 |
INTRODUCTION |
The TGF-
1 superfamily
comprises a large number of secreted signaling polypeptides implicated
in a diverse set of functions (1). TGF-
family members act by
binding to two classes of transmembrane serine-threonine kinases termed
the type I and type II receptors, thereby stabilizing their interaction
(2). Type II receptors are constitutively active kinases capable of
binding ligand alone, whereas type I receptors can only bind ligand
co-operatively in the presence of type II receptors. Following
ligand-induced association of the two receptor classes, the type II
receptor phosphorylates type I receptors on serine and threonine
residues in a critical regulatory region termed the GS domain (3, 4). This phosphorylation event activates the type I receptor, which then
mediates downstream signaling events (4).
The TGF-
family member activin induces animal pole tissue of
Xenopus embryos to form mesoderm rather than ectoderm (5), and recent evidence using a dominant-negative activin receptor suggests
that activin acts as an endogenous mesoderm-inducing factor in the
early amphibian embryo (6). We have shown previously that
constitutively active forms of the type I activin receptors ALK-2 and
ALK-4 (designated ALK-2* and ALK-4*) also induce prospective ectodermal
tissue to form mesoderm, but that the two receptors elicit different
responses (7). The effects of ALK-4* resemble those of activin itself,
in that it can, in a dose-dependent manner, induce
expression of the general mesodermal marker Xbra (8) as well
as the dorsoanterior marker goosecoid (9). The
response to ALK-2*, however, more resembles that of BMP-4 (10) in that it induces only ventral mesodermal markers and causes ventralization of
intact embryos (7). ALK-2* is also capable of counteracting the
dorsalizing effects of ALK-4*, an activity reminiscent of the ability
of BMP-4 to ventralize the response to activin (10), but ALK-2* and
BMP-4 differ in their ventralizing effects because the action of ALK-2*
is immediate (7), whereas that of BMP-4 is delayed (11).
Three regions were identified that might determine the specificity of
type I receptors, and use of chimeras-containing combinations of these
regions has led us to focus on a short loop on the small lobe of the
kinase, the
4-
5 loop. The 7 amino acids comprising this loop are
capable, when transferred from ALK-4* to ALK-2*, of carrying with them
the ability to induce dorsal markers. An analogous result has recently
been obtained using the TGF-
receptor TbR1/ALK-5 and Tsk7L/ALK-2 in
a tissue culture system (12). Significantly, however, the reverse
transfer of the
4-
5 loop from ALK-2* to ALK-4* does not
completely change the activity of the chimeric ALK-4* to that of
ALK-2*. Although the chimeric receptor retains the ability to induce
Xbra and loses the ability to induce dorsal genes, it is
unable effectively to counteract the dorsalizing effects of ALK-4*.
These observations suggest that there are several signaling pathways
downstream of type I receptors. One such pathway involves the Smad
proteins, which associate with type I-type II receptor complexes, are
phosphorylated on specific serine residues (13, 14), and translocate
from the cytoplasm to the nucleus after stimulation of cells with
exogenous TGF-
ligands (14, 15). The Smads possess sequence-specific
DNA binding properties (16), and Drosophila Mad (16) and
human Smad1 (17) have transcription activation domains, suggesting that
they might act directly in the regulation of gene expression.
In Xenopus development, overexpression of Smad proteins in
prospective ectodermal tissue causes mesoderm formation and, as with
the type I receptors, different Smads elicit different responses. Smad2, like ALK-4*, acts in a concentrationdependent manner
to induce the expression of dorsal genes (18), whereas Smad1 and Smad5,
like ALK-2*, induce ventral mesoderm (17, 19-22). These results
suggest that the specificity of the signal regulated by the
4-
5
loop is realized by activation of specific Smad signaling pathways.
However, Smad1, in contrast to ALK-2*, cannot counteract the early
effects of ALK-4* (or activin) at all, whereas Smad5 produces only a
very weak interference. The dominant ventralizing effects of ALK-2* are
therefore unlikely to be mediated by Smad competition, a conclusion
consistent with the observation that these ventralizing effects are not
mediated by the
4-
5 loop, which we suggest does act through
Smads. We have not yet identified the region of ALK-2* which causes
ventralization, but we do find that susceptibility to ventralization
resides in ALK-4* sequences that are not within the
4-
5 loop,
because dorsal gene induction mediated by a chimeric receptor with an
ALK-2* backbone cannot be inhibited by co-expressed ALK-2*.
 |
EXPERIMENTAL PROCEDURES |
Cloning and in Vitro Transcription of Chimeric Activin
Receptors--
Constitutively active forms of the ALK-2 and ALK-4
receptors (ALK-2* and ALK-4*) have been described previously (23).
Chimeras between the receptors were generated using a polymerase chain reaction technique in which DNA fragments were amplified using primers
possessing 5' extensions with homology to the sequence to which they
were to be fused. Reactions were then mixed and re-amplified. The
sequences that were exchanged are shown in Fig. 1. For the region IB1,
7 amino acids were exchanged between the receptors, namely residues 269 to 275 in ALK-4* and 270 to 276 in ALK-2* (NKDNGTW versus
MTSRHSST). For IB3, 15 amino acids were exchanged between the
receptors; that is, residues 359 to 373 in ALK-4* and residues 361 to
375 in ALK-2* (RHDAVTDTIDIAPNQ versus MHSQSTNQLDVGNNP). The
sequences exchanged in the GS domain regions were slightly more
extensive than those shown in Fig. 1, including some sequences more
N-terminal than those illustrated. Specifically these were residues 163 to 213 inclusive in ALK-4* (DMEDP to LQEII) and residues 158 to 214 inclusive in ALK-2* (NPRDV to LLECV). The clone containing
substitutions in the IB1 region of ALK-4* (ALK-4*IB1Mut)
was also generated by polymerase chain reaction, and the substitutions were NKDNGTW to NTGNGTW, one residue
(K to T) deriving from the equivalent position
of ALK-2 and the other (D to G) deriving from the equivalent position of ALK-3.
Clones were transcribed in vitro as described (24). RNAs
were synthesized side-by-side on the same day with the same reagents and quantified on agarose gels and by UV absorption. Xenopus
Smad1 and Smad2 (Xmad1 and Xmad2) were gifts of
Doug Melton (20). A mouse Smad5 cDNA (21) was a gift of
Ali Hemmati-Brivanlou.
Xenopus Embryos, Microinjection and
Dissection--
Fertilization, staging, culture, microinjection, and
dissection of Xenopus embryos were as described (24, 25). A
crude preparation of activin A was prepared from the conditioned medium of COS cells transfected with a human inhibin
A
cDNA. It was quantitated as described (26).
RNA Isolation and RNase Protection Assays--
RNase protection
analysis was carried out as described (24). Probes include cardiac
actin (27), Ef-1
(28), goosecoid (9), Xbra
(8), Pintallavis (29), and Xhox3 (30).
 |
RESULTS |
Identification by Sequence Analysis of Regions That May Determine
ALK-2* and ALK-4* Signaling Specificity--
The specificities of the
signals transduced by ALK-2* and ALK-4* are likely to be defined by
peptide binding sites within their intracellular domains. The
intracellular domains of all type I TGF-
receptors comprise a short
poorly conserved juxtamembrane domain, a highly conserved GS domain,
and finally a well conserved serine-threonine kinase. A multiple
sequence alignment was generated between the sequences of human ALK-1
to ALK-5 (31, 32) and Drosophila tkv, sax (33),
and Atr-1 (34). A number of divergent regions exist between the
vertebrate receptors, notably the juxtamembrane domain as well as a
number of regions within the kinase domain itself, but only three
divergent regions also retain homology with tkv, sax, and Atr-1 (Fig.
1). The most N-terminal region encompasses the GS domain itself; although the central core of this
motif is highly conserved, the sequences directly flanking it are
considerably more divergent. Other divergent regions were classified
interest boxes (IB), and two of these were also
conserved between the Drosophila orthologs. IB1 is a short
loop between kinase subdomains
4 and
5, and IB3 corresponds to
the activation loop of the enzyme.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 1.
Structure of the type I receptor kinase and
alignment of the three regions selected for analysis.
A, 3-dimensional representation of the type I receptor
kinase based on crystal structures of other kinases (44). The positions
of the IB1 region (the 4- 5 loop) on the small lobe of the kinase
and of the IB3 region (the activation loop of the enzyme) are marked
and colored. The position of the GS domain is also shown, although the
structure of this region is not known. B, representation of
the type I receptor kinase showing the locations of the GS domain, the
IB1 region, and the IB3 region. Sequences in the regions exchanged in
this analysis are aligned from the human ALK-1 to ALK-5 receptors and
the Drosophila thick veins and saxophone and Atr-1
receptors. Identical residues are shaded in the same
color. Regions showing almost 100% identity such as the
central core of the GS domain are not included.
|
|
The IB1 Sequence of ALK-4* Can Confer Activin-like Responses to
ALK-2*--
Activated ALK-2 and ALK-4 receptors (ALK-2* and ALK-4*)
elicit different responses in the Xenopus animal cap assay
(7). ALK-4* induces a wide range of tissues including notochord,
muscle, and endoderm, whereas ALK-2* induces only ventral mesodermal
derivatives. Furthermore, ALK-4* induces, like activin, expression of
the midline and anterior markers Pintallavis and
goosecoid, and at intermediate concentrations it activates
transcription of the more ventral marker Xbra. ALK-2*
induces expression only of Xbra. These markers are induced
by activin in an immediate-early fashion and are easily detectable by
the early gastrula stage, only 5 h after the mid-blastula transition (8, 9, 35). Expression of these genes is thus an ideal assay
for primary signaling events and allows us to distinguish between
signals deriving from ALK-2* and ALK-4*.
Chimeric receptors were generated in which each of the three candidate
specificity regions was replaced with that from the other receptor in
all possible combinations (Fig. 2).
Receptors C0 to C7 correspond to constructs that contain an ALK-2*
backbone, retaining either all ALK-2* sequences (C0) or containing
various portions of the ALK-4* receptor in C1 to C7. Previous work has shown that ALK-2* itself cannot induce significant levels of
Pintallavis or goosecoid, but it induces
Xbra efficiently. We show here that some of the chimeric
receptor constructs in the ALK-2* backbone behave very differently from
ALK-2* in this respect (Fig.
3A). Most strikingly, exchange
of the IB1 sequence from ALK-4* to ALK-2* results in strong expression
of both goosecoid and Pintallavis. This was the
case whether it was exchanged alone (C2) or in combination with the GS
domain and flanks (C4), in which case the response was slightly but
reproducibly stronger. By contrast, exchange of the GS domain and
flanks alone or exchange of the IB3 region did not reproduce an
ALK-4*-like response. These results indicate that the IB1 region of
ALK-4* is a critical determinant of signaling specificity that is
capable of transducing a signal that activates expression of
dorsal-specific genes. This conclusion was confirmed by assaying
expression of muscle-specific actin at stage 17; C4 was capable of
inducing strong expression of muscle actin, and C2 had this effect in
one of five experiments (not shown). The reason for this variability in
C2 behavior is not clear.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 2.
Schematic representation of the 16 chimeric
clones used in this study. Regions derived from ALK-4* are shown
in green, and regions from ALK-2* are shown in
red.
|
|

View larger version (53K):
[in this window]
[in a new window]
|
Fig. 3.
Induction of stage 10 markers by the chimeric
receptors. Embryos were injected at the one cell stage with 1 ng
of RNA encoding each receptor. Animal caps were cut at stage 8 and
cultured until stage 10 when they were processed for RNase protection
using probes specific for goosecoid, Pintallavis,
Xbra, and Ef-1 . Receptor constructs with an ALK-2*
backbone are shown in A and with an ALK-4* backbone are
shown in B. The order of lanes in B is
correct as marked.
|
|
Surprisingly, some chimeric receptors elicited no response. In
particular C7, containing all three regions of ALK-4*, was consistently
and reproducibly inactive in these assays and did not appear to
interfere with the normal development of whole embryos expressing it
(data not shown). In addition, the responses of animal caps expressing
C3, C5, and C6 were rather variable. Sometimes C5 caused induction of
Xbra, whereas on other occasions (such as the experiment
shown in Fig. 3A) it elicited no response. Most oddly, C6
usually showed no activity but occasionally caused a full ALK-4*-like
response, including induction of Pintallavis and
goosecoid. The reason for this variability is not known,
although additional experiments demonstrate that all chimeric receptors are capable, at least to some extent, of inducing Xhox3 at
stage 17, suggesting that they retain some signaling activity (not
shown). Despite these peculiarities, the consistency of the data
obtained with chimeras C2 and C4 highlights the importance of IB1 in
characterizing the receptor signal.
The
4/
5 Loop (IB1) of ALK-4* Is Required for the ALK-4*-like
Response--
Receptor constructs C8 to C15 contain the ALK-4*
receptor backbone with various combinations of ALK-2* sequences
replacing those of ALK-4*. ALK-4* induces expression of
Xbra, Pintallavis, and goosecoid, as
previously demonstrated (7) (Fig. 3B). Injection of RNA
encoding C9 to C15 demonstrated that only chimeras retaining the IB1
region of ALK-4* mediate induction of goosecoid and/or Pintallavis (Fig. 3B) or, when assayed at stage
17, muscle-specific actin (not shown). In contrast, receptors
containing the ALK-2* IB1 are capable only of an ALK-2*-like response
(C10) or of no response at all at this stage (C12, C14, and C15). In
contrast to the results obtained with chimeras possessing an ALK-2*
backbone, the results were consistent and reproducible in all cases,
and as with C0 to C7, the chimeras containing the ALK-4* backbone all
induced expression of Xhox3 at stage 17 (not shown).
Interestingly, chimeras C14 (containing the IB1 and IB3 regions of
ALK-2*) and C15 (containing all three regions) were similar to C6 and
C7 in their inability to induce expression of early mesoderm-specific
genes (Fig. 3B) and their concomitant failure to cause
severe defects to embryonic development (not shown). This suggests
either that progressive replacement of regions of one receptor with
another results in structural perturbation and receptor inactivity or
that there is a requirement for intramolecular compatibility between
receptor regions. Additional evidence for a need for intramolecular
compatibility comes from the observation that transfer of the ALK-2* GS
domain and its flanking sequences into ALK-4* (C9) or transfer of the
ALK-2* IB3 activation loop (C11), both, result in loss of
goosecoid activation. Simultaneous transfer of both regions,
however, (C13) has little effect on the receptor, which can both induce
goosecoid strongly and cause caps to invert at the early
gastrula stage.
The
4/
5 Loop (IB1) of ALK-4* Mediates Signals That Cause Cap
Inversion at the Early Gastrula Stage--
We have previously observed
that injection of 1 ng of ALK-4* RNA induces strong morphological
movements in Xenopus animal caps. A similar effect has been
observed following overexpression of activin by RNA injection (23) and
by overexpression of Smad2 (20). These movements, which occur at early
gastrula stage 10, precede those that occur when caps extend in
response to lower doses of activin when protrusions are formed at
mid-gastrula stage 11. Caps that invert at stage 10 do not form
protrusions and proceed to form tissue that we suspect is endodermal in
nature (7). In this case the cells may be mimicking the earliest
gastrulating cells on the dorsal side of the embryo, which form
pharyngeal endoderm and/or head mesoderm. Migration of these cells may
be temporally and mechanistically different from that of other
mesodermal precursors in vivo.
We tested whether any of the chimeric receptors were capable of
inducing morphological movements characteristic of ALK-4* overexpression. Fig. 4 shows that the
chimeric receptors C2 and C4 both elicit cap inversion like that
induced by ALK-4*. This response is also seen with the ALK-4*
backbone-derived chimera C13. All the constructs that cause this effect
therefore contain the IB1 region of ALK-4*, with transfer of this
region alone into ALK-2* (C2) being sufficient to mediate the response.
Because the rapid cap inversion effect is also seen in response to
Smad2 overexpression (20), these results suggest that the signal
specified by the IB1 region of the receptor feeds directly into the
Smad signaling pathway.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 4.
Morphological responses of stage 10 caps to
the chimeric receptors. Animal caps were dissected at stage 8 from
embryos injected with 1 ng of the indicated RNAs. They were
photographed at mid-gastrula stage 11.
|
|
Mutation of the IB1 Region of ALK-4* Disrupts Receptor
Signaling--
To confirm that the
4/
5 loop (IB1) region of
ALK-4* is responsible for the activin-like responses of the receptor,
we generated a receptor clone containing a 2-amino acid substitution in
the IB1 region of ALK-4, with one amino acid substituted from the equivalent position in the ALK-2* receptor and the other from the ALK-3
receptor, hence generating a chimeric sequence within the IB1 domain
itself (NKDNGTW to NTGNGTW). We predict that
this would disturb interaction of candidate downstream signaling
components with the peptide sequence. Indeed this substitution resulted
in complete ablation of the early gastrula responses seen normally with
ALK-4*, consistent with a requirement for this region for effective
signaling (Fig. 5).

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 5.
Mutation of the IB1 region of ALK-4*
abolishes inducing activity. Animal caps were dissected at stage 8 from embryos injected with 1 ng of ALK-4* or ALK-4*IB1Mut,
which carries a 2-amino acid substitution within the IB1 region. At
stage 10, the caps were processed for RNase protection using probes
specific for goosecoid, Pintallavis, and
Xbra. Ef-1 was used as a loading control.
|
|
The Ability of ALK-2* to Interfere with ALK-4*-mediated Gene
Induction Cannot Be Recapitulated by Transfer of the IB1 Region of
ALK-2* into the ALK-4* Backbone--
ALK-2* interferes with the
ability of ALK-4* to induce expression of goosecoid and
Pintallavis (7). This is unlikely to result purely from
competition for common signaling components at the membrane; low doses
of ALK-2* will interfere very efficiently with ALK-4*-like responses,
and kinase-inactive ALK-2* constructs interfere only at much higher
doses, producing a qualitatively different type of interference (7). We
asked whether the
4/
5 loop (IB1) from ALK-2* would confer this
seemingly active inhibitory property to ALK-4*, a reasonable hypothesis
as the equivalent region of ALK-4* is so potent in signal specificity.
Surprisingly, this did not appear to be the case. The chimeras C3 and
C5 (which contain the ALK-2* IB1 in the ALK-2* backbone) and C10, C12,
C14, and C15 (which contain the ALK-2* IB1 in the ALK-4* backbone) all
failed to demonstrate effective suppression of ALK-4* induction of
goosecoid and Pintallavis (Fig.
6). Co-injection of these constructs lowered goosecoid and increased Xbra levels
slightly, but effective extinction of dorsal markers did not occur, and
the observed effects were reminiscent of the partial suppression
mediated by co-injection of kinase inactive ALK-2* (7). We therefore
believe that the active inhibitory action of ALK-2* derives
predominantly from signals elsewhere in the ALK-2* receptor.

View larger version (70K):
[in this window]
[in a new window]
|
Fig. 6.
Chimeric receptors do not interfere with
dorsal gene activation by ALK-4*. Animal caps were dissected at
stage 8 from embryos co-injected with 1 ng of ALK-4* and 1 ng of ALK-2*
or of other chimeric receptors. Caps were harvested at early gastrula
stage 10 and processed for RNase protection.
|
|
The Ability of ALK-2* to Interfere with Dorsal Gene Induction
Requires the ALK-4* Receptor Backbone--
The ability of ALK-2* to
interfere with dorsal gene induction by ALK-4* requires sequences that
are not contained within the ALK-2
4/
5 loop, because transfer of
this region to ALK-4* does not confer interference activity. If other
regions of ALK-2* are responsible for interference with dorsal gene
induction, however, the question of how the C2 chimeric receptor
activates dorsal genes is raised. With the exception of the ALK-4
4/
5 loop (which comprises just 7 amino acids), C2 is identical to
ALK-2* (Fig. 2). Why does it not inhibit its own dorsalizing activity?
One possibility is that susceptibility to inhibition requires ALK-4 sequences that are not present in C2. This was tested by co-expressing C2 with ALK-2*. Fig. 7A
(lanes 1 and 2) shows that ALK-2* is unable to
inhibit induction of goosecoid and Pintallavis by
C2, indicating that ALK-4 sequences are indeed required to respond to
the inhibitory effects of ALK-2*. This result also indicates that the
inhibitory effects of ALK-2* do not involve competition for molecules
downstream of type I receptors; if they did, ALK-2* should inhibit
dorsal gene induction by C2.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 7.
ALK-4 sequences confer susceptibility to
inhibition of dorsal gene activation by ALK-2*. A,
ALK-2* inhibits dorsal gene induction by ALK-4* but not by C2 (which is
based on an ALK-2* backbone). B, ALK-2* inhibits dorsal gene
activation by ALK-4*, C9, C11, and C13 (all of which contain an ALK-4*
backbone). Animal caps were dissected at stage 8 from embryos injected
with RNA (500 pg) encoding ALK-4*, ALK-2*, C2, C9, C11, or C13, either
alone or in the indicated combinations. Caps were cultured until early
gastrula stage 10 and then analyzed by RNase protection.
|
|
To confirm that susceptibility to inhibition requires sequences present
in ALK-4, ALK-2* was co-expressed with C9, C11, and C13, all of which
are capable of activating expression of Pintallavis and (to
some extent) goosecoid, and all of which are based on an
ALK-4* backbone (Figs. 2 and 3). Fig. 7B shows that ALK-2* is capable of inhibiting dorsal gene induction by all these constructs.
Overexpression of Smad1 or Smad5 Does Not Interfere with Induction
of Dorsal Genes by ALK-4*--
We suggest above that the IB1 region of
the type I receptor feeds directly into the Smad signaling pathway. If
this is so, then because the dominant ventralizing effects of ALK-2*
are not specified by the IB1 region, overexpression of ventralizing
Smad family members like Smad1 and Smad5 should also fail to counteract the effects of ALK-4*. Consistent with this suggestion, Fig.
8A shows that Smad1 is
completely ineffective at suppressing the ability of ALK-4* to induce
Pintallavis and goosecoid at stage 10.5. Smad1 is
also unable to suppress the ability of activin to induce expression of
goosecoid and Pintallavis (Fig. 8B),
although a recent report suggests that Smad1 is capable of reducing the induction of goosecoid by activin by stage 11 (17).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 8.
Smad1 does not interfere with dorsal gene
activation by ALK-4* or activin. Smad5 elicits slight interference,
comparable with that obtained with chimera C10. A, Smad1,
unlike ALK-2*, does not interfere with dorsal gene induction by ALK-4*.
B, Smad1, unlike ALK-2*, does not interfere with dorsal gene
induction by activin. C, Smad5 causes only slight
interference with dorsal gene induction by ALK-4*, comparable with that
caused by C10. This experiment was carried out at the same time as that
shown in Fig. 7A, and lanes 1, 2,
3, and 5 of C are identical to those
shown in lanes 6, 5, 3, and
4 of Fig. 7A. Animal caps were dissected at stage
8 from embryos injected with RNA encoding ALK-4* (500 pg), ALK-2* (500 pg), Smad1 (2 ng), Smad5 (2 ng), or chimera C10 (500 pg), either alone
or in the indicated combinations. Caps in lanes 3,
5, and 7 of panel B were treated with
8 units/ml activin. All animal caps were cultured until early gastrula
stage 10 and then analyzed by RNase protection. All lanes
shown are from the same gel, but their order has been changed for
clarity of presentation.
|
|
Similar experiments reveal that Smad5 is also capable of eliciting
little interference with dorsal gene induction, producing only a weak
inhibition that resembles that obtained with the C10 chimera (Fig.
8C). We conclude that the rapid ventralizing effects of
ALK-2* are not mediated solely through Smad competition.
 |
DISCUSSION |
Classification of Type I TGF-
Receptors--
Our analysis
allows the type I TGF-
superfamily receptors to be grouped into
three classes (Fig. 1). ALK-4 and ALK-5, receptors for activin and
TGF-
, respectively, fall into one group and are highly related. The
IB1 domains of these receptors are identical to that of
Drosophila Atr-1, although the GS and IB3 regions of Atr-1
differ from ALK-4 and ALK-5. ALK-3 (a BMP 2/4 receptor) and tkv fall
into another class, defined particularly by sequences in their IB1
regions as well as in the C-terminal flanks of their GS domains.
Finally ALK-1, ALK-2, and saxophone can be grouped together by similar
criteria, although this class is also characterized by strong homology
in the N-terminal flank of the GS domain and the C-terminal half of the
IB3 activation loop. Because, with the exception of Atr-1, the
receptors fall into the same classes at each of the three regions, it
seems likely that tkv and sax are derived from genes duplicated before
the divergence of insect and vertebrate lineages and that they may be
true orthologs of ALK-3 (and ALK-6, not shown here) and ALK-1/ALK-2,
respectively. We note that neither the daf-1 receptor (36) nor another
putative type I activin receptor from Caenorhabditis elegans
(SwissProt data base entry Q09488) possess homology with the
Drosophila and vertebrate type I receptors in the IB1 region
of the kinase. This is slightly unexpected as other components of the
TGF-
signaling system appear to be intact in C. elegans,
including the ligands, the type I and type II receptors, and the Smads.
Finally, although our sequence analysis defines three groups of type I
TGF-
superfamily receptor, the L3 loop of the Smad proteins, which
determines the specificity of receptor-mediated Smad activation, falls
into only two classes (37). It is possible that additional Smads remain
to be discovered or that other regions of the Smads also define
receptor specificity.
The
4/
5 Loop Confers Activin- and Smad2-like Responses to
ALK-2*--
The dorsal-inducing properties of constitutively active
ALK-4 can be conferred on ALK-2 by transfer of the
4/
5 loop from the kinase domain of one receptor to the other (creating C2). The
dorsal-inducing effects of the chimeric ALK-2 receptor resemble those
of Smad2, and we postulate that the
4/
5 loop interacts with the
Smad signaling pathway. Attempts to confirm this by immunoprecipitation of tagged Smad2 after receptor activation has not, however, revealed a
simple relationship between receptor subtype and Smad2 phosphorylation. ALK-2* and ALK-4*, both, cause phosphorylation of Smad2 (data not
shown). The functional significance of this phosphorylation is not yet
clear, because the sites of phosphorylation are unknown.
Analysis of other chimeric receptors suggests that there is a
requirement for compatibility between sequences in the IB3 activation loop and sequences in the juxtamembrane region, in or around the GS
domain. In particular, we note that transfer into ALK-4* of the ALK-2*
GS domain and its flanking sequences (creating C9) or transfer of the
ALK-2* IB3 activation loop (creating C11), both, result in loss of
goosecoid activation. Simultaneous transfer of both regions,
however, (creating C13) has little effect on the receptor, which can
both induce goosecoid strongly and cause caps to invert at
the early gastrula stage. It is possible that these regions lie close
to each other in the tertiary structure of the receptor, with the
activation loop being required for phosphorylation of the juxtamembrane
region. Consistent with this idea, point mutation of threonines in a
region N-terminal to the juxtamembrane sequences of TbR1/ALK-5 results
in defective receptor signaling (38). If true the idea would explain
why the two regions of the type I receptor show orthologous conservation.
The
4/
5 Loop Does Not Define All Signaling Properties of Type
I TGF-
Receptors--
Although transfer of the
4/
5 loop from
ALK-4* to ALK-2* causes the latter to acquire dorsal gene inducing
properties and to behave apparently identically to ALK-4*, the same is
not true of the reverse transfer. The C10 chimera, which contains the
ALK-2*
4/
5 loop in the ALK-4* backbone, is capable of inducing
expression of Xbra but is unable to counteract the
dorsalizing effects of ALK-4*. It differs in this respect from ALK-2*,
and the results therefore demonstrate that the
4/
5 loop is not
sufficient to account for all the signaling properties of the type I receptors.
Our data suggest that the
4/
5 loop regulates the activation of
specific Smad family members, and consistent with this idea, we observe
that the "ventralizing" Smads 1 and 5, like C10, cannot inhibit
dorsal gene induction by ALK-4* (Fig. 8). This conforms with previous
work demonstrating that although Smad1 can induce expression of ventral
mesodermal markers, it causes little reduction in levels of
goosecoid (17). Together, these observations support a
growing body of evidence suggesting that type I TGF-
receptors employ additional Smad-independent signaling pathways (39-42).
The mechanism by which ALK-2* inhibits dorsal gene induction by ALK-4*
is under investigation. The effect is interesting because it is
immediate and therefore differs from the ventralizing effects both of
BMP-4 and of the BMP receptor ALK-3, both of which are delayed (11,
43). The effect is also highly specific, because very low levels of
ALK-2* are sufficient to inhibit dorsal induction by ALK-4*, and
ventralization requires the kinase activity of ALK-2* (7). We also note
that chimeras C3 and C5 do not inhibit dorsal gene induction by ALK-4*,
despite the fact that they contain sequences derived predominantly from
ALK-2* and, significantly, that the chimeras C2 and C4, which contain
the ALK-4*
4/
5 loop in the ALK-2 backbone, are refractory to the
effects of ALK-2*. The latter result indicates that ventralization
requires sequences present in the ALK-4 backbone and cannot be due
simply to competition between ALK-2* and ALK-4* for Smad4 (43). It is
also unlikely that ALK-2* sequesters Smad2 but fails to phosphorylate
it, because we cannot detect stable interactions between ALK-2* and
tagged Smad2 (data not shown).