An activin mutant with disrupted ALK4 binding blocks signaling via type II receptors

Activins control many physiologic and pathophysiologic processes in multiple tissues and, like other TGF-beta superfamily members, signal via type II (ActRII/IIB) and type I (ALK4) receptor serine kinases. ActRII/IIB are promiscuous receptors known to bind at least a dozen TGF-beta superfamily ligands including activins, myostatin, several BMPs, and nodal. Here we utilize a new screening procedure to rapidly identify activin-A mutants with loss of signaling activity. Our goal was to identify activin-A mutants able to bind ActRII but unable to bind ALK4 and which would be, therefore, candidate type II activin receptor antagonists. Using the structure of BMP-2 bound to its type I receptor (ALK3) as a guide, we introduced mutations in the context of the inhibin betaA cDNA and assessed the signaling activity of the resulting mutant proteins. We identified several mutants in the finger (M91E, I105E, M108A) and wrist (activin A/activin C chimera, S60P, I63P) regions of activin-A with reduced signaling activity. Of these the M108A mutant displayed the lowest signaling activity while retaining wild-type-like affinity for ActRII. Unlike wild-type activin-A, the M108A mutant was unable to form a cross-linked complex with ALK4 in the presence of ActRII indicating that its ability to bind ALK4 was disrupted. This data suggested that the M108A mutant might be capable of modulating signaling of activin and related ligands. Indeed, the M108A mutant antagonized activin-A and myostatin, but not TGF-beta, signaling in 293T cells, indicating it may be generally capable of blocking ligands that signal via ActRII/IIB.     

Activin A/bone morphogenetic protein (BMP) chimeras exhibit BMP-like activity and antagonize activin and myostatin

Activins and bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family of growth and differentiation factors that induce signaling in target cells by assembling type II and type I receptors at the cell surface. Ligand residues involved in type II binding are located predominantly in the C-terminal region that forms an extended beta-sheet, whereas residues involved in type I binding are located in the alpha-helical and preceding loop central portion of the molecule. To test whether the central residues are sufficient to determine specificity toward type I receptors, activin A/BMP chimeras were constructed in which the central residues (45-79) of activin A were replaced with corresponding residues of BMP2 and BMP7. The chimeras were assessed for activin type II receptor (Act RII) binding, activin-like bioactivity, and BMP-like activity as well as antagonistic properties toward activin A and myostatin. ActA/BMP7 chimera retained Act RII binding affinity comparable with wild type activin A, whereas ActA/BMP2 chimera showed a slightly reduced affinity toward Act RII. Both the chimeras were devoid of significant activin bioactivity in 293T cells in the A3 Lux reporter assay up to concentrations 10-fold higher than the minimal effective activin A concentration (approximately 4 nM). In contrast, these chimeras showed BMP-like activity in a BRE-Luc assay in HepG2 cells as well as induced osteoblast-like phenotype in C2C12 cells expressing alkaline phosphatase. Furthermore, both the chimeras activated Smad1 but not Smad2 in C2C12 cells. Also, both the chimeras antagonized ligands that signal via activin type II receptor, such as activin A and myostatin. These data indicate that activin residues in the central region determine its specificity toward type I receptors. ActA/BMP chimeras can be useful in the study of receptor specificities and modulation of transforming growth factor-beta members, activins, and BMPs.     

Identification of a functional binding site for activin on the type I receptor ALK4

Activins, like other members of the transforming growth factor-beta (TGF-beta) superfamily, initiate signaling by assembling a complex of two types of transmembrane serine/threonine receptor kinases classified as type II (ActRII or ActRIIB) and type I (ALK4). A kinase-deleted version of ALK4 can form an inactive complex with activin and ActRII/IIB and thereby acts in a dominant negative manner to block activin signaling. Using the complex structure of bone morphogenetic protein-2 bound to its type I receptor (ALK3) as a guide, we introduced extracellular domain mutations in the context of the truncated ALK4 (ALK4-trunc) construct and assessed the ability of the mutants to inhibit activin function. We have identified five hydrophobic amino acid residues on the ALK4 extracellular domain (Leu40, Ile70, Val73, Leu75, and Pro77) that, when mutated to alanine, have substantial effects on ALK4-trunc dominant negative activity. In addition, eleven mutants partially affected activin binding to ALK4. Together, these residues likely constitute the binding surface for activin on ALK4. Cross-linking studies measuring binding of 125I-activin-A to the ALK4-trunc mutants in the presence of ActRII implicated the same residues. Our results indicate that there is only a partial overlap of the binding sites on ALK4 and ALK3 for activin-A and bone morphogenetic protein-2, respectively. In addition three of the residues required for activin binding to ALK4 are conserved on the type I TGF-beta receptor ALK5, suggesting the corresponding region on ALK5 may be important for TGF-beta binding.     

Cripto is a noncompetitive activin antagonist that forms analogous signaling complexes with activin and nodal

Cripto plays critical roles during embryogenesis and has been implicated in promoting the growth and spread of tumors. Cripto is required for signaling by certain transforming growth factor-beta superfamily members, such as Nodal, but also antagonizes others, such as activin. The opposing effects of Cripto on Nodal and activin signaling seem contradictory, however, because these closely related ligands utilize the same type I (ALK4) and type II (ActRII/IIB) receptors. Here, we have addressed this apparent paradox by demonstrating that Cripto forms analogous receptor complexes with Nodal and activin and functions as a noncompetitive activin antagonist. Our results show that activin-A and Nodal elicit similar maximal signaling responses in the presence of Cripto that are substantially lower than that of activin-A in the absence of Cripto. In addition, we provide biochemical evidence for complexes containing activin-A, Cripto, and both receptor types and show that the assembly of such complexes is competitively inhibited by Nodal. We further demonstrate that Nodal and activin-A share the same binding site on ActRII and that ALK4 has distinct and separable binding sites for activin-A and Cripto. Finally, we show that ALK4 mutants with disrupted activin-A binding retain Cripto binding and prevent the effects of Cripto on both activin-A and Nodal signaling. Together, our data indicate that Cripto facilitates Nodal signaling and inhibits activin signaling by forming receptor complexes with these ligands that are structurally and functionally similar.     

Identification of a binding site on the type II activin receptor for activin and inhibin

Type II activin receptors (ActRII and ActRIIB) are single-transmembrane domain serine/threonine kinase receptors that bind activin to initiate the signaling and cellular responses triggered by this hormone. Inhibin also binds type II activin receptors and antagonizes many activin effects. Here we describe alanine scanning mutagenesis of the ActRII extracellular domain. We identify a cluster of three hydrophobic residues (Phe(42), Trp(60), and Phe(83)) that, when individually mutated to alanine in the context of the full-length receptor, cause the disruption of activin and inhibin binding to ActRII. Each of the alanine-substituted ActRII mutants retaining activin binding maintains the ability to form cross-linked complexes with activin and supports activin cross-linking to the type I activin receptor ALK4. Unlike wild-type ActRII, the three mutants unable to bind activin do not cause an increase in activin signaling when transiently expressed in a corticotroph cell line. Together, our results implicate these residues in forming a critical binding surface on ActRII required for functional interactions with both activin and inhibin. This first identification of a transforming growth factor-beta family member binding site may provide a general basis for characterizing binding sites for other members of the superfamily.     

Inhibin α-subunit N terminus interacts with activin type IB receptor to disrupt activin signaling

Inhibin is a heterodimeric peptide hormone produced in the ovary that antagonizes activin signaling and FSH synthesis in the pituitary. The inhibin β-subunit interacts with the activin type II receptor (ActRII) to functionally antagonize activin. The inhibin α-subunit mature domain (N terminus) arose relatively early during the evolution of the hormone, and inhibin function is decreased by an antibody directed against the α-subunit N-terminal extension region or by deletion of the N-terminal region. We hypothesized that the α-subunit N-terminal extension region interacts with the activin type I receptor (ALK4) to antagonize activin signaling in the pituitary. Human or chicken free α-subunit inhibited activin signaling in a pituitary gonadotrope-derived cell line (LβT2) in a dose-dependent manner, whereas an N-terminal extension deletion mutant did not. An α-subunit N-terminal peptide, but not a control peptide, was able to inhibit activin A signaling and decrease activin-stimulated FSH synthesis. Biotinylated inhibin A, but not activin A, bound ALK4. Soluble ALK4-ECD bioneutralized human free α-subunit in LβT2 cells, but did not affect activin A function. Competitive binding ELISAs with N-terminal mutants and an N-terminal region peptide confirmed that this region is critical for direct interaction of the α-subunit with ALK4. These data expand our understanding of how endocrine inhibin achieves potent antagonism of local, constitutive activin action in the pituitary, through a combined mechanism of competitive binding of both ActRII and ALK4 by each subunit of the inhibin heterodimer, in conjunction with the co-receptor betaglycan, to block activin receptor-ligand binding, complex assembly, and downstream signaling.     

The 1.1 A crystal structure of human TGF-beta type II receptor ligand binding domain

Transforming growth factor beta (TGF-beta) is involved in a wide range of biological functions including development, carcinogenesis, and immune regulation. Here we report the 1.1 A resolution crystal structure of human TGF-beta type II receptor ectodomain (TBRII). The overall structure of TBRII is similar to that of activin type II receptor ectodomain (ActRII) and bone morphogenic protein receptor type IA (BRIA). It displays a three-finger toxin fold with fingers formed by the beta strand pairs beta1-beta2, beta3-beta4, and beta5-beta6. The first finger in the TBRII is significantly longer than in ActRII and BRIA and folds tightly between the second finger and the C terminus. Surface charge distributions and hydrophobic patches predict potential TBRII binding sites.     

Reconstitution and analysis of soluble inhibin and activin receptor complexes in a cell-free system

Activins and inhibins compose a heterogeneous subfamily within the transforming growth factor-beta (TGF-beta) superfamily of growth and differentiation factors with critical biological activities in embryos and adults. They signal through a heteromeric complex of type II, type I, and for inhibin, type III receptors. To characterize the affinity, specificity, and activity of these receptors (alone and in combination) for the inhibin/activin subfamily, we developed a cell-free assay system using soluble receptor-Fc fusion proteins. The soluble activin type II receptor (sActRII)-Fc fusion protein had a 7-fold higher affinity for activin A compared with sActRIIB-Fc, whereas both receptors had a marked preference for activin A over activin B. Although inhibin A and B binding was 20-fold lower compared with activin binding to either type II receptor alone, the mixture of either type II receptor with soluble TGF-beta type III receptor (TbetaRIII; betaglycan)-Fc reconstituted a soluble high affinity inhibin receptor. In contrast, mixing either soluble activin type II receptor with soluble activin type I receptors did not substantially enhance activin binding. Our results support a cooperative model of binding for the inhibin receptor (ActRII.sTbetaRIII complex) but not for activin receptors (type II + type I) and demonstrate that a complex composed of activin type II receptors and TbetaRIII is both necessary and sufficient for high affinity inhibin binding. This study also illustrates the utility of this cell-free system for investigating hypotheses of receptor complex mechanisms resulting from crystal structure analyses.     

Analysis and characterization of the functional TGFβ receptors required for BMP6-induced osteogenic differentiation of mesenchymal progenitor cells

Although BMP6 is highly capable of inducing osteogenic differentiation of mesenchymal progenitor cells (MPCs), the molecular mechanism involved remains to be fully elucidated. Using dominant negative (dn) mutant form of type I and type II TGFβ receptors, we demonstrated that three dn-type I receptors (dnALK2, dnALK3, dnALK6), and three dn-type II receptors (dnBMPRII, dnActRII, dnActRIIB), effectively diminished BMP6-induced osteogenic differentiation of MPCs. These findings suggested that ALK2, ALK3, ALK6, BMPRII, ActRII and ActRIIB are essential for BMP6-induced osteogenic differentiation of MPCs. However, MPCs in this study do not express ActRIIB. Moreover, RNA interference of ALK2, ALK3, ALK6, BMPRII and ActRII inhibited BMP6-induced osteogenic differentiation in MPCs. Our results strongly suggested that BMP6-induced osteogenic differentiation of MPCs is mediated by its functional TGFβ receptors including ALK2, ALK3, ALK6, BMPRII, and ActRII. [BMB Reports 2013; 46(2): 107-112]     

A flexible activin explains the membrane-dependent cooperative assembly of TGF-beta family receptors

A new crystal structure of activin in complex with the extracellular domain of its type II receptor (ActRIIb-ECD) shows that the ligand exhibits an unexpected flexibility. The motion in the activin dimer disrupts its type I receptor interface, which may account for the disparity in its affinity for type I versus type II receptors. We have measured the affinities of activin and its antagonist inhibin for ActRIIb-ECD and found that the affinity of the 2-fold symmetric homodimer activin for ActRIIb-ECD depends on the availability of two spatially coupled ActRIIb-ECD molecules, whereas the affinity of the heterodimer inhibin does not. Our results indicate that activin's affinity for its two receptor types is greatly influenced by their membrane-restricted setting. We propose that activin affinity is modulated by the ligand flexibility and that cooperativity is achieved by binding to two ActRII chains that immobilize activin in a type I binding-competent orientation.     

Alantolactone inhibits cell proliferation by interrupting the interaction between Cripto-1 and activin receptor type II A in activin signaling pathway

It has been suggested that deregulation of activin signaling contributes to tumor formation. Activin signaling is blocked in cancer cells due to the complex formed by Cripto-1, activin, and activin receptor type II (ActRII). In this study, the authors used a mammalian two-hybrid system to construct a drug screening model to obtain a small molecular inhibitor capable of interrupting the interaction between Cripto-1 and ActRII. They screened 300 natural components and identified alantolactone. Data suggested that alantolactone induced activin/SMAD3 signaling in human colon adenocarcinoma HCT-8 cells. The authors also found that alantolactone exhibited antiproliferative function specific to tumor cells, with almost no toxicity to normal cells at a concentration of 5 μg/mL. Furthermore, they proved that the antiproliferative function of alantolactone was activin/SMAD3 dependent. These results suggest that alantolactone performs its antitumor effect by interrupting the interaction between Cripto-1 and the activin receptor type IIA in the activin signaling pathway. Moreover, screening for inhibitors of Cripto-1/ActRII is a potentially beneficial approach to aid in discovering novel cancer treatment.     

High resolution structures of the bone morphogenetic protein type II receptor in two crystal forms: implications for ligand binding

BMPRII is a type II TGF-beta serine threonine kinase receptor which is integral to the bone morphogenetic protein (BMP) signalling pathway. It is known to bind BMP and growth differentiation factor (GDF) ligands, and has overlapping ligand specificity with the activin type II receptor, ActRII. In contrast to activin and TGF-beta type ligands, BMPs bind to type II receptors with lower affinity than type I receptors. Crystals of the BMPRII ectodomain were grown in two different forms, both of which diffracted to high resolution. The tetragonal form exhibited some disorder, whereas the entire polypeptide was seen in the orthorhombic form. The two structures retain the basic three-finger toxin fold of other TGF-beta receptor ectodomains, and share the main hydrophobic patch used by ActRII to bind various ligands. However, they present different conformations of the A-loop at the periphery of the proposed ligand-binding interface, in conjunction with rearrangement of a disulfide bridge within the loop. This particular disulfide (Cys94-Cys117) is only present in BMPRII and activin receptors, suggesting that it is important for their likely shared mode of binding. Evidence is presented that the two crystal forms represent ligand-bound and free conformations of BMPRII. Comparison with the solved structure of ActRII bound to BMP2 suggests that His87, unique amongst TGF-beta receptors, may play a key role in ligand recognition.     

An Antibody Blocking Activin Type II Receptors Induces Strong Skeletal Muscle Hypertrophy and Protects from Atrophy

The myostatin/activin type II receptor (ActRII) pathway has been identified to be critical in regulating skeletal muscle size. Several other ligands, including GDF11 and the activins, signal through this pathway, suggesting that the ActRII receptors are major regulatory nodes in the regulation of muscle mass. We have developed a novel, human anti-ActRII antibody (bimagrumab, or BYM338) to prevent binding of ligands to the receptors and thus inhibit downstream signaling. BYM338 enhances differentiation of primary human skeletal myoblasts and counteracts the inhibition of differentiation induced by myostatin or activin A. BYM338 prevents myostatin- or activin A-induced atrophy through inhibition of Smad2/3 phosphorylation, thus sparing the myosin heavy chain from degradation. BYM338 dramatically increases skeletal muscle mass in mice, beyond sole inhibition of myostatin, detected by comparing the antibody with a myostatin inhibitor. A mouse version of the antibody induces enhanced muscle hypertrophy in myostatin mutant mice, further confirming a beneficial effect on muscle growth beyond myostatin inhibition alone through blockade of ActRII ligands. BYM338 protects muscles from glucocorticoid-induced atrophy and weakness via prevention of muscle and tetanic force losses. These data highlight the compelling therapeutic potential of BYM338 for the treatment of skeletal muscle atrophy and weakness in multiple settings.     

Localization of an activin/activin receptor system in the porcine ovary.

The aim of this study was to locate a possible activin/activin receptor system within porcine ovaries containing functional corpora lutea. In situ hybridization was used to assess the gene expression of beta(A)- and beta(B)-activin subunits, and immunohistochemical studies were done to detect activin-A protein and activin receptor type II. mRNA expression of the beta(A)- and beta(B)-activin subunits was found in the granulosa from the unilaminar follicle stage onward, in the developing thecal layer of multilaminar and small antral follicles, in the theca interna of mid-sized antral follicles, in corpora lutea, and in the ovarian surface epithelium. Immunoreactive activin A protein could be detected at the same ovarian sites, but in thecal tissue of small antral follicles only. This protein was also demonstrated at the peripheral zone of oocytes from multilaminar and antral follicles. A positive immunoreaction for activin receptor was found in granulosa cells from multilaminar and older follicles and in oocytes from the earliest stages of follicular development onward. In late multilaminar follicles and in antral follicles, the oolemma was stained. Except for small antral follicles, a positive activin receptor immunoreaction was absent in the follicular theca. Activin receptor immunoreaction was furthermore present in corpora lutea and in the ovarian surface epithelium. It is concluded that, within porcine ovaries containing functional corpora lutea, an activin/activin receptor system is present in all intact follicles, the corpora lutea and the surface epithelium. Within follicles, granulosa and theca cells are the main sites of activin synthesis, while oocytes and granulosa cells are the main activin binding sites.     

Characterization of the Ligand Binding Functionality of the Extracellular Domain of Activin Receptor Type IIB

The single transmembrane domain serine/threonine kinase activin receptor type IIB (ActRIIB) has been proposed to bind key regulators of skeletal muscle mass development, including the ligands GDF-8 (myostatin) and GDF-11 (BMP-11). Here we provide a detailed kinetic characterization of ActRIIB binding to several low and high affinity ligands using a soluble activin receptor type IIB-Fc chimera (ActRIIB.Fc). We show that both GDF-8 and GDF-11 bind the extracellular domain of ActRIIB with affinities comparable with those of activin A, a known high affinity ActRIIB ligand, whereas BMP-2 and BMP-7 affinities for ActRIIB are at least 100-fold lower. Using site-directed mutagenesis, we demonstrate that ActRIIB binds GDF-11 and activin A in different ways such as, for example, substitutions in ActRIIB Leu⁷⁹ effectively abolish ActRIIB binding to activin A yet not to GDF-11. Native ActRIIB has four isoforms that differ in the length of the C-terminal portion of their extracellular domains. We demonstrate that the C terminus of the ActRIIB extracellular domain is crucial for maintaining biological activity of the ActRIIB.Fc receptor chimera. In addition, we show that glycosylation of ActRIIB is not required for binding to activin A or GDF-11. Together, our findings reveal binding specificity and activity determinants of the ActRIIB receptor that combine to effect specificity in the activation of distinct signaling pathways.     

Activin-A up-regulates type I activin receptor mRNA levels in human immortalized extravillous trophoblast cells.

Activin is known to play an important regulatory role in reproduction, including pregnancy. To further examine the role and signaling mechanism of activin in regulating placental function, the steady-state level of activin type I receptor (ActRI) mRNA in immortalized extravillous trophoblasts (IEVT) cells was measured using competitive PCR (cPCR). An internal standard of ActRI cDNA for cPCR was constructed for the quantification of ActRI mRNA levels in IEVT cells. ActRI mRNA levels were increased in a dose-dependent manner by activin-A with the maximal effect observed at the dose of 10 ng/ml. Time course studies revealed that activin-A had maximal effects on ActRI mRNA levels at 6 hours after treatment. The effects of activin-A on ActRI mRNA levels was blocked by follistatin, an activin binding protein, in a dose-dependent manner. In addition, inhibin-A inhibited basal, as well as activin-A-induced ActRI mRNA levels. These findings provide evidence, for the first time, that activin-A modulates ActRI mRNA levels in human trophoblast cells.     

Epidermal growth factor contains both positive and negative determinants for interaction with ErbB-2/ErbB-3 heterodimers

Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are potent activators of the ErbB-1 receptor, but, unlike TGF-alpha, EGF is also a weak activator of ErbB-2/ErbB-3 heterodimers. To understand the specificity of EGF-like growth factors for binding to distinct ErbB members, we used EGF/TGF-alpha chimeras to examine the requirements for ErbB-2/ErbB-3 activation. Here we show that in contrast to these two wild-type ligands, distinct EGF/TGF-alpha chimeras are potent activators of ErbB-2/ErbB-3 heterodimers. On the basis of differences in the potency of these various chimeras, specific residues in the linear N-terminal region and the so-called B-loop of these ligands were identified to be involved in interaction with ErbB-2/ErbB-3. A chimera consisting of human EGF sequences with the linear N-terminal region of human TGF-alpha was found to be almost as potent as the natural ligand neuregulin (NRG)-1beta in activating 32D cells expressing ErbB-2/ErbB-3 and human breast cancer cells. Binding studies revealed that this chimera, designated T1E, has high affinity for ErbB-2/ErbB-3 heterodimers, but not for ErbB-3 alone. Subsequent exchange studies revealed that introduction of both His2 and Phe3 into the linear N-terminal region was already sufficient to make EGF a potent activator of ErbB-2/ErbB-3 heterodimers, indicating that these two amino acids contribute positively to this receptor binding. Analysis of the B-loop revealed that Leu26 in EGF facilitates interaction with ErbB-2/ErbB-3 heterodimers, while the equivalent Glu residue in TGF-alpha impairs binding. Since all EGF/TGF-alpha chimeras tested have maintained high binding affinity for ErbB-1, it is concluded that the diversity of the ErbB signaling network is determined by specific amino acids that facilitate binding to one receptor member, in addition to residues that impede binding to other ErbB family members.     

Extracellular domain chimeras of the TSH and LH/CG receptors reveal the mid-region (amino acids 171-260) to play a vital role in high affinity TSH binding

We constructed a series of TSH-LH/CG receptor chimeras by homologous substitution of relatively small regions of the TSH receptor extracellular domain for the corresponding region of the extracellular domain of the LH/CG receptor. Constructs were stably expressed in Chinese hamster ovary cells. Of the five chimeric receptors, only TSH-LHR-14, which contains mid-region domain C (amino acid residues 171-260) of the extracellular component of the TSH receptor, exhibited TSH binding of relatively high affinity. Consistent with this TSH binding, chimera TSH-LHR-14 was the only one that demonstrated a functional response to TSH stimulation in terms of intracellular cAMP generation. These data indicate that domain C plays a vital role in TSH receptor function.