 µl of
phosphate-buffered saline (137 mM NaCl, 2.7 mM
KCl, 6.5 mM Na2HPO4 and 1.5 mM KH2PO4). To initiate the
radioiodination, 10 µl of chloramine-T (100 µg/ml in 50 mM sodium phosphate (pH 7.5); Sigma) was added. After 2 min, the iodination was stopped by addition of 20 µl of 50 mM N-acetyl-L-tyrosine (Sigma), 200 µl of 60 mM sodium iodide, and 200 µl of 10 M ultrapure urea (Life Technologies). Subsequently, the
reaction mixture was passed over a Sephadex G-25 column (Amersham Pharmacia Biotech), which was equilibrated and eluted with
phosphate-buffered saline containing 0.1% (w/v) hemoglobin (Sigma).
Peak fractions, with specific activities of 30-100 µCi/µg of
protein, were routinely obtained, pooled, and stored at
80 °C.
Activin/Follistatin Cross-link--
Radioiodinated
follistatin288 (FS288) was cross-linked to cold wild type and mutant
activins using bis-sulfo-succinimidyl suberate (BS3;
Pierce) (33). Approximately 2 ng of iodinated FS288 (5 µl) was
incubated with 500 µl of activin-containing conditioned medium prepared as described above. After 2 h of incubation at 4 °C on a rotary shaker, 125 µl of 5 mM BS3 in
HEPES-buffered saline (150 mM NaCl and 20 mM
HEPES; Life Technologies, Inc.) was added and the reaction was
incubated for 1 h at 4 °C. Activin/follistatin complexes were
purified using wheat germ agglutinin-agarose (Sigma) beads (33). They
were separated by SDS-PAGE under reducing conditions and visualized by autoradiography.
Receptor Binding Studies--
PK15 cells (28-cm2
dishes) were transfected with different combinations of activin
receptors using the vaccinia virus-T7 system as described above. On the
second day, the cells were washed with ice-cold binding medium
(HEPES-buffered Dulbecco's modified Eagle's medium (pH 7.5)
containing 0.2% (w/v) bovine serum albumin) for 10 min. Cells were
incubated with 150 pM labeled activin A in 1.5 ml of
binding medium for 2 h at 4 °C. For competition studies, cells
were incubated with a constant amount (150 pM) of
125I-labeled wild type activin and (simultaneously added)
different amounts of cold wild type or mutant activins. Then, iodinated activin was removed by gently and repeatedly washing the cells with
ice-cold HEPES-buffered saline containing 0.9 mM
CaCl2. Activin was cross-linked by incubation in 1.5 ml of
HEPES-buffered saline containing 1 mM BS3 for
30 min at 4 °C. The reaction was then quenched for 5 min at 4 °C
by addition of 150 µl of 10× quench solution (10×: 10 mM Tris (pH 7.5), 2 mM EDTA, and 200 mM glycine). The cells were scraped from the plates in 1 ml
of detachment buffer (10 mM Tris (pH 7.4), 1 mM
EDTA, 10% (v/v) glycerol, 0.5 µg of aprotinin/ml, 0.5 µg of
leupeptin/ml, and 0.3 mM phenylmethylsulfonyl fluoride), and collected by centrifugation (5 min at 4 °C). The pellet was then
dissolved in 50 µl of solubilization buffer (10 mM Tris
(pH 7.4), 1 mM EDTA, 125 mM NaCl, 1% (v/v)
Triton X-100, 0.5 µg of aprotinin/ml, 0.5 µg of leupeptin/ml, and
0.3 mM phenylmethylsulfonyl fluoride), followed by
incubation for 40 min on ice. Proteins were separated by SDS-PAGE and
visualized by autoradiography.
Large Scale Production and Purification of Activins--
The
large scale production of wild type and mutant activins was performed
using a baculovirus expression system. The mutant cDNA was inserted
in the baculotransfer vector pVL1393 under transcriptional control of
the baculoviral polyhedrin promotor. Recombinant baculovirus was
generated by homologous recombination in Spodoptera
frugiperda cells (Sf9) cotransfected with the recombinant
transfer construct and BaculogoldTM virus AcNPV DNA
(PharMingen). Recombinant virus was plaque-purified and amplified to
high titer stock for production. Activin was purified from conditioned
medium of recombinant baculovirus infected Sf9 cells, harvested
72 h after infection.
Purification of wild type and mutant activins was performed by use of
an optimized four-step purification protocol in which the conditioned
medium is diafiltrated and concentrated in the presence of 6 M urea and loaded onto an anion exchange column (Fractogel-(EMD)-TMAE, Merck). The flow-through is then loaded on a
Protein Pack Sulfonyl (Millipore) cation exchange column. The 150 mM NaCl fraction is then adjusted to 10% acetonitrile, 0.1% trifluoroacetic acid (v/v) and separated by RPC-4
(Fractogel-butyl) reversed phase chromatography. Mutant activins are
recovered in the 30-34% acetonitrile fraction (34) and further
purified on a RPC-8 (Brownlee octyl) column run as a polishing step.
Quantification of these pure activins was obtained through amino acid
composition analysis.
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RESULTS |
Mutagenesis--
Some mutations in structurally important regions
of TGF-
family members have been reported to lead to improper
biosynthesis of these ligands (15, 16, 20, 35, 36). In our study, this
was also the case when severe changes in activin A were introduced. For
example, the substitution of loop 1 of activin A by the equivalent region of TGF-
2 led to undetectable protein expression levels in the
vaccinia virus-T7 system, both in the secreted and in the intracellular
fraction (data not shown). It is likely that intracellular degradation
occurred, as has been suggested for most cysteine mutants of activin A
and TGF-
1 (20, 36). To avoid synthesis and intracellular trafficking
problems due to structural changes, we anticipated that the majority of
mutant activins used in this study should be generated by single amino
acid substitution.
Four solvent-accessible regions can be deduced from the
three-dimensional structure of TGF-
2 and BMP-7 (17-19): the N
terminus, loop 1, loop 2, and the C-terminal segment of the long
-helix (18; see also Fig. 1). These
regions are the most flexible structures in the dimer, and their
sequences are divergent throughout the family, which marks them as good
candidates for receptor interaction. Most of the mutations introduced
are single alanine substitutions at charged residues in these domains
(Fig. 1A). A large panel of 39 activin A mutants was
constructed by oligonucleotide-directed mutagenesis (Fig.
1B).

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Fig. 1.
A, schematic diagram of the activin A
monomer (taken from Ref. 20). The proposed structure is based on the
known structures of TGF- 2 and BMP-7 (17-19). Residues that, when
mutated individually or in combination (as indicated in panel
B), did not significantly alter wild type activity, are
highlighted in light gray. Mutant activins with
higher or lower activity compared with wild type are highlighted in
dark gray (i.e. Asp-27 and Lys-102,
respectively). Cysteine residues are boxed and cysteine
bonds are presented as solid, gray
bars. The cysteine bond at residue 80 represents the
intermolecular disulfide bond. B, list of mutations
introduced in activin A. TGF- 2L1, chimeric activin/TGF- protein
in which amino acid V18-P32 from activin is replaced by the
corresponding residues of TGF- 2 (loop 1:
22IDFKRDLGWKWIHEP36, TGF- numbering). The
quadruple activin mutant protein containing D27G was obtained because
of misincorporation during oligonucleotide synthesis.
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Synthesis and Secretion of the Mutant Polypeptides--
Synthesis
of this large panel of activin polypeptides was first analyzed in HeLa
cells using the T7 vaccinia virus-based expression system. As suggested
previously, these cells have sufficient levels of endogenous furin to
support correct and efficient processing of the activin A precursor
(13). Synthesized proteins were visualized by metabolic labeling
followed by SDS-PAGE. We assessed both the maturation of activin A
mutants to a 25-kDa dimer as well as their capacity to heterodimerize
with zebrafish activin B; a secreted activin AB heterodimer can be
resolved in SDS-PAGE because activin A homodimers have a slower
migration than activin B homodimers. Nearly all activin A mutant
polypeptides were processed like the wild type precursor and they
heterodimerized efficiently (and predominantly) with activin B, as
observed previously (Ref. 37 and data not shown; only activin dimers of
D27K, K102A, K102E and K102R, respectively, are shown in Fig.
2). This indicates that the overall
structure of the precursor polypeptides and their intracellular folding
and dimerization in the rough endoplasmic reticulum are not altered. As
well as analyzing their ability to dimerize, the ability of the mutants
to bind follistatin (FS), an antagonistic binding protein of activin,
was tested by cross-linking. All mutant activins tested (including
K102A and K102E), formed complexes with FS288 like wild type activin
(shown for K102A and K102E in Fig. 3).
This again suggests that their overall structure is not dramatically
altered, if at all.

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Fig. 2.
Detection of metabolically labeled activins
(wild type and Asp-27 and Lys-102 mutants shown only; data not shown
for other mutants) secreted from HeLa cells, using the vaccinia
virus-T7 system. ActA-ActB heterodimers
( A- B) as well as homodimers
( A- A and
B- B) can be distinguished (37).
[35S]Methionine- and [35S]cysteine-labeled
proteins secreted from the cells were separated by SDS-PAGE under
non-reducing conditions on 15% gels. Mock, non-infected
cells; T7, vaccinia virus-infected but not DNA-transfected
cells; +, cotransfection with T7 plasmids containing the respective
inserts.
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Fig. 3.
Binding of mutant activins to
follistatin. Approximately 2 ng of iodinated FS288 (in 5 µl of
phosphate-buffered saline) was incubated with 500 µl of vaccinia
virus-T7 produced proteins. After cross-linking of the
activin/follistatin complexes, they were purified using wheat germ
agglutinin-Sepharose (33) and separated by SDS-PAGE under reducing
conditions followed by autoradiography. FS, follistatin;
FS A, complex of one follistatin molecule and
one activin monomer (33).
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A modified version of the vaccinia virus-T7 expression system was used
to produce all mutant activins (see "Experimental Procedures"). De novo synthesized proteins were pulse-labeled early in
infection and then chased with excess cold amino acids. Assuming that
their production rates do not differ, this allowed us to normalize the different mutant activin concentrations in the conditioned medium relative to wild type by detecting and quantifying the signal of the
25-kDa dimer using PhosphorImager analysis. The concentration of
activins (± 30 ng/ml) was high enough in the conditioned medium for
use in different sensitive bioassays.
Biological Activities of vv-T7 Produced Mutant Activins--
The
crude conditioned media described above were used for a preliminary
characterization of the bio-activities of the different mutant
activins. The first bioassay was based on the stimulatory effect of
activin on the production of FSH by pituitary cells (38). Wild type
activin A stimulated FSH production in a dose-dependent manner with a maximum stimulation of 2-2.5-fold compared with non-stimulated cells at concentrations of 2.5-5 ng of activin/ml. Purified activin A exhibited a half-maximal stimulation
(ED50) of FSH production at a concentration of 0.4 ng/ml in
our modified FSH assay (25), which is more sensitive than previously
used assays (38). Most of the unpurified mutant activin preparations behaved like wild type activin A in this assay (data not shown). However, mutant K102E was not significantly active, while K102A consistently displayed a lower activity than wild type activin A. Loss
of bio-activity of K102E was restored to wild type levels when this
lysine (Lys-102) was replaced with another positively charged residue
(mutant K102R). Mutant activin A bearing a D27K substitution appeared
to be more active in this assay (data not shown, but see below).
Activin causes animal cap explants of early Xenopus embryos
to undergo a rapid and dramatic morphogenetic response (39), allowing a
provisional assessment of the mesoderm-inducing activities of different
activin mutants. Mesoderm-inducing activities of the crude vv-T7
produced mutant activins were first assessed by observing the
elongation of Xenopus animal caps cultured in
activin-containing media. The animal cap assay is very sensitive, since
it has an ED50 (50% of the animal caps show elongation) of
0.2 ng of activin/ml. Nearly all activin variants induced weak to
strong elongation (like wild type activin) of animal caps, but mutant
K102E showed no elongation (data not shown).
Based on these two biological assays, we selected four activin mutants
for further analysis. Three mutants bear amino acid substitutions at
Lys-102 (Ala, Glu, and Arg), and the positive charge at this position
is apparently critical for biological activity (see also below). The
D27K mutant was also selected because it displayed higher specific activity.
Biological Activities of Purified Mutant Activins--
In order to
analyze the selected mutant proteins in more detail, they were produced
in large amounts using the baculovirus expression system. Four
baculovirus recombinants were generated and used to infect insect cells
(Spodoptera frugiperda (Sf9) cells). The secreted
25-kDa dimer was purified from 1.5-3 liters of conditioned medium. The
mutant proteins were purified to homogeneity in four steps using a
modification of a previously published purification protocol for
activin A (34). This yielded pure activins, as judged by SDS-PAGE
followed by silver staining (data not shown). Quantification of these
pure activins was obtained through amino acid analysis.
In order to confirm the data obtained with conditioned media, the
mesoderm-inducing activities of the purified mutant activins were
studied using the animal cap assay. At a concentration of 2.5 ng/ml,
all mutant activins and wild type activin A caused clear elongation of
the animal caps, except for mutant K102E (data not shown). The
mesoderm-inducing activities of the activins were confirmed by studying
expression of Xenopus Brachyury (Xbra), which is
induced in an immediate-early fashion in amphibian embryos by activin.
Both D27K and K102R induced expression of Xbra to levels
similar to those induced by wild type ligand, whereas virtually no
Xbra expression was detected in animal caps incubated with K102E activin (Fig. 4). Mutant K102A was
less potent than wild type activin A in this assay.

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Fig. 4.
Analysis of Xenopus Brachyury
(Xbra) expression induced by different
activins. Animal caps were cultured in 2.5 ng/ml mutant or wild
type activin, and frozen for analysis at the equivalent of stage 11. RNA was isolated and hybridized with radioactive probes specific for
Xbra and ornithine decarboxylase (ODC) as a loading
control.
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The FSH release assay was also repeated with purified activins.
Different dilutions of the mutant proteins were tested in order to
generate a dose-response curve for each mutant activin. Mutant D27K
stimulated FSH levels to 240% of the unstimulated control (100%
level), whereas maximum stimulation with wild type activin A was 200%
(Fig. 5). This indicates that the D27K
mutant has a higher intrinsic biological activity in this assay, a
conclusion consistent with the observation that the onset of
stimulation occurred with a lower dose of D27K compared with wild type.
The ED50 values (i.e. the concentration of
protein that results in half of the maximum stimulation) are 0.4 ng/ml
for wild type versus 0.2 ng/ml for D27K, suggesting that
D27K has a higher affinity for the receptor complex.

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Fig. 5.
FSH release from pituitary cells by wild type
activin A and mutants. Rat pituitary cells were seeded in 24-well
plates, and the medium was changed to fresh medium containing purified
wild type or mutant activins. The cells were incubated for 2 days, and
FSH released into the medium was measured by RIA. Results are
represented as the mean values with standard deviations. Shown is one
representative experiment with triplicate incubations (with each
dilution) of the cells. , wild type activin; , D27K; ×, K102E;
, K102A; , K102R.
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The K102A and K102E mutants were less efficient at FSH stimulation.
Their maximal stimulation was 160% of the unstimulated control and
this required very high concentrations of ligand, which are known for
wild type activin to result in nonspecific effects in the assay
(including luteinizing hormone stimulation; Ref. 13). Also, the onset
of stimulation occurred at higher doses for K102A and K102E compared
with wild type activin A. Overall, K102A and K102E were about 50-fold
less potent than wild type activin in this assay. However, the K102R
mutant had FSH stimulatory capacities comparable with wild type activin.
These data confirm those obtained with the unpurified mutant activins
produced in HeLa cells with the vaccinia virus-T7 system. Mutants K102A
and K102E had little or no biological activity, while mutant K102R was
as active as wild type activin. The improved agonistic properties of
mutant D27K became obvious in the FSH assay. Mutant D27K is active at a
4-fold lower concentration compared with wild type and this mutant
activin also generated a higher level of FSH stimulation than wild type.
Receptor Binding of Purified Mutant Activins--
In order to test
the binding of mutant activins to type I and type II receptors,
affinity cross-linking experiments were performed on cells
overexpressing different (mouse) activin receptor combinations. These
receptors (IIA, IIB, ALK-2, and ALK-4) were expressed in kidney (PK15)
cells using the vaccinia virus-T7 expression system (40). Radiolabeled
wild type activin, and mutants D27K and K102R, all bound to the activin
receptors tested (type II receptors and type II-I receptor
combinations, respectively), whereas no binding could be detected with
the K102E mutant (Fig. 6). K102A binds to
ActRIIA/ALK-4 and ActRIIB/ALK-4, but interacts only very weakly with
type II/ALK-2 combinations. The fact that activins bind better to
complexes containing ALK-4 than those containing ALK-2 suggests that
ALK-4 is the type I receptor that responds to activin in vivo, a conclusion consistent with previous observations on the binding of activin to primary pituitary cells (25). The lack of
detectable cross-linked complexes with the K102E mutant was not due to
the method of cross-linking itself. BS3 uses lysine
residues to cross-link, and Lys-102 in activin A is not essential for
cross-linking by BS3, since mutant K102R could still be
cross-linked to receptor complexes in a manner similar to wild
type.

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Fig. 6.
Binding of radiolabeled wild type and mutant
activins to different combinations of activin receptors. PK15
cells were transfected (using the vaccinia virus-T7 system) with
cDNAs encoding different activin receptors: ActRIIA
(IIA), ActRIIB (IIB), ActRIA (ALK-2),
and/or ActRIB (ALK-4), as indicated. The cells were
affinity-labeled using 125I-wild type (WT),
125I-D27K, 125I-K102E, 125I-K102A
or 125I-K102R activins, and then incubated with
BS3 cross-linking agent. Samples were analyzed by SDS-PAGE
(8% gels; composite figure, only the K102A lane is taken from another
experiment) followed by autoradiography (data not shown), and analysis
using a PhosphorImager.
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In order to compare the binding affinities of the different mutant
proteins with that of wild type activin, competition cross-linking experiments were performed on PK15 cells transfected with ActRIIA and
ALK-4. These transfected cells were affinity-labeled using a constant
amount of 125I-activin A (150 pM) in the
presence of increasing concentrations (5-, 10-, and 20-fold excess) of
cold mutant or wild type activin. Wild type activin A and mutant D27K
competed for binding to the ActRIIA/ALK-4 receptor complex
efficiently, whereas mutants K102A and K102E did not (Fig.
7). Interestingly, K102R competed
efficiently for binding to ALK-4 in the ActRIIA/ALK-4 receptor
combination, but very poorly for binding to ActRIIA alone (data not
shown).

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Fig. 7.
Binding of iodinated wild type activin to
transfected PK15 cells and competition with cold wild type or mutant
activins. ActRIIA was cotransfected with ALK-4 using the vaccinia
virus-T7 system. The cells were affinity-labeled using
125I-wild type activin in the presence of different
concentrations of cold competitor (wild type or mutant activin A),
followed by cross-linking with BS3. Samples were analyzed
by SDS-PAGE (8% gels) followed by autoradiography (data not shown) and
analysis using a PhosphorImager. Competition of 125I-wild
type binding by cold wild type or mutant activins was quantitated by
the amounts of radioactivity in the ActRII complex (II)
using a PhosphorImager. Experiments were repeated using different
concentrations of cold activins (both wild type and mutant), and
representative data are shown. , no competitor added.
|
|
Quantification (using a PhosphorImager) indicates that D27K
(KD 350 pM) has a 2-fold higher affinity
than wild type activin (KD 600 pM, a
figure confirmed by Scatchard analysis; data not shown) for binding to
ActRIIA/ALK-4 receptor complexes. The affinity of K102A for this
receptor complex is lower than that of wild type activin; although this
mutant is able to bind to ActRIIA/ALK-4 (Fig. 6), it is not able to
compete with wild type activin, at least at the concentrations of
competitor tested. The 2-fold stronger binding of D27K for the
ActRIIA/ALK-4 receptor complex is consistent with the results from the
FSH assay, where mutant D27K had a 2-fold lower
ED50 than wild type activin. The relative binding
affinities of mutants K102A, K102E, and K102R for the
ActRIIA-ALK-4 receptor complex are also in agreement with their activities in the FSH assay.
 |
DISCUSSION |
In the present study, we have identified two individual amino
acids in activin A that are important for biological activity as
assessed by their ability to stimulate FSH release by gonadotropic pituitary cells and to induce mesoderm in Xenopus animal cap
assays: residue K102, located in loop 2 of each subunit of the dimer, and D27, in loop1. Substitution of the positively-charged amino acid
(K102) with a neutral (A) or negatively charged (E) residue greatly
reduces activin function, whereas mutant K102R has no effect on activin
bio-activity, suggesting that a positive charge at position 102 is
crucial for activity in these assays. Substitution of D27 with K
results in a mutant protein with a 2-fold higher specific activity than
wild type activin. This study adds important new results to previously
obtained data concerning the structure and function of activins, which
have demonstrated that phenylalanine 21 of zebrafish activin B and 2 cysteine residues (Cys-4 and Cys-12 in the mature protein) of human
activin A are important for biological activity (16, 20). However, the
precise level at which the phenylalanine 21 mutant affects the
biological activity of activin has not been determined.
Affinity cross-linking experiments indicate that Lys-102 is crucial for
interaction with the type II receptor and, as predicted by the current
model of receptor activation, also for binding to a type II (A/B)-type
I (ALK-2/4) receptor complex while mutant D27K can be cross-linked to
the ActRIIA/ALK-4 receptor complex with a 2-fold higher efficiency than
wild type activin. Since D27K displays a higher biological activity, we
do not believe that this more efficient cross-linking occurs because of
the introduction of an additional lysine, but that it rather reflects a
higher binding affinity of D27K for the receptor combination tested
here. The latter could be the result of a higher rate of association and/or a lower rate of dissociation.
Lys-102 is positioned in a region (loop 2) of the ligand previously
shown to be important for high affinity binding of TGF-
1 to T
RII
(21). Other approaches, using antagonistic peptides that block binding
of TGF-
to its receptors, have defined the W/RXXD motif
of the N-terminal segment of the long
-helix of TGF-
s as a
primary determinant for receptor binding (41). However, the
W/RXXD motif does not seem to be involved specifically in type II receptor binding, as a peptide containing this motif also blocks binding of TGF-
to the high molecular weight type III and
type V receptors (42, 43). Moreover, such peptide studies usually need
high concentrations of peptides to interfere with the function of the
wild type molecule, which can lead to nonspecific effects, as reported
previously (44).
All mutant activins, including K102A and K102E, can be cross-linked to
the activin-binding and inhibitory protein follistatin (FS288). Many
conclusions can be drawn from this observation. First of all, together
with the fact that all activin A variants can form mature homodimers
and heterodimers with activin B, this indicates that their overall
three-dimensional structure is not dramatically altered. However, at
this stage it is not clear whether Lys-102 or Asp-27 introduces local
structural changes in the receptor binding pocket of the ligand or
whether these mutations are directly involved in interaction with the
receptor. This is difficult to assess, since conformation-specific
monoclonal antibodies for ligands of the TGF-
family are not
available. In addition, it is significant that Lys-102 and Asp-27 are
located in the most flexible and solvent-accessible loop regions of the
ligand, which favor the hypothesis that they interact directly with the
receptors. Strikingly, Lys-102 and Asp-27 are conserved in BMP-7 and
GDF-5, which have been shown to bind to and signal through ActRIIA or ActRIIB containing receptor complexes (25, 45). In contrast, TGF-
s have, respectively, a lysine (Lys) and a glutamic acid (Glu)
residue at these positions, and TGF-
s do not bind ActRIIA or IIB.
Both these observations with BMP-7 and GDF-5 (25, 45) supported the
notion that these amino acids (Asp-27 and Lys-102) are important
for (type II) receptor recognition.
Second, since K102E does not bind to the activin type IIA and IIB
receptors, this suggests that the receptor binding determinant of
activin is (at least in part) distinct from the follistatin binding
determinant. Consistent with this idea, we note that BMPs can also bind
follistatin and that follistatin can form a trimeric complex with BMP
and its receptor (46). A peptide approach has defined two contact sites
in activin that are necessary for interaction with follistatin (47).
These sites encompass amino acids 15-29 and 99-116 in activin A. Although Lys-102 is localized in one of these regions, it is not
necessary for binding to follistatin, suggesting that follistatin may
act by masking this amino acid and thus prevents activin from binding
to the activin type II receptors, as reported previously (33).
An ideal antagonistic ligand would be able to interfere with wild type
ligand function by binding to its receptor(s) without activating the
signal transduction cascade. In the TGF-
family, such an antagonist
would bind with a normal or even higher affinity to a type II receptor,
but not at all to a type I receptor. Although we performed an extensive
mutagenesis study, such an antagonistic activin variant was not found.
Possibly, a more drastic change is needed to interfere with binding to
type I receptors or no strict separate binding determinants exist for
binding to type II and type I receptors. A dominant-negative ligand,
distinct from an antagonist, might interfere with wild type function in two ways: either by altering the affinity of the mutant/wild type heterodimer for its receptor(s) or by interfering with the processing of the wild type ligand. Co-translation of the wild type and
dominant-negative ligands would thereby deplete the endogenous pool of
activin. Such dominant-negative variants of activin B, BMP-7 and BMP-4, have been described, and in these the consensus cleavage site for the
protein convertase is modified into a noncleavable sequence (16, 35).
In addition, certain cysteine mutants of different TGF-
members have
been identified as dominant negative; however, such mutations may
result in nonspecific inhibition of ligand secretion (20, 23). The
K102E mutant is likely to act as a dominant negative construct of the
first kind, because homodimers and heterodimers with activin B are
still secreted but, at least in the case of the K102E homodimer, cannot
interact with the type II receptor. Future studies, for example using
RNA injection experiments in Xenopus embryos, can
investigate this question in more detail. Future work should also try
to extrapolate our data to other ligands of the TGF-
family. In this
way, our work will contribute to the design of agonistic, dominant
negative, and antagonistic variants of TGF-
members. These variants
might help in the development of new therapeutic agents,
e.g. for use in bone repair, wound healing, fibrosis, immune
modulation, and acute kidney insufficiency.