From the Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California 92037
Received for publication, February 25, 2003
, and in revised form, March 20, 2003.
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ABSTRACT |
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INTRODUCTION |
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The signaling events initiated by activin require binding of two types of transmembrane serine/threonine receptor kinases classified as type II (ActRII or ActRIIB) and type I (ALK4). Both receptors are transmembrane proteins with ligand binding activity in the extracellular domain and serine/threonine kinase activity in the intracellular domain (15). The activin type II receptors are the primary ligand-binding proteins and can bind ligand with high affinity in the absence of type I receptors (18). The type I receptor, however, is unable to bind ligand in the absence of the type II receptors (19, 20). In the receptor complex, the constitutively active type II receptor kinase phosphorylates ALK4 in the regulatory GS domain, a glycine- and serine-rich segment on the membrane-proximal side of the kinase domain, and this phosphorylation leads to activation of ALK4 (21). Once activated, ALK4 binds and then phosphorylates cytoplasmic Smad proteins, which form part of the post-receptor signal transduction system (22).
Recently obtained crystal structure data have greatly advanced our understanding of how members of the TGF- superfamily interact with their receptors. The complex structure of BMP-7 dimer bound to the ActRII-ECD shows that the ActRII-ECD makes contact with only one of the dimer subunits (23). The binding interface revealed by the x-ray structure agrees with the binding affinities available for mutants of ActRII (24), activin-A (25, 26), and BMP-2 (27). The structure of BMP-2 in complex with BMP receptor IA (ALK3, BR1A) was also solved recently (28), revealing a type I receptor ECD fold similar to that of the ActRII-ECD. In the structure of the complex, ALK3 binds to the finger-helix groove of the BMP-2 dimer in such a way that each ALK3-ECD molecule is in contact with both BMP-2 monomers. The ligand binding interface on ALK3 for BMP-2 is characterized by a groove on the concave surface of the ECD and by residues in the short
helix.
Based on the crystal structure of the BMP2·ALK3-ECD complex and homology modeling, we have subjected the ALK4-ECD to alanine scanning mutagenesis with the goal of identifying the amino acid residues required for activin binding. We predicted that by individually mutating ALK4-ECD residues to alanine we would be able to identify amino acids that are important for activin binding while minimizing the structural changes caused by mutation. Using this approach, we have identified five hydrophobic amino acid residues (Leu40, Ile70, Val73, Leu75, and Pro77) that, when individually mutated to alanine, had substantial effects on activin binding to ALK4. In addition, eleven other mutants moderately or weakly disrupted activin binding. Homology modeling suggests that these residues interact with each other to form a contiguous surface on the concave face of ALK4 that is a likely activin binding interface.
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EXPERIMENTAL PROCEDURES |
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Mutagenesis of ALK4A kinase-deleted ALK4 construct (ALK4-trunc) that encodes the first 206 amino acids of ALK4 was generated using standard PCR techniques. To incorporate the amino-terminal FLAG tag (following glycine 28) and to generate mutations in the ECD of ALK4-trunc or full-length ALK4, we utilized an overlapping PCR strategy (24). Primers were constructed to incorporate a 5'-HindIII site and a 3'-EcoRI site, and PCR products were gel-purified and digested with both enzymes and then subcloned into HindIII/EcoRI-digested pcDNA3 vector to yield mutant receptor constructs. For each construct, the mutated amino-terminal ECD region was confirmed by DNA sequencing.
Transfection and Detection of Cell Surface Expression of ALK4 in Intact HEK293T CellsHEK293T cells were grown in 5% CO2 to 4060% confluence on poly-D-lysine-coated 6-well plates in complete Dulbecco's modified Eagle's medium (with 10% bovine calf serum, penicillin, streptomycin, and L-glutamine). Cells were transfected with wild-type ALK4, ALK4-trunc, or ALK4-trunc ECD mutants using Perfectin (Gene Therapy Systems). For co-transfection of ActRII and ALK4, a 2:1 ratio of their respective cDNAs was used. For Western blotting, cells were solubilized in 200 µl of 1% Triton X-100, and protein concentrations were determined using the BCA method according to the manufacturer's instructions. SDS-PAGE and electrotransfer to nitrocellulose were carried out using NuPAGE gels and a NOVEX X-cell II apparatus as described previously (24). To detect FLAG-tagged ALK4 constructs expressed at the cell surface, intact cells were fixed in paraformaldehyde, incubated with anti-FLAG antibody, washed, and treated with peroxidase-conjugated anti-mouse Ig. Specific antibody staining was measured using 3,3',5,5'-tetramethyl-benzidine peroxidase substrate as described previously (24).
Covalent Cross-linkingCovalent cross-linking was performed by incubating transfected HEK293T cells (4 x 106) with 125I-activin-A (106 cpm/well) for 2 h at room temperature in binding buffer (24) with gentle rocking. Cells were washed, resuspended in 1 ml of Hepes buffer (HDB; 12.5 mM Hepes, pH 7.4, 140 mM NaCl, and 5 mM KCl), brought to 0.5 mM disuccinimidyl suberate, and incubated for 30 min on ice. Cross-linking reactions were quenched with TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl), cells were solubilized in 1% Triton X-100 lysis buffer for 30 min on ice, and the resultant supernatant was subjected to immunoprecipitation using anti-FLAG antibody. Immune complexes were analyzed by SDS-PAGE and autoradiography.
Luciferase Assays in HEK293T and Mink Lung Epithelial (Mv1Lu) CellsThe activin/TGF-responsive luciferase reporter plasmid A3-Lux (30) and the FAST2 transcription factor (30) were used as described (31). HEK293T cells were plated on poly-D-lysine-coated 24-well plates at a density of
150,000 cells per well. Approximately 24 h later each well was transfected with ALK4-trunc constructs (200 ng) and/or empty vector (200 or 400 ng; pcDNA3), FAST-2 (50 ng), A3-lux (25 ng), and cytomegalovirus-
-galactosidase (25 ng). Transfections were performed under optimized conditions using Perfectin transfection reagent (Gene Therapy Systems). The cells were treated 68 h post-transfection with activin-A (1 nM) or activin-B (1 nM) for
16 h and harvested in solubilization buffer (1% Triton X-100, 25 mM glycylglycine, pH 7.8, 15 mM MgSO4, 4 mM EGTA, and 1 mM dithiothreitol), and luciferase reporter activity was measured and normalized relative to
-galactosidase activities. Transfection and luciferase assays were performed in essentially the same manner for Mv1Lu cells as described above for HEK293T cells, but 3TP-lux was used instead of A3-Lux, and FAST2 was not used.
Computer Modeling of ALK4-ECDComputer modeling and molecular mechanics employed Discover/Insight II (Accelrys, San Diego, CA) using the cff91 force field hosted on a Silicon Graphics Octane work station running IRIX64, version 6.5. Construction of an interactive computer graphical model of ALK4 employed homology modeling based on the x-ray crystallographic structure of ALK3 reported by Kirsch et al. (28) in their structure of the BMP-2·ALK3 complex (Protein Data Bank entry 1ES7
[PDB]
). Based on the primary sequence alignment of ALK3 and ALK4, 55 residues were identified for single-residue substitution; eight deletions of one residue or more and a single insertion of five residues were similarly identified. Single residue substitutions were performed with retention of side chain orientation where possible; in the event of severe overlap, manual rotation was conducted to minimize steric hindrance. Local perturbations caused by deletion were allowed to anneal by restrained minimization wherein the backbone atoms of the nascent ALK4 model were tethered in place with the exception that the residues on either side of a deletion were allowed to move. A simple turn structure was imposed on the five-residue insertion spanning ALK4 residues Lys72-Val76. This insertion attempted to maximize the solvent-accessible area of Lys72 and Glu74 and to exploit a naturally arising salt bridge involving Asp56 and Lys72. Final overlap of the experimentally observed ALK3 and theoretically derived ALK4 structures was 1.25 Å root mean square over the common backbone.
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RESULTS |
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Expression of ALK4-trunc in HEK293T CellsTo facilitate detection of ALK4-trunc expressed in HEK293T cells, we used PCR to introduce a FLAG epitope tag (DYKDDDDK) at the extreme amino terminus immediately following the putative signal peptide (after Gly28). To demonstrate that the FLAG-tagged ALK4-trunc constructs were expressed in 293T cells, we initially performed Western blot experiments. Fig. 2A shows that wild-type and selected mutant ALK4-trunc proteins are expressed at comparable levels in 293T cells. An antibody directed against the FLAG epitope tag recognizes an 26-kDa protein (expected size 22.6 kDa) expressed in cells transfected with ALK4-trunc constructs but not vector alone (Fig. 2A). To demonstrate that the FLAG-tagged ALK4-trunc constructs were expressed at the cell surface we used an intact cell enzyme-linked immunosorbent assay (24) (Fig. 2B). Together, these results demonstrate that the FLAG epitope is present on the ALK4-trunc constructs, that the wild-type and mutant receptors are expressed in this system at similar levels, and that the receptors are expressed at the cell surface.
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Covalent Cross-linking of Activin to ActRII and Wild-type or Mutant ALK4-trunc ConstructsTo identify ALK4-trunc mutants with altered activin binding, we co-expressed the mutants in HEK293T cells together with ActRII, and performed affinity labeling experiments. Immunoprecipitation of cross-linked complexes with anti-FLAG antibodies directed against ALK4-FLAG led to the identification of a number of ALK4 residues important for activin-A binding (Fig. 3). In cells transfected with ActRII-Myc and wild-type ALK4-trunc, the predominant species of 40 kDa represented ALK4-trunc cross-linked to a single activin subunit (the
55-kDa species presumably represented ALK4-trunc cross-linked to the activin dimer). The ActRII·activin cross-linked complex (
80 kDa) was ineffectively immunoprecipitated using anti-FLAG antibodies consistent with previous studies (23) that have shown the activin·ActRII·ALK4-trunc complex to be unstable following solubilization. Of the 26 ALK4-trunc mutants tested, five had significant effects on activin binding whereas several others had intermediate effects. Mutants L40A and V73A formed cross-linked complexes with 125I-activin-A more efficiently than wild-type ALK4-trunc (Fig. 3). In contrast, mutants I70A, L75A, and P77A cross-linked poorly to 125I-activin-A (Fig. 3). Mutants P71A, K72A, V76A, G79A, F82A, R91A, and T93A showed some decrease in binding to 125I-activin-A compared with wild-type ALK4-trunc (Fig. 3). These results were supported by immunoprecipitation using antibodies directed against ActRII-Myc (data not shown). The Myc antibody effectively immunoprecipitated activin·ActRII complexes but was less efficient in isolating activin bound to ALK4-trunc. Nevertheless, the same pattern of 125I-activin-A cross-linking to the ALK4-trunc mutants was evident.
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Mutation of Amino Acids Required for Activin Binding Affects the Dominant Negative Activity of ALK4-trunc in HEK293T CellsWe tested the relative abilities of wild-type and mutant ALK4-trunc constructs to block the activin-A induction of the TGF-/activin responsive luciferase reporter construct A3-Lux in HEK293T cells. As shown in Fig. 4, activin-A induced A3-Lux reporter activity when this plasmid was co-transfected with FAST2 (31) into HEK293T cells. Activin-induced A3-Lux activation was decreased
65% when wild-type ALK4-trunc was co-transfected (Fig. 4). However, consistent with the cross-linking data, receptors with alanine substitutions at any one of the three residues identified to be essential for activin binding (Ile70, Leu75, or Pro77) were not able to exhibit dominant negative activity (Fig. 4). In addition, the ALK4-trunc mutants with increased activin-A binding (L40A and V73A; see Fig. 3) were more effective dominant negative inhibitors than wild-type ALK4-trunc (Fig. 4), despite equal expression at the cell surface (Fig. 2B). Mutant L40A decreased activin-induced A3-Lux activation by
80% relative to vector-transfected cells whereas mutant V73A decreased activation by
90% relative to vector. The behavior of most of the other ALK4-trunc mutants in the functional assay (Fig. 4) corresponded to their ability to cross-link activin (Fig. 3). Mutants P71A, V76A, G79A, F82A, R91A, and T93A, which showed decreased binding to 125I-activin-A compared with wild-type ALK4-trunc (Fig. 3), also displayed impaired dominant negative activity (suppressing the activin-induced luciferase response by 3550%; see Fig. 4). Mutants S55A, A78G, and S87A, although displaying activin binding comparable with wild-type ALK4-trunc (Fig. 3), were less effective dominant negative inhibitors (Fig. 4). All other mutants suppressed activin-induced luciferase activity to the same extent as wild-type ALK4-trunc. Together, these results provide further evidence in intact cells demonstrating the importance of the Leu40-Ile70-Val73-Leu75-Pro77 hydrophobic residues in mediating activin/ALK4 binding and activin signaling.
ALK4 Binding Determinants Are Conserved for Activin-A and Activin-BWe tested whether wild-type and selected mutant ALK4-trunc constructs could also block activin-B induced luciferase activity. Fig. 5B shows that activin-B-induced A3-Lux activity was suppressed 55% upon overexpression of wild-type ALK4-trunc (compared with
75% suppression of activin-A induced A3-Lux activity; see Fig. 5A). ALK4-trunc constructs with alanine substitutions at Ile70, Leu75, or Pro77 were poor dominant negative inhibitors of both activin-A- and activin-B-induced luciferase activity (Fig. 5, A and B). In addition, the V73A mutant was a more effective dominant negative inhibitor than wild-type ALK4 for both activin-A and activin-B. Mutations at residues Ser38 and Phe82 had little effect on ALK4 dominant negative activity for either activin isoform (Fig. 5). Overall, these results indicate that activin-A and activin-B interact with the same residues on ALK4.
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Mutation of Amino Acids Required for Activin Binding Affect the Ability of ALK4 to Mediate Activin Signaling in Mink Lung Epithelial CellsMv1Lu mink lung epithelial cells have low levels of activin type I and type II receptors and very limited responsiveness to activin (19, 20). However, these cells show strong transcriptional and antiproliferative responses to activin when transfected with appropriate activin receptors (19, 20) and therefore provide an ideal system to compare the effects of wild-type and mutant ALK4. Mv1Lu cells co-transfected with the TGF-/activin responsive luciferase reporter construct 3TP-Lux and empty vector responded poorly to treatment with 1 nM activin-A, showing a 3-fold induction in luciferase activity (Fig. 6). In contrast, cells transfected with wild-type ALK4 or the M53A mutant (which had no effect on ALK4-trunc function in the dominant negative assay) mediated a 12-fold induction in luciferase activity. Alanine mutations at residues Ile70 and Leu75 were shown previously to be essential for activin binding and resulted in full-length ALK4 mutants that were unable to mediate activin signaling (Fig. 6).
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Structure-Function Relationship Analysis of ALK4The three-dimensional atomic structure of ALK4 has yet to be determined. Therefore, we constructed a homology model of the ALK4-ECD (Fig. 7B) based on the ALK3-ECD structure (Fig. 7A) (28). Despite relatively low sequence homology (30% in the structurally defined ECD regions) of these type I receptors, the ten cysteine residues can be aligned and are thought to confer the same general fold (three-finger toxin fold) in ALK4 as observed in the ALK3 structure. Our modeling was also instructed by the ALK5-ECD model Guimond et al. (33) produced based on the ALK3-ECD structure. ALK4 and ALK5 have higher sequence homology (
38% in the structurally defined ECD regions) than that shared between ALK4 and ALK3 (Fig. 1). In addition, there are fewer non-homologous regions (i.e. gaps in sequence alignments) observed between ALK4 and ALK5 compared with ALK4 and ALK3. Overall, the modeled ALK4-ECD structure preserves the open left hand topology of the ALK3-ECD structure, with a concave face and a convex face stemming from the curvature of the central
-sheet (Fig. 7, A and B). However, the pre-helix extension in the ALK4-ECD model significantly alters the conformation of this region with respect to the ALK3-ECD structure (Fig. 7C). In addition, predicted groove-forming residues of ALK4, including Met53, Ser55, and Thr93, are only partially exposed on the concave surface of the receptor suggesting a possible distortion and masking of the groove in the ALK4-ECD compared with the ALK3-ECD (Fig. 7B).
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Mapping of the ALK4 residues mutated in this study onto the modeled structure of the ALK4-ECD is shown in Fig. 7B. Our mutagenesis studies have identified five hydrophobic amino acid residues (Leu40, Ile70, Val73, Leu75, and Pro77) that are each important for activin binding and that, together with contributions from several other residues, likely provide an interface for ligand binding and responsiveness. Based on the ALK3 structure (28), residues Leu40 and Ile70 are predicted to be groove-forming residues on ALK4 (Fig. 7). Residues Val73, Leu75, and Pro77, however, are in the region of ALK4 that has low homology with ALK3 (Fig. 1). Modeling predicts that this region of ALK4 (Pro71-Ala78; see Fig. 7) would form a largely hydrophobic loop prior to the short helix. Fig. 7 shows that residues Val73, Leu75, and Pro77 of this loop are in close proximity to residues Leu40 and Ile70, and this hydrophobic patch likely mediates activin binding. Other residues that were mutated in this study are also indicated on the surface of the model of the ALK4-ECD. Residues Val76, Ala78, Lys80, Phe82, Tyr83, Arg91, and Thr93, which when mutated partially affected activin signaling, surround the hydrophobic patch. In contrast, residues that when mutated had no effect on ALK4 function (e.g. Ser38, Met53, Lys72, Pro81, Glu88, Asp89, and Leu90) are, in general, distant to the putative binding surface.
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DISCUSSION |
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In recent years, crystal structure data have greatly advanced our understanding of how members of the TGF- superfamily interact with their receptors. The structure of BMP-2 in complex with BMP receptor IA (ALK3) revealed a type I receptor fold similar to that of the ActRII-ECD (28, 37). This structure shows that each ALK3-ECD molecule makes contacts with both subunits of the BMP-2 dimer. The ligand binding interface on ALK3 is characterized by a groove on the concave surface similar to the ligand binding surface on ActRII (28, 37). The groove is formed by residues His43 and Pro45 on one side and Lys79, Met78, and Gln86 on the other side; the floor of the groove is largely hydrophobic because of the side chains of Ile99, Phe60, and Met78, and the groove ends in a partly polar and partly hydrophobic hollow surrounded by side chains of Ile62, Ile99, and Arg97. The groove is filled by residues from the pre-helix loop of BMP-2. Outside the groove, Phe85 of ALK3 fits with knob-into-hole packing into a hydrophobic pocket on BMP-2 and was proposed to be a key determinant for BMP-2 binding (28).
Based primarily on information from the crystal structure of the BMP2·ALK3-ECD complex, we subjected the ALK4-ECD to alanine scanning mutagenesis in an effort to identify the amino acid residues required for activin binding and function. We selected 26 residues on the ALK4-ECD that we individually mutated to alanine in the context of the kinase-deleted receptor. These mutants were expressed at similar levels as determined by Western blot experiments and were targeted to the cell surface based on detection of their respective amino-terminal FLAG epitopes on intact cells.
Our results indicate that there is only a partial overlap of the binding sites on ALK4 and ALK3 for activin-A and BMP-2, respectively. Of the predicted groove-forming residues on ALK4 (Ser38, Leu40, Met53, Ser55, Ile70, Pro71, Tyr83, Ser86, Arg91, and Thr93) only mutation of residues Leu40 and Ile70 had pronounced effects on ALK4 function. Surprisingly, Phe82, which corresponds to Phe85 on ALK3, and which was predicted to be a key feature generally required for type I receptor-ligand interactions (28), is not absolutely required for activin-A binding to ALK4. Of greater importance are residues (Val73, Leu75, and Pro77) in the region preceding the short helix (Lys80-Leu85) where there are five residues in ALK4 that do not have corresponding residues on the ALK3-ECD. Modeling predicts that this stretch of amino acids would form a largely hydrophobic loop prior to the short
helix. This loop, together with contributions from residues Leu40 and Ile70, likely forms a functional activin binding surface. Guimond et al. (33) in their recent study of the ALK5-ECD discussed the probable functional importance of non-homologous regions of type I receptors. Here we show that a region of low structural similarity between ALK4 and the BMP type I receptors (ALK2, ALK3, and ALK6) determines ligand binding.
Interestingly, of the seven ALKs, only ALK4 and ALK5 (type I TGF receptor) contain the pre-helix loop extension. In addition, three of the ALK4 residues we have shown to be required for activin binding (Ile70, Leu75, and Pro77; the ILP motif) are conserved in ALK5 (Ile72, Leu77, and Pro79). Although limited mutagenesis of the ALK5-ECD has been performed (33), residues Ile72, Leu77, and Pro79 were not targeted. Based on structural homology between activin and TGF-
and their respective type I receptor ECDs, we predict that Ile72, Leu77, and Pro79 on the ALK5-ECD are likely to play an important role in TGF-
binding. The additional pre-helix residues present in ALK4 and ALK5 may contribute to the inability of activin and TGF-
to bind to their type I receptors in the absence of their type II receptors (19, 20). BMP type I receptors lack this pre-helix loop extension and can directly associate with their respective ligands (38).
Because activin does not bind ALK5, and TGF- does not bind ALK4, residues on the ALK4-ECD other than the conserved ILP motif presumably determine ligand specificity. Mutation of residue Val73 (Ile75 on ALK5) to alanine resulted in a 2-fold increase in activin binding to ALK4 in the presence of ActRII, relative to wild-type ALK4. Moreover, in functional assays, mutant V73A was more efficient than wild-type ALK4 in mediating activin signaling. The mechanism by which mutant V73A mediates increased activin activity is unclear. As described previously (19), the affinity of activin for ActRII was not increased significantly by co-expression with wild-type ALK4, and it also appeared to be unaffected by the V73A mutant (data not shown). In addition, the V73A mutant does not bind ligand in the absence of ActRII (data not shown). Val73 is in close proximity to the ILP motif and, based on the BMP-2·ALK3 structure, likely contacts residues in the wrist epitope of the activin dimer following activin binding to ActRII. It is possible that the conservative valine to alanine mutation at position 73 increases the affinity of ALK4 for the activin·ActRII complex. Other residues, including Leu40 (Thr42 on ALK5), are probably involved in determining ligand specificity; however, the identification of an activating mutation at residue 73 further highlights the importance of the pre-helix loop extension of ALK4 for activin binding.
Three of the hydrophobic residues required for activin binding and signaling (the ILP motif) are conserved between ALK4 and ALK5 and may mediate the similar manner in which activin and TGF- interact with their type I receptors. The pre-helix extension that is integral to the activin binding site on ALK4 is absent in ALK2, ALK3, and ALK6 and may explain the differences in type I receptor binding observed between activins and BMPs. Future studies could address these questions of ligand specificity and mechanisms of type I receptor binding by generating ALK4/ALK3 (or ALK5) chimeras. Changing important ALK4 residues individually or in combination to the corresponding residues on ALK3 may yield chimeric ALK4 receptors that could bind activin and/or BMP-2 in the absence of type II receptors. The identification of residues on ALK4 that restrict activin binding in the absence of type II receptors could provide insight into the structural basis for the type I receptor recruitment model that describes activin signaling.
The importance of characterizing the ALK4 binding interface is further highlighted by the roles this receptor plays in development. Recent evidence indicates that signaling by other TGF- ligands, including nodal, GDF-1, and Vg1, is mediated by ActRII/IIB and ALK4 (39, 41). Nodal, GDF1, and Vg1 have been implicated in early embryonic patterning events (39, 40). However, unlike activin, these other TGF-
ligands require additional co-receptors such as the EGF-CFC protein Cripto to generate signals (42). Current evidence suggests that Cripto, or other EGF-CFC proteins, bind to ALK4 and form a complex with nodal-related ligands and ActRII/IIB (40). It will be interesting to determine whether the Cripto binding site on ALK4 is distinct from the activin binding site. If the binding sites do not overlap, then specific activin and nodal agonists or antagonists targeting ALK4 could be generated. Such compounds could be used in the regulation of a wide array of diverse biological processes including hormone release, cell proliferation, differentiation, and pattern formation during embryogenesis (16, 43).
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FOOTNOTES |
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These authors contributed equally to this work.
Foundation for Medical Research Senior Investigator. To whom correspondence should be addressed: Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA 92037. Tel.: 858-453-4100 (ext. 1307); Fax: 858-552-1546; E-mail: vale{at}salk.edu.
1 The abbreviations used are: TGF-, transforming growth factor-
; BMP, bone morphogenetic protein; ECD, extracellular domain; HEK, human embryonic kidney; ALK4-trunc, truncated ALK4; EGF-CFC, epidermal growth factor-cripto, FRL-1, cryptic.
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REFERENCES |
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